Method for specifically mapping antigens to antibody-secreting cells

Abstract

The present disclosure provides antibody capture complexes and methods of capturing target antibodies secreted by antibody secreting cells.

Description

[0001] References to sequence listings

[0002] This application includes a sequence listing submitted electronically as an XML file, named 381203733SEQ, created on March 10, 2023, and 824 kilobytes in size. The sequence listing is incorporated herein by reference. Technical Field

[0003] The present disclosure relates in part to antibody capture complexes and methods of mapping antigen specificity to antibody secreting cells. Background Art

[0004] B cells express B cell receptors (BCR) on their surfaces, allowing the determination of antigen-specific B cell repertoires. Although there are a variety of platforms available for discovering antibodies from B cells expressing cell surface BCR, the affinity of separated antibodies varies from low to high. After activation by homologous antigens, B cells undergo fine-tuning of their BCRs and eventually differentiate into antibody secreting cells (ASCs). Antibody secreting cells are a specialized cell type that represents the end of the B cell differentiation program and includes plasmablasts, short-lived plasma cells, and long-lived plasma cells (or more commonly, referred to as "plasma cells" (PC); Tellier and Nutt, Eur. J. Immunol., 2019, 49, 30-37). These stem cells can produce high-affinity antibodies with potential for treatment or prevention. However, ASC may also be the source of antibody-mediated pathology.

[0005] While antigen-specific antibodies from B cells that express a BCR on their cell surface can be easily cloned and sequenced following flow cytometric isolation, PCs, which are ASCs that do not express a cell surface BCR, cannot currently be easily profiled in a high-throughput manner. Although PCs may be a source of very high-affinity antibodies, the specificity of any given secreted antibody cannot be rapidly determined. Because current platforms have limitations in their application to PCs, there has long been a need to efficiently map a given antibody to the BCR that secretes that antibody. - PC pairing, and effectively determine the antigen specificity of the antibody. Summary of the invention

[0006] The present disclosure provides an antibody capture complex comprising a first component of a binding pair connected to an antibody capture molecule; wherein the first component of the binding pair is capable of binding to a second component of the binding pair; wherein the antibody capture molecule is capable of binding to a target antibody, wherein the target antibody is secreted by an antibody secreting cell. In some embodiments, when the second component of the binding pair is biotin and the first component of the binding pair is streptavidin, the antibody capture molecule does not bind to the κ light chain of the target antibody.

[0007] The present disclosure also provides a method for capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting an antibody-secreting cell population with a second component of a binding pair so that the second component of the binding pair binds to a cell surface of the antibody-secreting cell population, wherein the antibody-secreting cell population comprises antibody-secreting cells that secrete the target antibody; contacting the antibody-secreting cell population with an antibody capture complex, wherein the antibody capture complex comprises a first component of the binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to the second component of the binding pair on the cell surface of the antibody-secreting cell, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cell; and contacting the antibody-secreting cell population with an antigen, whereby the target antibody secreted by the antibody-secreting cell and captured by the antibody capture complex binds to the antigen.

[0008] The present disclosure also provides a method for capturing target antibodies secreted by antibody-secreting cells, the method comprising: contacting an antibody-secreting cell population with an antibody capture complex, wherein the antibody-secreting cell population comprises antibody-secreting cells that secrete the target antibody, wherein the antibody capture complex comprises a first component of a binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to a second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; and contacting the antibody-secreting cell population with an antigen, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the antigen.

[0009] The present disclosure also provides a method for capturing IgE antibodies secreted by antibody-secreting cells, wherein the IgE antibodies are directed against allergens, the method comprising: contacting an antibody-secreting cell population with NHS-biotin to bind biotin to the cell surface, wherein the antibody-secreting cell population comprises antibody-secreting cells that secrete IgE antibodies; contacting the antibody-secreting cell population with an antibody capture complex, wherein the antibody capture complex comprises streptavidin linked to the extracellular domain of a high-affinity IgE receptor (FcεRIa), whereby the antibody capture molecule binds to the IgE antibodies secreted by the antibody-secreting cells; and contacting the antibody-secreting cell population with an antigen, wherein the antigen is an antigenic portion of an allergen or a mixture of multiple antigenic portions, whereby the IgE antibodies captured by the antibody capture complex bind to the antigen.

[0010] In some embodiments of the methods disclosed herein, the method further comprises: after contacting the antibody secreting cell population with the antigen, sorting the antibody secreting cells to collect the antibody secreting cell pool, wherein the antibody secreting cells in the pool each secrete antibodies captured by the antibody capture molecule and bound by the antigen, wherein the collected antibody secreting cell pool comprises antibody secreting cells that secrete the target antibody. In some embodiments of the methods disclosed herein, the method further comprises separating the collected antibody secreting cell pool into single cells and separating the antibody secreting cells that secrete the target antibody. In some embodiments, nucleic acids encoding antibodies are separated from antibody secreting cells that secrete the target antibody.

[0011] In some embodiments of the methods disclosed herein, the step of contacting a population of antibody-secreting cells with an antigen comprises: (a) contacting the population of antibody-secreting cells with a first labeled form of the antigen so that the antigen binds to antibodies captured at the cell surface of the antibody-secreting cell population including the target antibodies captured on the cell surface of the antibody-secreting cells, wherein the first labeled form of the antigen is conjugated to a first detectable label; (b) washing the population of antibody-secreting cells to remove unbound antigen; (c) contacting the population of antibody-secreting cells with: (i) an unlabeled form of the antigen, (ii) a second labeled form of the antigen, or (iii) an unlabeled form of the antigen and a second labeled form of the antigen; (d) washing the population of antibody-secreting cells to remove unbound antigen, and (e) collecting a pool of antibody-secreting cells that remain bound to the first labeled form of the antigen, wherein the collected pool of antibody-secreting cells contains antibody-secreting cells that secrete the target antibody, and wherein the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex remains bound to the first labeled form of the antigen.

[0012] In some embodiments of the methods disclosed herein, the method further comprises: after collecting the antibody-secreting cell pool that retains the antigen binding in the first labeled form, separating the collected antibody-secreting cell pool into single cells and isolating the antibody-secreting cells that secrete the target antibody. In some embodiments, nucleic acids encoding antibodies are isolated from the antibody-secreting cells that secrete the target antibody.

[0013] The present disclosure also provides a method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by a cell comprising a modified cell surface, and wherein the target antibody is subsequently captured on the modified cell surface, the method comprising: a) providing an antibody-secreting cell comprising a modified cell surface, wherein the antibody-secreting cell secretes the target antibody, wherein the target antibody is subsequently captured on the modified cell surface, wherein the target antibody captured on the modified cell surface binds to an antigen, wherein the antigen is linked to a barcode nucleic acid molecule, and wherein the antibody-secreting cell further comprises a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of the target antibody captured on the modified cell surface; b) subjecting the barcode nucleic acid molecule to a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of the target antibody captured on the modified cell surface; The invention relates to a method for preparing an antigen barcode nucleic acid molecule comprising a first amplicon library for gene expression in antibody-secreting cells, a second amplicon library for variable regions, diversity regions, and joining regions (VDJ) in antibody-secreting cells, and a third amplicon library for antigen barcode nucleic acid molecules; e) sequencing each of the three amplicon libraries; and f) identifying the region of the gene encoding the antigen binding fragment of the target antibody captured on the surface of the modified cell.

[0014] The present disclosure also provides a method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with a second component of a binding pair so that the second component of the binding pair binds to a cell surface, wherein the population of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody; b) contacting the population of antibody-secreting cells with an antibody capture complex, wherein the antibody capture complex comprises a first component of a binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to the second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cell; c) contacting the population of antibody-secreting cells with a second anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cell and captured by the antibody capture complex binds to the second anti-Ig antibody; d) contacting the population of antibody-secreting cells with an antigen comprising a barcode nucleic acid molecule, whereby the target antibody secreted by the antibody-secreting cell and captured by the antibody capture complex binds to the antigen; e) collecting the antibody-secreting a cell pool, wherein each cell in the pool secretes an antibody captured by an antibody capture complex and bound by a second anti-Ig antibody and an antigen comprising a barcode nucleic acid molecule, and wherein the antibody-secreting cell pool comprises antibody-secreting cells that secrete a target antibody; f) separating the antibody-secreting cell pool into single antibody-secreting cells, and for each single antibody-secreting cell: 1) hybridizing a portion of the barcode nucleic acid molecule with a portion of a first nucleic acid molecule attached to a solid surface, wherein the solid surface and the first nucleic acid molecule attached thereto are unique to each single antibody-secreting cell; 2) hybridizing a portion of a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of the target antibody with a portion of a second nucleic acid molecule attached to the solid surface; 3) preparing a first amplicon library of gene expression, a second amplicon library of variable regions, diversity regions, and joining regions (VDJ), and a third amplicon library of antigen barcode nucleic acid molecules; and 4) sequencing each of the three amplicon libraries; and g) identifying a region of a gene encoding an antigen-binding fragment of the target antibody.

[0015] The present disclosure also provides a method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with an antibody capture complex, wherein the population of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody, wherein the antibody capture complex comprises a first component of a binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to a second component of the binding pair on the cell surface of the population of antibody-secreting cells, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cell; b) contacting the population of antibody-secreting cells with a second anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cell and captured by the antibody capture complex binds to the second anti-Ig antibody; c) contacting the population of antibody-secreting cells with an antigen comprising a barcode nucleic acid molecule, whereby the target antibody secreted by the antibody-secreting cell and captured by the antibody capture complex binds to the antigen; d) collecting a pool of antibody-secreting cells, wherein each cell in the pool secretes a second component of the binding pair linked to an antibody capture molecule; The method comprises the steps of: obtaining antibodies captured by a complex and bound by a second anti-Ig antibody and an antigen comprising a barcode nucleic acid molecule, and wherein the pool of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody; e) separating the pool of antibody-secreting cells into single antibody-secreting cells, and for each single antibody-secreting cell: 1) hybridizing a portion of the barcode nucleic acid molecule with a portion of a first nucleic acid molecule attached to a solid surface, wherein the solid surface and the first nucleic acid molecule attached thereto are unique to each single antibody-secreting cell; 2) hybridizing a portion of a nucleic acid molecule encoding a region of a gene encoding an antigen-binding fragment of the target antibody with a portion of a second nucleic acid molecule attached to the solid surface; 3) preparing a first amplicon library of gene expression, a second amplicon library of variable regions, diversity regions, and joining regions (VDJ), and a third amplicon library of antigen barcode nucleic acid molecules; and 4) sequencing each of the three amplicon libraries; and f) identifying the region of the gene encoding the antigen-binding fragment of the target antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] Figure 1 A representative workflow of the Ig secretion capture method is shown. Step 1 shows biotinylation of cells using N-hydroxysuccinimide biotin (NHS-biotin). Step 2 shows the Ig secretion capture reagent, which contains streptavidin coupled to anti-Fc. αIgK is used to express antibodies to the κ light chain or FcεRIα extracellular domain for specific IgE capture. Step 3 shows the secreted αIgE or αIgG antibody binding to an oligonucleotide barcoded and fluorescently labeled antigen bound by an anti-IgG or anti-IgE antibody.

[0018] Figure 2 A representative workflow of the IgE secretion capture method is shown. Step 1 shows biotinylation of PC using NHS-biotin. Step 2 shows a reagent comprising the extracellular domain of the high affinity IgE receptor (ectoFcεRIα) conjugated to streptavidin ("Secretory IgE Capture Reagent") and streptavidin bound to biotin on PC. Step 2 also shows the ectoFcεRIα of the Secretory IgE Capture Reagent capturing IgE secreted by PC. Step 3 shows captured IgE bound to barcoded antigen and bound by anti-IgE antibody.

[0019] Figure 3 A representative workflow of the IgG secretion capture method is shown. Step 1 shows biotinylation of cells using NHS-biotin. Step 2 shows the IgG secretion capture reagent, which contains streptavidin-conjugated anti-mouse IgK assembled on biotinylated cells. Step 3 shows secreted IgG antibodies bound to barcoded antigens and bound by anti-IgG antibodies.

[0020] 4A to 4F Display, in Figure 4A In the present study, secreted IgG captured on the surface of ARH77 cells was detected. Figure 4B Detection of secretory IgE captured on the surface of U266 cells is shown. Figure 4C It was shown that, although secretory IgE captured on the surface of U266 cells was detected, surface IgE as a B cell receptor was not detected. Figure 4D Titration of streptavidin-αIgk (StAv-αIgk) on ARH77 cells is shown. Figure 4E Titration of Streptavidin-FceRIα (StAv-FcεRIα) on U266 cells is shown. Figure 4F High specificity was shown for detecting secretory IgE captured on the surface of U266 cells, but not IgM secreted by Ramos cells.

[0021] FIG. 5A to FIG. 5G Display, in Figure 5A , a representative workflow of the procedure for determining the sequence of the V(D)J regions of antibodies specific for a barcoded antigen and a barcoded antigen, and then confirming the antigen specificity of the antibodies by enzyme-linked immunosorbent assay (ELISA). Figure 5B Flow cytometry of antigen-specific IgG captured on the surface of PCs from hIL-4Rα-immunized mice is shown. Figure 5CVisualization of antigen specificity based on barcode signals of hIL-6Rα (non-specific) and hIL-4Rα (specific) in bone marrow (BM) PCs and spleen-draining lymph node (dLN) B cells and PCs is shown. Figure 5D Shown are UMAPs of B cells and PCs isolated from spleen / dLN and bone marrow of challenged mice. Figure 5E Shown are graphs indicating the levels of total IgG, anti-hIL-4Rα ELISA antibody, and the ratio of anti-hIL-4Rα to total IgG in BM-PCs, SP-PCs, and SP-B cells. Fig. 5F and Figure 5G Shown are graphs from two separate experiments illustrating the binding affinity of antibodies cloned from BM-PCs, SP-PCs, and SP-B cells.

[0022] 6A to 6F Display, in Fig. 6A , a graph illustrating the percent similarity of hIL-4Rα-specific and nonspecific cells isolated from BM-PCs, spleen / dLN-PCs, and spleen / dLN-B cells of challenged mice to germline V regions. Figure 6B Shown is a Venn diagram illustrating the overlap of clones based on the CDR3 nucleotide sequences of the heavy and light chains of clones isolated from BM-PCs, spleen / dLN-PCs, and spleen / dLN-B cells. Figure 6C Volcano plot comparison of differential gene expression between hIL-4Rα-specific and non-antigen-specific BM-PCs is shown. Fig.6D Shown is a heat map illustrating the transcriptional profiles of differentially expressed genes in hIL-4Rα-specific and non-antigen-specific BM-PCs. Fig. 6E Shown is a UMAP illustrating the localization and binding affinity (Kd) of hIL-4Rα-specific antibodies from BM-PCs on total BM-PCs. Fig. 6F UMAP illustrating proportional gene expression of Cd24a, Ppob, Fkbp11, Ssr2, Tmem176a, Tmem176b, Ly6d, and CD74 in BM-PCs is shown.

[0023] 7A to 7H Display, in Fig. 7A , a representative workflow of the procedure for determining the sequence of the V(D)J regions of antibodies specific for a barcoded antigen and a barcoded antigen, and then confirming the antigen specificity of the antibodies by enzyme-linked immunosorbent assay (ELISA). Figure 7B From left to right, it shows cells, single cells, living cells, and DUMP - Cells and CD138 + Blimp-1 +Gating of cells' PCs. Figure 7C IgE secretion Venus+ PCs were derived from BM and LDLN of mice that had been challenged with house dust mite (HDM) extract. Fig.7D IgE based on VDJ-BCR expression on non-IgE PCs is shown + UMAP for PC. Fig. 7E The proportions of IgE, IgG, IgA, and IgM PCs and the average expression of the top 4 genes expressed in the different isotypes in dLN and BM are shown. Figure 7F Visualization of antigen specificity based on barcode expression of Der p 1, Der p 2, Der f 1 relative to the negative control Ole e 1 is shown. Figure 7G Representative binding curves of IgE antibodies to Der p 1, Der p 2, Der f 1, and the negative control antigen Ole e 1 are shown. Figure 7H Antigen specificity verified by ELISA for a subset of IgE antibodies produced by IgE-secreting cells is shown.

[0024] FIG. 8A to FIG. 8G Display, in Fig. 8A Flow chart of BM from allergic bone marrow donors, BM from non-allergic bone marrow control donors, and non-streptavidin FcεR1α capture control. Figure 8B Results of tests performed on serum to detect IgE levels specific for cat dander e1, dog dander e5, common birch t3, house dust mite d1, Timothy g6, and Alternaria m6 are shown. Figure 8C Expression dot plots illustrating proportional expression of immunoglobulin genes, as well as PC and B cell markers in total PCs are shown. Fig.8D Shown are graphs illustrating the proportional expression of IGHE transcripts in IgE, IgG, IgA and IgM PCs. Fig. 8E Shown is a table of clustered BCR clonotypes indicating the five IgE clonotypes, their clonal sizes, and the number of IgE versus non-IgE cells in each clonotype. Figure 8F An alignment diagram providing a comparison between the IgE sequences from IgE PC and the IgG sequences in clonotype 21 is shown. Figure 8G The antigen specificity of one human IgE antibody isolated from allergic BM by the IgE capture complex of the present disclosure as tested by ELISA is shown. Representative binding curves of human IgE antibodies to FeI d 1, Can e 1, Der p 1 and DNP-IgE are shown.

[0025] Fig. 9Single cell capture and library preparation of mRNA, V(D)J regions, and antigen barcodes are shown. Fig. 9 : (i) the light blue rectangle represents the template switch oligonucleotide; (ii) the beige rectangle represents a sequence of three consecutive riboguanosine residues; (iii) the orange rectangle represents a unique molecular identifier (UMI); (iv) the purple rectangle represents a surface barcode; and (iv) the black square immediately to the right of the purple rectangle represents a sequencing primer.

[0026] Fig.10 Representative flow charts for the analysis of single-cell mRNA, V(D)J region, and antigen barcode libraries are shown.

[0027] Fig.11 Visualization of antigen specificity is shown. Cells with higher antigen specificity scores (i.e., strong signals for the target antigen and low signals for the control antigen) are preferentially used to select antibody candidates. The target antigens are the HDM allergens Der p 1, Der p 2, and Der f 1, and the negative control is the olive antigen Ole e 1. The x-power quantile rank (qC) of the control antigen is subtracted from the quantile rank (qT) of the target antigen, i.e., qT-qC x , to determine the antigen-specific score for each target antigen. Cells with the highest antigen-specific score were selected as antigen-specific candidates.

[0028] Fig.12 A table of IgE candidates based on antigen specificity scores is shown. For each candidate, the table records the sample from which the cells were obtained, the raw antigen barcode counts for each target antigen, the normalized antigen barcode counts, and the antigen specificity score. 1,069 IgE + Cell data.

[0029] FIG. 13A to FIG. 13B Binding of IgE antibodies derived from secretory IgE of PCs captured with a secretory IgE capture reagent, where the PCs were derived from mice challenged with HDM extracts, is shown. Fig.13A Representative binding curves of IgE antibodies to Der p 1 (left panel), Der p 2 (middle panel), and Der f 1 (right panel) are shown. Fig. 13B Representative binding curves of IgE antibodies to the negative control antigen Ole e 1 are shown.

[0030] FIG. 14A to FIG. 14C It was shown that antigen-specific secretory IgG can be expressed in challenged captured on the PC surface of mice. Fig.14A Shown to detect antigen-specific IgG + Continuous flow cytometric gating strategy for splenic B cells. Fig. 14B Shown is a sequential flow cytometric gating strategy to identify BM-derived PCs on which antigen-specific secretory IgG has been captured. Fig. 14C Shown from left to right are four controls used to precisely define the antigen-specific PC population derived from BM and the spleen positive control.

[0031] FIG. 15A to FIG. 15D Shown on the y-axis are normalized values ​​of target antigen barcode counts compared to normalized barcode counts of control antigen barcode counts in all PCs derived from immunized mice and stained by isotype. Fig.15A and Fig. 15B Data from bone marrow cells are shown, whereas Fig. 15C and Fig.15D Data from splenocytes are shown.

[0032] FIG. 16A to FIG. 16C Display, in Fig.16A Middle, flow cytometry images of ARH77 cells stained with αIgG. Fig. 16B Shown are flow cytometric images of U266 cells stained with αIgE. Fig. 16C Shown are flow cytometric analyses of Ramos, U266, or a 100:1 mixture of these cells in the presence of embodiments of the StAv-FceRIα capture complex.

[0033] FIG. 17A to FIG. 17D Display, in Fig.17A In Figure 2, mice underwent an immunization regimen before tissues were collected for downstream antibody specificity mapping via antibody secretion TRAP and sequencing efforts. Fig. 17B IgG-secreting PCs gated as live cells, lymphocytes, monocytes, DUMP- cells (TCRβ, CD200R3, Ly6G, CD49b, CD11b and IgM), CD98+TACI+ and IgG+Ag+ cells are shown. Fig. 17C Ag+IgG+ cells from spleen and dLN gated as live cells, lymphocytes, monocytes, CD19+CD3-, IgD-IgM- and IgG+Ag+ cells are shown. Fig.17D The gate identifying Ag+IgG+BMPCs is shown.

[0034] 18A to 18D Display, in Fig.18A Combined transcriptional profiling and unbiased clustering analysis of splenic plasma cells, BM plasma cells, and splenic B cells. Fig.18B and Fig.18CThree distinct cell populations generated by scRNA-seq are shown: bone marrow plasma cells (BMPCs), spleen / dLN plasmablasts / plasmacytoid cells (Sp / dLN PB / PCs), and spleen / dLN B cells (Sp / dLN B cells). Fig.18D Biacore analysis of supernatants and purified antibodies of the present disclosure is shown, showing affinity for BMPC, Sp / dLN PB / PC, and Sp / dLN B cells as measured by the negative log10 of the binding Kd.

[0035] FIG. 19A to FIG. 19B Display, in Fig.19A Representative binding curves of IgE antibodies to Der p 1, Der p 2, Der f 1, and negative control Olee1. Fig.19B A FeI d 1 specific ELISA testing the binding of human IgE mAb12_2 and its cross-reactivity with FeI d 1 is shown.

[0036] FIG. 20A to FIG. 20B Display, in Fig. 20A Figure 2. Gating strategy for isolating plasma cells from human bone marrow using CD38 staining. Fig. 20B Exemplary sera from allergic donors are shown The results of the test.

[0037] FIG. 21A to FIG. 21D It was shown that the antibody secretion TRAP and sequencing methods disclosed herein allow for the detection of vaccine-specific IgG-secreting plasma cells from bone marrow donors. Fig.21A , Representative workflow of the procedure for determining the gating of PCs as cells, single cells, live cells, DUMP- cells, and CD20-CD38++ cells. Fig. 21B IgG-secreting cells and antigen-positive cells found among IgG-secreting cells are shown. Fig. 21C Lack of detection of IgG-secreting PCs in the absence of the NHS-biotin component, or StAv-Igkappa, or anti-IgG antibodies is shown. Fig.21D Demonstrated is the lack of detection of antigen-specific IgG-secreting cells in the absence of antigen staining.

[0038] Fig. 22The workflow of the secretion trap combined with antigen tracking (STAC) is depicted. Step 1: Biotinylate antibody-secreting cells with amine-reactive NHS-biotin. Step 2: Capture secreted antibodies on the surface of antibody-secreting cells using streptavidin coupled to anti-hIgG. Anti-hIgG was used here because all antibodies have human constants. Step 3: Incubate cells with monomeric hIL-4R conjugated to AlexaFluor647 for 30 min at room temperature. Wash cells and then incubate with hIL-4R antigen pre-incubated with StAv-PE for 2 × 45 min at room temperature.

[0039] FIG. 23A to FIG. 23D Flow cytometric analysis of cells subjected to the STAC protocol is shown. Fig.23A A table of antibodies used in the experiments is shown. Antibodies 1-3 are specific for hIL-4Rα with different affinities (KD), and antibody 4 is specific for a different target and served as a negative control for the experiments. Fig. 23B A model B cell line without a surface BCR (A25 cell line) was incubated with NHS-biotin and anti-hIgG trap reagent ( Fig. 22 These cells were then mixed with Fig.23A The cells were incubated with antibodies from the table in the table, followed by antigen tracking incubation ( Fig. 22 Step 3). Fig.23C Flow cytometric analysis of cells subjected to the STAC protocol is shown, demonstrating that antibody secreting cells can be isolated based on affinity. Fig.23A Antibodies 1-4 are color coded on the flow cytometry plots. Higher affinity antibodies (ab_1, ab_2) favor monomeric hIL-4Rα-AlexaFluor647 (y-axis), lower affinity antibody (ab_3) favors tetrameric hIL-4Rα-biotin-StAv-PE (x-axis), and the negative control antibody (ab_4) is negative for both antigens. Fig.23D Flow cytometry analysis of cells subjected to the STAC protocol is shown. All cells are hIgK+, indicating that all antibodies, including the negative control (ab_4), are bound to the surface via the secretion trap. Anti-hIgK FMO is shown in grey, all other colors are Fig.23A The colors in are consistent. DETAILED DESCRIPTION

[0040] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0041] Various terms related to aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, these terms should be given their ordinary meanings in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0042] Unless otherwise expressly stated, it is not intended that any method or aspect described herein be construed as requiring that its steps be performed in a particular order. Therefore, in the absence of a method claim specifically stating in the claim or description that the steps are to be restricted to a particular order, it is not intended that an order be inferred in any respect. This applies to any possible non-explicit basis for interpretation, including: logical issues regarding the arrangement of steps or operational flow; plain meaning derived from grammatical composition or punctuation; the number or type of aspects described in the specification.

[0043] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0044] As used herein, the term "about" means that the numerical value cited is approximate and that small variations will not significantly affect the practice of the disclosed embodiments. Where numerical values ​​are used, unless the context indicates otherwise, the term "about" means that the numerical value may vary by 10% and remain within the scope of the disclosed embodiments.

[0045] As used herein, in certain embodiments, the terms "comprising" may be replaced with "consisting of" or "consisting essentially of" as needed.

[0046] As used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" or "oligonucleotide" may include polymeric forms of nucleotides of any length, may include DNA and / or RNA, and may be single-stranded, double-stranded or multi-stranded. A strand of a nucleic acid also refers to its complementary strand.

[0047] As used herein, the term "antibody" may include intact immunoglobulin molecules, fragmented immunoglobulin molecules including antigen binding fragments, antigen binding fragments without any other fragments of immunoglobulin molecules.

[0048] The present disclosure provides an antibody capture complex comprising a first component of a binding pair connected to an antibody capture molecule; wherein the first component of the binding pair is capable of binding to a second component of the binding pair; wherein the antibody capture molecule is capable of binding to a target antibody, wherein the target antibody is secreted by an antibody secreting cell. In some embodiments, when the second component of the binding pair is biotin and the first component of the binding pair is streptavidin, the antibody capture molecule does not bind to the κ light chain of the target antibody.

[0049] In some embodiments, the first component of the binding pair comprises a first reagent and the second component comprises a second reagent, wherein the first reagent and the second reagent form a bond.

[0050] In some embodiments, the first component of the binding pair comprises avidin, streptavidin or avidin, and the second component of the binding pair comprises biotin. In some embodiments, the first component of the binding pair comprises biotin, and the second component of the binding pair comprises avidin and / or streptavidin. In some embodiments, the first component of the binding pair comprises biotin, and the second component of the binding pair comprises avidin. In some embodiments, the first component of the binding pair comprises one of jun and fos, and the second component of the binding pair comprises the other of jun and fos. In some embodiments, the first component of the binding pair comprises one of mad and max, and the second component of the binding pair comprises the other of mad and max. In some embodiments, the first component of the binding pair comprises one of myc and max, and the second component of the binding pair comprises the other of myc and max. In some embodiments, the first component of the binding pair comprises one of azide and alkyne, and the second component of the binding pair comprises the other of azide and alkyne. In some embodiments, the first member of the binding pair comprises one of an azide and a phosphine, and the second member of the binding pair comprises the other of an azide and a phosphine.

[0051] In some embodiments, the second component of the binding pair comprises a surface marker of an antibody-secreting cell, and the first component of the binding pair comprises an antibody bound to a surface marker. In some embodiments, the cell surface marker comprises CD27, CD38, CD45, CD138, CD98, CD78, CD319, CXCR4, BCMA, GPRC5D, FCRL5, CD19, Ly6D, CD52 or a transmembrane activator calcium regulator and cyclophilin ligand interactor (TACI). In some embodiments, the cell surface marker comprises CD27. In some embodiments, the cell surface marker comprises CD38. In some embodiments, the cell surface marker comprises CD45. In some embodiments, the cell surface marker comprises CD138. In some embodiments, the cell surface marker comprises CD98. In some embodiments, the cell surface marker comprises CD78. In some embodiments, the cell surface marker comprises CD319. In some embodiments, the cell surface marker comprises CXCR4. In some embodiments, the cell surface marker comprises BCMA. In some embodiments, the cell surface marker comprises GPRC5D. In some embodiments, the cell surface marker comprises FCRL5. In some embodiments, the cell surface marker comprises CD19. In some embodiments, the cell surface marker comprises Ly6D. In some embodiments, the cell surface marker comprises CD52. In some embodiments, the cell surface marker comprises TACI.

[0052] In some embodiments, the second component of the binding pair comprises an interleukin-6 receptor (IL-6R), and the first component of the binding pair comprises an anti-IL-6R antibody, interleukin-6 (IL-6), and / or a fragment of IL-6 that can bind to IL-6R. In some embodiments, the second component of the binding pair comprises CD27, and the first component of the binding pair comprises an anti-CD27 antibody, CD70, and / or a fragment of CD70 that can bind to CD27.

[0053] In some embodiments, the second component of the binding pair comprises fluorescein isothiocyanate (FITC), and the first component of the binding pair comprises an anti-FITC antibody. In some embodiments, the second component of the binding pair comprises fluorescein, and the first component of the binding pair comprises an anti-fluorescein antibody. In some embodiments, the second component of the binding pair comprises phycoerythrin (PE), and the first component of the binding pair comprises an anti-PE antibody. In some embodiments, the second component of the binding pair comprises APC, and the first component of the binding pair comprises an anti-APC antibody. In some embodiments, the second component of the binding pair comprises BV421, and the first component of the binding pair comprises an anti-BV421 antibody. In some embodiments, the second component of the binding pair comprises BV510, and the first component of the binding pair comprises an anti-BV510 antibody. In some embodiments, the second component of the binding pair comprises BV605, and the first component of the binding pair comprises an anti-BV605 antibody. In some embodiments, the second component of the binding pair comprises BV650, and the first component of the binding pair comprises an anti-BV650 antibody. In some embodiments, the second component of the binding pair comprises BV711 and the first component of the binding pair comprises an anti-BV711 antibody. In some embodiments, the second component of the binding pair comprises BV786 and the first component of the binding pair comprises an anti-BV786 antibody.

[0054] In some embodiments, the antibody capture molecule comprises a capture antibody.

[0055] In some embodiments, the capture antibody comprises an anti-Fc antibody. In some embodiments, the capture antibody comprises an anti-Fcγ antibody, an anti-Fcα antibody, or an anti-Fcε antibody. In some embodiments, the capture antibody comprises an anti-Fcγ antibody. In some embodiments, the capture antibody comprises an anti-Fcα antibody. In some embodiments, the capture antibody comprises an anti-Fcε antibody. In some embodiments, the anti-Fcγ antibody comprises an anti-FcγRI antibody, an anti-FcγRIIA antibody, an anti-FcγRIIB antibody, an anti-FcγRIIB1 antibody, an anti-FcγRIIB2 antibody, an anti-FcγRIIIA antibody, and / or an anti-FcγRIIIB antibody. In some embodiments, the anti-Fcγ antibody comprises an anti-FcγRI antibody. In some embodiments, the anti-Fcγ antibody comprises an anti-FcγRIIA antibody. In some embodiments, the anti-Fcγ antibody comprises an anti-FcγRIIB antibody. In some embodiments, the anti-Fcγ antibody comprises an anti-FcγRIIB1 antibody. In some embodiments, the anti-Fcγ antibody comprises an anti-FcγRIIB2 antibody. In some embodiments, the anti-Fcγ antibody comprises an anti-FcγRIIIA antibody. In some embodiments, the anti-Fcγ antibody comprises an anti-FcγRIIIB antibody. In some embodiments, the anti-Fcα antibody comprises an anti-FcαRI antibody. In some embodiments, the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody. In some embodiments, the anti-Fcε antibody comprises an anti-FcεRI antibody. In some embodiments, the anti-Fcε antibody comprises an anti-FcεRII antibody.

[0056] In some embodiments, the capture antibody comprises an anti-light chain κ antibody and / or an anti-light chain λ antibody. In some embodiments, the capture antibody comprises an anti-light chain κ antibody. In some embodiments, the capture antibody comprises an anti-light chain λ antibody.

[0057] In some embodiments, the capture antibody comprises an anti-Ig antibody.

[0058] In some embodiments, the capture antibody comprises an anti-IgM antibody.

[0059] In some embodiments, the capture antibody comprises an anti-IgG antibody. In some embodiments, the anti-IgG antibody comprises an anti-IgG1 antibody, an anti-IgG2 antibody, an anti-IgG2a antibody, an anti-IgG2b antibody, an anti-IgG3 antibody and / or an anti-IgG4 antibody. In some embodiments, the anti-IgG antibody comprises an anti-IgG1 antibody. In some embodiments, the anti-IgG antibody comprises an anti-IgG2 antibody. In some embodiments, the anti-IgG antibody comprises an anti-IgG2a antibody. In some embodiments, the anti-IgG antibody comprises an anti-IgG2b antibody. In some embodiments, the anti-IgG antibody comprises an anti-IgG3 antibody. In some embodiments, the anti-IgG antibody comprises an anti-IgG4 antibody.

[0060] In some embodiments, the capture antibody comprises an anti-IgA antibody. In some embodiments, the anti-IgA antibody comprises an anti-IgA1 antibody, an anti-IgA2 antibody, an anti-secretory IgA antibody, or a polymeric anti-IgA antibody. In some embodiments, the anti-IgA antibody comprises an anti-IgA1 antibody. In some embodiments, the anti-IgA antibody comprises an anti-IgA2 antibody. In some embodiments, the anti-IgA antibody comprises an anti-secretory IgA antibody. In some embodiments, the anti-IgA antibody comprises a polymeric anti-IgA antibody.

[0061] In some embodiments, the capture antibody comprises an anti-IgE antibody.

[0062] In some embodiments, the antibody capture molecule comprises an Fc receptor or an extracellular domain of an Fc receptor. In some embodiments, Fc receptors include Fcγ receptors, Fcα receptors and / or Fcε receptors. In some embodiments, Fc receptors include Fcγ receptors. In some embodiments, Fc receptors include Fcα receptors. In some embodiments, Fc receptors include Fcε receptors. In some embodiments, Fcγ receptors include FcγRI receptors, FcγRIIA receptors, FcγRIIB receptors, FcγRIIB1 receptors, FcγRIIB2 receptors, FcγRIIIA receptors, FcγRIIIB receptors and / or FcRn receptors. In some embodiments, Fcγ receptors include FcγRI receptors. In some embodiments, Fcγ receptors include FcγRIIA receptors. In some embodiments, Fcγ receptors include FcγRIIB receptors. In some embodiments, Fcγ receptors include FcγRIIB1 receptors. In some embodiments, Fcγ receptors include FcγRIIB2 receptors. In some embodiments, Fcγ receptors include FcγRIIIA receptors. In some embodiments, Fcγ receptors include FcγRIIIB receptors. In some embodiments, Fcγ receptors include FcRn receptors. In some embodiments, Fcα receptors include FcαRI receptors and / or Fcα / μR receptors. In some embodiments, Fcα receptors include FcαRI receptors. In some embodiments, Fcα receptors include Fcα / μR receptors. In some embodiments, Fcε receptors include FcεRI receptors and / or FcεRII receptors. In some embodiments, Fcε receptors include FcεRI receptors. In some embodiments, Fcε receptors include FcεRII receptors. In some embodiments, Fc receptors include fragments of Fc receptors capable of binding to Fc.

[0063] In some embodiments, the antibody capture molecule comprises protein A, a fragment of protein A capable of binding to a portion of the Fc region, protein G and / or a fragment of protein G capable of binding to a portion of the Fc region. In some embodiments, the antibody capture molecule comprises protein A. In some embodiments, the antibody capture molecule comprises a fragment of protein A capable of binding to a portion of the Fc region. In some embodiments, the antibody capture molecule comprises protein G. In some embodiments, the antibody capture molecule comprises a fragment of protein G capable of binding to a portion of the Fc region. In some embodiments, the antibody capture molecule comprises protein L and / or a fragment of protein L capable of binding to a portion of the light chain. In some embodiments, the antibody capture molecule comprises protein L. In some embodiments, the antibody capture molecule comprises a fragment of protein L capable of binding to a portion of the light chain.

[0064] In some embodiments, the antibody capture complex comprises avidin or streptavidin linked to the extracellular domain of the high affinity IgE receptor (FcεRIa). In some embodiments, the antibody capture complex comprises avidin linked to FcεRIa.

[0065] In some embodiments, the antibody capture complex comprises streptavidin linked to FcεRIa.

[0066] Target Antibody

[0067] In some embodiments, the target antibody comprises an IgM antibody.

[0068] In some embodiments, the target antibody comprises an IgG antibody. In some embodiments, the anti-IgG antibody comprises an IgG1 antibody, an IgG2 antibody, an IgG2a antibody, an IgG2b antibody, an IgG3 antibody, and / or an IgG4 antibody. In some embodiments, the anti-IgG antibody comprises an IgG1 antibody. In some embodiments, the anti-IgG antibody comprises an IgG2 antibody. In some embodiments, the anti-IgG antibody comprises an IgG2a antibody. In some embodiments, the anti-IgG antibody comprises an IgG2b antibody. In some embodiments, the anti-IgG antibody comprises an IgG3 antibody. In some embodiments, the anti-IgG antibody comprises an IgG4 antibody.

[0069] In some embodiments, the target antibody comprises an IgA antibody. In some embodiments, the IgA antibody comprises an IgA1 antibody, an IgA2 antibody, a secretory IgA antibody, or a polymeric IgA antibody. In some embodiments, the IgA antibody comprises an IgA1 antibody. In some embodiments, the IgA antibody comprises an IgA2 antibody. In some embodiments, the IgA antibody comprises a secretory IgA antibody. In some embodiments, the IgA antibody comprises a polymeric IgA antibody.

[0070] In some embodiments, the target antibody comprises an IgE antibody.

[0071] Target Antigen

[0072] In some embodiments, the target of the target antibody (i.e., the antigen to which the target antibody binds) is an allergen. In some embodiments, the allergen comprises a component of an animal product, a drug, a food, a substance present in venom, a substance present in the saliva of a biting insect, a mold spore, a cosmetic, a metal, latex, wood, and / or plant pollen. In some embodiments, the allergen comprises a component of an animal product. In some embodiments, the allergen comprises a drug. In some embodiments, the allergen comprises a food. In some embodiments, the allergen comprises a substance present in venom. In some embodiments, the allergen comprises a substance present in the saliva of a biting insect. In some embodiments, the allergen comprises mold spores. In some embodiments, the allergen comprises cosmetics. In some embodiments, the allergen comprises a metal. In some embodiments, the allergen comprises latex. In some embodiments, the allergen comprises wood. In some embodiments, the allergen comprises plant pollen.

[0073] In some embodiments, the animal product comprises Bla g, Bla g 1, Bla g 2, Bla g 3, Bla g 3, Bla g 4, Bla g 5, Bla g 6, Bla g 7, Bla g 8, Bla g 9, Bla g 11, Per a, Per a 1, Per a 2, Per a 3, Per a 6, Per a 7, Per a 9, Per a 10, Per a 11, Per a 12, Der p 1, Der p 2, Der f 1, Eur m 1, Pso o 1, Equ c 1, Fe d 1, Fe d 4, Mus m 1, rat n 1, dust mite tropomyosin, shellfish tropomyosin, and / or cockroach tropomyosin. In some embodiments, the animal product comprises Bla g. In some embodiments, the animal product comprises Bla g 1. In some embodiments, the animal product comprises Bla g 2. In some embodiments, the animal product comprises Bla g 3. In some embodiments, the animal product comprises Bla g 3. In some embodiments, the animal product comprises Bla g 4. In some embodiments, the animal product comprises Bla g 5. In some embodiments, the animal product comprises Bla g 6. In some embodiments, the animal product comprises Bla g 7. In some embodiments, the animal product comprises Bla g 8. In some embodiments, the animal product comprises Bla g 9. In some embodiments, the animal product comprises Bla g 11. In some embodiments, the animal product comprises Per a. In some embodiments, the animal product comprises Per a 1. In some embodiments, the animal product comprises Per a 2. In some embodiments, the animal product comprises Per a 3. In some embodiments, the animal product comprises Per a 6. In some embodiments, the animal product comprises Per a 7. In some embodiments, the animal product comprises Per a 9. In some embodiments, the animal product comprises Per a 10. In some embodiments, the animal product comprises Per a 11. In some embodiments, the animal product comprises Per a 12. In some embodiments, the animal product comprises Der p 1. In some embodiments, the animal product comprises Der p 2. In some embodiments, the animal product comprises Der f 1. In some embodiments, the animal product comprises Eur m 1. In some embodiments, the animal product comprises Pso o 1. In some embodiments, the animal product comprises Equc 1. In some embodiments, the animal product comprises Fel d 1. In some embodiments, the animal product comprises Fel d 4.In some embodiments, the animal product comprises Mus m 1. In some embodiments, the animal product comprises Rat n 1. In some embodiments, the animal product comprises dust mite tropomyosin. In some embodiments, the animal product comprises shellfish tropomyosin. In some embodiments, the animal product comprises cockroach tropomyosin.

[0074] In some embodiments, the animal product comprises animal fur, animal dander, cockroach calyx, wool, and / or dust mite secretions. In some embodiments, the animal product comprises animal fur. In some embodiments, the animal product comprises animal dander. In some embodiments, the animal product comprises cockroach calyx. In some embodiments, the animal product comprises wool. In some embodiments, the animal product comprises dust mite secretions.

[0075] In some embodiments, the drug comprises penicillin, sulfonamides, salicylates, and / or neomycin. In some embodiments, the drug comprises penicillin. In some embodiments, the drug comprises sulfonamides. In some embodiments, the drug comprises salicylates. In some embodiments, the drug comprises neomycin.

[0076] In some embodiments, the food comprises celery, celeriac, corn, maize, eggs, fruit, pumpkin, eggplant, beans, milk, seafood, sesame, soy, tree nuts, wheat and / or balsam of Peru. In some embodiments, the food comprises celery. In some embodiments, the food comprises celeriac. In some embodiments, the food comprises corn. In some embodiments, the food comprises maize. In some embodiments, the food comprises eggs. In some embodiments, the food comprises fruit. In some embodiments, the food comprises pumpkin. In some embodiments, the food comprises eggplant. In some embodiments, the food comprises beans. In some embodiments, the food comprises milk. In some embodiments, the food comprises seafood. In some embodiments, the food comprises sesame. In some embodiments, the food comprises soy. In some embodiments, the food comprises tree nuts. In some embodiments, the food comprises wheat. In some embodiments, the food comprises balsam of Peru. In some embodiments, beans comprise kidney beans, peas, peanuts or soybeans. In some embodiments, beans comprise kidney beans. In some embodiments, beans comprise peas. In some embodiments, beans comprise peanuts. In some embodiments, the legumes comprise soybeans. In some embodiments, the tree nuts comprise pecans and / or almonds. In some embodiments, the tree nuts comprise pecans. In some embodiments, the tree nuts comprise almonds.

[0077] In some embodiments, the substance present in the venom comprises a substance present in bee sting venom and / or a substance present in wasp sting venom. In some embodiments, the substance present in the venom comprises a substance present in bee sting venom. In some embodiments, the substance present in the venom comprises a substance present in wasp sting venom.

[0078] In some embodiments, the biting insect is a mosquito or a tick. In some embodiments, the biting insect is a mosquito. In some embodiments, the biting insect is a tick. In some embodiments, the allergen in the biting insect is present in saliva. In some embodiments, the allergen comprises an oligosaccharide. In some embodiments, the allergen comprises galactose-alpha-1,3-galactose or galactose-alpha-1,3-galactose-beta-1,4-N-acetylglucosamine.

[0079] In some embodiments, the cosmetic comprises fragrance and / or quaternium-15. In some embodiments, the cosmetic comprises fragrance. In some embodiments, the cosmetic comprises quaternium-15.

[0080] In some embodiments, the metal comprises nickel and / or chromium. In some embodiments, the metal comprises nickel. In some embodiments, the metal comprises chromium.

[0081] In some embodiments, plant pollen comprises grass pollen, weed pollen and / or tree pollen. In some embodiments, plant pollen comprises grass pollen. In some embodiments, plant pollen comprises weed pollen. In some embodiments, plant pollen comprises tree pollen. In some embodiments, grass pollen comprises ryegrass pollen and / or timothy grass pollen. In some embodiments, grass pollen comprises ryegrass pollen. In some embodiments, grass pollen comprises timothy grass pollen. In some embodiments, weed pollen comprises ragweed pollen, plantain pollen, nettle pollen, pollen of Artemisia vulgaris species, pollen of Chenopodium album species or acidleaf grass pollen. In some embodiments, weed pollen comprises ragweed pollen. In some embodiments, weed pollen comprises plantain pollen. In some embodiments, weed pollen comprises nettle pollen. In some embodiments, weed pollen comprises pollen of Artemisia species. In some embodiments, weed pollen comprises pollen of Chenopodium species. In some embodiments, weed pollen comprises acid grass pollen. In some embodiments, tree pollen comprises birch pollen, alder pollen, hazel pollen, hornbeam pollen, pollen of the genus Aesculus, willow pollen, poplar pollen, pollen of the genus Platanus, pollen of the genus Tilia, pollen of the species Olea, pollen of Ashe juniper, or pollen of the species Alstoniascholaris. In some embodiments, tree pollen comprises birch pollen. In some embodiments, tree pollen comprises alder pollen. In some embodiments, tree pollen comprises hazel pollen. In some embodiments, tree pollen comprises hornbeam pollen. In some embodiments, tree pollen comprises pollen of the genus Aesculus. In some embodiments, tree pollen comprises willow pollen. In some embodiments, tree pollen comprises poplar pollen. In some embodiments, tree pollen comprises pollen of the genus Lycopodiella. In some embodiments, tree pollen comprises pollen of the genus Tilia. In some embodiments, tree pollen comprises pollen of the species Olea europaea. In some embodiments, tree pollen comprises pollen of Juniperus juniper. In some embodiments, tree pollen comprises pollen of the species Altissima. In some embodiments, plant pollen comprises olive tree pollen. In some embodiments, the allergen comprises Ole e 1.

[0082] In some embodiments, the allergen comprises an allergen from an oak tree (Quercus albus), a Japanese cedar (Cryptomeria japonica), or an elm or hickory tree.

[0083] In some embodiments, the allergen comprises a dog allergen, such as Can f protein. In some embodiments, the allergen comprises a milk allergen or a beef protein, such as Bos d 2-13. In some embodiments, the allergen comprises a chicken allergen (e.g., Gal d protein), a pig allergen (i.e., Sus s protein), or serum albumin. In some embodiments, the allergen comprises a fish allergen (e.g., Gad c / Gad m / Pan h / Sals protein). In some embodiments, the allergen comprises parvalbumin.

[0084] In any of the embodiments described herein, the allergen can be replaced with a non-allergenic compound, such as, for example, a foreign antigen. In some embodiments, the foreign antigen comprises a spike protein of a coronavirus. In some embodiments, the foreign antigen comprises a mutated peptide from a host. In some embodiments, the foreign antigen comprises a protein from another species. In some embodiments, the foreign antigen comprises a compound from an infectious disease, such as SARS-CoV-2 or influenza virus.

[0085] In some embodiments, the antibody secreting cell is a plasma cell. In some embodiments, the antibody secreting cell is a plasmablast. In some embodiments, the plasma cell is a short-lived plasma cell. In some embodiments, the plasma cell is a long-lived plasma cell.

[0086] Method for capturing target antibodies secreted by antibody-secreting cells

[0087] The present disclosure also provides a method for capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting an antibody-secreting cell population with a second component of a binding pair so that the second component of the binding pair binds to the cell surface, wherein the antibody-secreting cell population comprises antibody-secreting cells that secrete the target antibody; contacting an antibody-secreting cell population with an antibody capture complex, wherein the antibody capture complex comprises a first component of a binding pair connected to an antibody capture molecule, whereby the first component of the binding pair binds to the second component of the binding pair on the cell surface of the antibody-secreting cell, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cell; and contacting an antibody-secreting cell population with an antigen so that the target antibody secreted by the antibody-secreting cell and captured by the antibody capture complex binds to the antigen. In some embodiments, the method further comprises, after contacting the antibody-secreting cell population with the antibody capture complex, contacting the antibody-secreting cell population with a second anti-Ig antibody. The step of contacting the second anti-Ig antibody can be performed before, simultaneously with, or after the step of contacting the antibody-secreting cell population with the antigen.

[0088] The present disclosure also provides a method for capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting an antibody-secreting cell population with an antibody capture complex, wherein the antibody-secreting cell population comprises antibody-secreting cells that secrete the target antibody, wherein the antibody capture complex comprises a first component of a binding pair connected to an antibody capture molecule, whereby the first component of the binding pair binds to the second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cell; and contacting the antibody-secreting cell population with an antigen, whereby the target antibody secreted by the antibody-secreting cell and captured by the antibody capture complex binds to the antigen. In some embodiments, the method further comprises, after contacting the antibody-secreting cell population with the antibody capture complex, contacting the antibody-secreting cell population with a second anti-Ig antibody. The step of contacting the second anti-Ig antibody can be performed before, simultaneously with, or after the step of contacting the antibody-secreting cell population with the antigen.

[0089] In some embodiments, after contacting the antibody-secreting cell population with the antigen, the method may further include sorting the antibody-secreting cell population to collect an antibody-secreting cell pool, wherein the antibody-secreting cells in the pool each secrete an antibody that is captured by an antibody capture molecule and bound by the antigen, wherein the collected antibody-secreting cell pool contains antigen-secreting cells that secrete the target antibody; and in some such embodiments, the method may further include separating the collected antibody-secreting cell pool into single cells and separating the antigen-secreting cells that secrete the target antibody.

[0090] In some embodiments, the antigen is a barcoded antigen. In some embodiments, the antigen comprises an allergen. In some embodiments, the barcoded antigen comprises a barcode nucleic acid molecule. In some embodiments, a portion of the nucleotide sequence of the barcode nucleic acid molecule is unique to the antigen. In some embodiments, a portion of the barcode nucleic acid molecule comprises a sequencing primer. In some embodiments, the sequencing primer is located upstream of the portion of the nucleotide sequence of the barcode nucleic acid molecule that is unique to the antigen. In some embodiments, a portion of the barcode nucleic acid molecule is complementary to a template switch oligonucleotide (TSO). In some embodiments, a portion of the nucleotide sequence of the barcode nucleic acid molecule is unique to the antigen, is located upstream of the portion of the barcode nucleic acid molecule, and is complementary to the TSO. In some embodiments, the 3' end of the barcode nucleic acid molecule comprises three consecutive cytidine or ribocytidine residues. In some embodiments, the barcode nucleic acid molecule comprises DNA. In some embodiments, the barcode nucleic acid molecule comprises RNA.

[0091] In some embodiments, the antibody secreting cell population is contacted with a plurality of barcoded antigens, wherein each barcoded antigen in the plurality of barcoded antigens comprises a unique antigen linked to a unique nucleic acid molecule. In some embodiments, the unique antigen comprises an allergen. In some embodiments, the unique nucleic acid molecule comprises a barcode nucleic acid molecule.

[0092] In some embodiments, each barcoded antigen in the plurality of barcoded antigens is detectably labeled. In some embodiments, each barcoded antigen in the plurality of barcoded antigens is labeled with a radioactive compound, a fluorescent compound, or an enzyme. In some embodiments, each barcoded antigen in the plurality of barcoded antigens is labeled with a radioactive compound. In some embodiments, each barcoded antigen in the plurality of barcoded antigens is labeled with a fluorescent compound. In some embodiments, each barcoded antigen in the plurality of barcoded antigens is labeled with an enzyme. In some embodiments, the radioactive compound comprises 3 H. 14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 In some embodiments, the radioactive compound comprises 3 H. In some embodiments, the radioactive compound comprises 14 C. In some embodiments, the radioactive compound comprises 19 F. In some embodiments, the radioactive compound comprises 35 S. In some embodiments, the radioactive compound comprises 125 1. In some embodiments, the radioactive compound comprises 131 1. In some embodiments, the radioactive compound comprises 111 In. In some embodiments, the radioactive compound comprises 99Tc. In some embodiments, the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin or fluorescent protein. In some embodiments, the fluorescent compound comprises fluorescein. In some embodiments, the fluorescent compound comprises fluorescein isothiocyanate. In some embodiments, the fluorescent compound comprises rhodamine. In some embodiments, the fluorescent compound comprises 5-dimethylamine-1-naphthalenesulfonyl chloride. In some embodiments, the fluorescent compound comprises phycoerythrin. In some embodiments, the fluorescent compound comprises fluorescent protein. In some embodiments, the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase or glucose oxidase. In some embodiments, the enzyme comprises alkaline phosphatase. In some embodiments, the enzyme comprises horseradish peroxidase. In some embodiments, the enzyme comprises luciferase. In some embodiments, the enzyme comprises glucose oxidase.

[0093] In embodiments where a second anti-Ig antibody is used, in some such embodiments, the second anti-Ig antibody comprises an anti-IgM antibody, an anti-IgG antibody, an anti-IgA antibody, and / or an anti-IgE antibody. In some embodiments, the second anti-Ig antibody comprises an anti-IgM antibody. In some embodiments, the second anti-Ig antibody comprises an anti-IgG antibody. In some embodiments, the second anti-Ig antibody comprises an anti-IgA antibody. In some embodiments, the second anti-Ig antibody comprises an anti-IgE antibody.

[0094] In some embodiments, the second anti-Ig antibody is detectably labeled. In some embodiments, the second anti-Ig antibody is labeled with a radioactive compound, a fluorescent compound, or an enzyme. In some embodiments, the second anti-Ig antibody is labeled with a radioactive compound. In some embodiments, the second anti-Ig antibody is labeled with a fluorescent compound. In some embodiments, the second anti-Ig antibody is labeled with an enzyme. In some embodiments, the radioactive compound comprises 3 H. 14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 In some embodiments, the radioactive compound comprises 3 H. In some embodiments, the radioactive compound comprises 14 C. In some embodiments, the radioactive compound comprises 19 F. In some embodiments, the radioactive compound comprises 35 S. In some embodiments, the radioactive compound comprises 125 1. In some embodiments, the radioactive compound comprises 1311. In some embodiments, the radioactive compound comprises 111 In. In some embodiments, the radioactive compound comprises 99 Tc. In some embodiments, the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin or fluorescent protein. In some embodiments, the fluorescent compound comprises fluorescein. In some embodiments, the fluorescent compound comprises fluorescein isothiocyanate. In some embodiments, the fluorescent compound comprises rhodamine. In some embodiments, the fluorescent compound comprises 5-dimethylamine-1-naphthalenesulfonyl chloride. In some embodiments, the fluorescent compound comprises phycoerythrin. In some embodiments, the fluorescent compound comprises fluorescent protein. In some embodiments, the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase or glucose oxidase. In some embodiments, the enzyme comprises alkaline phosphatase. In some embodiments, the enzyme comprises horseradish peroxidase. In some embodiments, the enzyme comprises luciferase. In some embodiments, the enzyme comprises glucose oxidase.

[0095] In some embodiments, the antibody secreting cell population is obtained by enriching the cell population obtained from a human for immune cells. In some embodiments, the cell population obtained from a human is enriched for plasma cells or plasmablasts.

[0096] In some embodiments, the antibody secreting cell population is obtained from human lymph nodes, lungs, bone marrow and / or blood. In some embodiments, the antibody secreting cell population is obtained from lymph nodes. In some embodiments, the antibody secreting cell population is obtained from lungs. In some embodiments, the antibody secreting cell population is obtained from bone marrow. In some embodiments, the antibody secreting cell population is obtained from blood.

[0097] In some embodiments, the antigen and / or the second anti-Ig antibody are detected. In some embodiments, the antibody-secreting cells are sorted to collect a pool of antibody-secreting cells that bind to the antigen and / or the second anti-Ig antibody, wherein the collected pool of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody.

[0098] In some embodiments, the antibody-secreting cells are contacted with an Fc blocker prior to contacting the population of antibody-secreting cells with the second member of the binding pair.

[0099] The present disclosure also provides a method for capturing IgE antibodies secreted by antibody-secreting cells, wherein the IgE antibodies are directed against allergens, the method comprising: contacting a population of antibody-secreting cells with NHS-biotin to bind biotin to the cell surface, wherein the population of antibody-secreting cells comprises antibody-secreting cells that secrete IgE antibodies; contacting the population of antibody-secreting cells with an antibody capture complex, wherein the antibody capture complex comprises streptavidin linked to the extracellular domain of FcεRIa, and whereby the antibody capture molecule binds to the IgE antibodies secreted by the antibody-secreting cells; and contacting the population of antibody-secreting cells with an antigen, wherein the antigen is an antigenic portion of an allergen or a mixture of multiple antigenic portions, whereby the IgE antibodies secreted by the antibody-secreting cells and captured by the antibody capture complex bind to the antigen. In some embodiments, the antigen comprises a plurality of barcoded antigens, wherein each of the plurality of barcoded antigens is a different antigenic portion of an allergen. In some embodiments, the method further comprises sorting the antibody-secreting cell population to collect an antibody-secreting cell pool, wherein the antibody-secreting cells in the pool each secrete an IgE antibody captured by the antibody-capture complex and bound by the antigen, wherein the collected antibody-secreting cell pool comprises antigen-secreting cells that secrete IgE antibodies.

[0100] In some embodiments, the method further comprises contacting the antibody secreting cell population with an anti-IgE antibody so that the IgE antibody secreted by the antibody secreting cell and captured by the antibody capture complex binds to the anti-IgE antibody. In some embodiments, the method may further comprise sorting the antibody secreting cell population to collect an antibody secreting cell pool, wherein the antibody secreting cells in the pool each secrete an IgE antibody captured by the antibody capture complex and bound by an antigen and an anti-IgE antibody, wherein the collected antibody secreting cell pool comprises antigen secreting cells that secrete IgE antibodies.

[0101] In some embodiments, the method further comprises separating the collected pool of antibody-secreting cells into single cells and isolating antigen-secreting cells that secrete IgE antibodies.

[0102] Antigen tracking

[0103] The antibody secreting cell group may include multiple antibody secreting cells, each of which secretes antibodies captured by the antibody capture complex, wherein the multiple antibody secreting cells include antibody secreting cells that secrete target antibodies. Some antibodies secreted by multiple antibody secreting cells bind to the antigen bound by the target antibody, although the antibodies may have different affinities for the antigen. Antigen tracking can be used to obtain a cell group rich in antibody secreting cells that secrete antibodies with high affinity for the antigen, which allows the separation of antibody secreting cells that secrete target antibodies with high affinity. Therefore, in some embodiments of the methods disclosed herein, the step of contacting the antibody secreting cell group with the antigen includes antigen tracking. In these embodiments, as an initial binding step, the antibody secreting cell group is contacted with the first labeled form of antigen so that the antigen binds to the target antibody captured at the surface of the antibody secreting cell and forms an antigen-antibody complex, followed by a "tracking" step, wherein the cell is contacted with one of several forms of antigen: (i) unlabeled form of antigen ("cold tracking"), (ii) second labeled form of antigen ("hot tracking"), or a combination of unlabeled form of antigen and second labeled form of antigen ("combined tracking"). Cells that still bind to the first labeled form of the antigen after tracking represent cells that secrete antibodies with high binding affinity. Thus, antigen tracking allows selection among cells that secrete antibodies with different affinities to enrich for cells that secrete antibodies with high affinity and subsequently isolate antibody-secreting cells that secrete target antibodies with high affinity.

[0104] The term "enrichment" refers to increasing the frequency or percentage of desired cells in a cell population, for example, increasing the percentage of antibody-secreting cells that secrete high-affinity antibodies in an antibody-secreting cell population that secretes high-affinity antibodies, and the antibody-secreting cell population contains cells that secrete antibodies with various affinities (e.g., high affinity, medium affinity, and low affinity). Therefore, the antibody-secreting cell population enriched in cells that secrete high-affinity antibodies covers a cell population of antibody-secreting cells that secrete high-affinity antibodies with a higher frequency and / or a higher percentage due to the enrichment process. In this article, the enrichment process is a process including antigen tracking, thereby selecting cells that secrete antibodies with high affinity to antigens from a cell population that secretes antibodies with various affinities to antigens, and separating cells that secrete high-affinity antibodies from cells that secrete non-high-affinity antibodies.

[0105] The cell population obtained as a result of tracking is rich in antibody-secreting cells that secrete antibodies with high binding affinity to the antigen of interest. In other words, compared with the cell population before tracking or without tracking, the enriched cell population contains a greater percentage of cells that secrete antibodies that bind to the antigen with high binding affinity. In some embodiments, at least 40% of the collected cells secrete high-affinity antibodies, such as antibodies with a KD of 0.1pM to 25nM. In some embodiments, the enriched cell population can be a population in which at least 50% of the cells in the population secrete antibodies that bind to the antigen of interest with high binding affinity, such as antibodies with a KD of 0.1pM to 25nM. In some embodiments, the enriched cell population can be a population in which at least 60% of the cells in the population secrete antibodies that bind to the antigen of interest with high binding affinity, such as antibodies with a KD of 0.1pM to 25nM. In some embodiments, the enriched cell population can be a population in which at least 70% of the cells in the population secrete antibodies that bind to the antigen of interest with high binding affinity, such as antibodies with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population can be a population in which at least 80% of the cells in the population secrete antibodies that bind to the antigen of interest with high binding affinity, such as antibodies with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population can be a population in which at least 90% of the cells in the population secrete antibodies that bind to the antigen of interest with high binding affinity, such as antibodies with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population can be a population in which at least 95% of the cells in the population secrete antibodies that bind to the antigen of interest with high binding affinity, such as antibodies with a KD of 0.1 pM to 25 nM. In some embodiments, the frequency of cells secreting high-affinity antibodies (e.g., antibodies with KD of 0.1 pM to 25 nM) in the cell population after tracking increases by at least 30% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high-affinity antibodies (e.g., antibodies with KD of 0.1 pM to 25 nM) in the cell population after tracking increases by at least 40% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high-affinity antibodies (e.g., antibodies with KD of 0.1 pM to 25 nM) in the cell population after tracking increases by at least 50% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high-affinity antibodies (e.g., antibodies with KD of 0.1 pM to 25 nM) in the cell population after tracking increases by at least 75% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high affinity antibodies (eg, antibodies with a KD of 0.1 pM to 25 nM) in the cell population increases by at least 100% after chasing compared to the frequency in the cell population before or without chasing.In some embodiments, the frequency of cells secreting high-affinity antibodies (e.g., antibodies with a KD of 0.1 pM to 25 nM) in the cell population after tracking increases by at least 200% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high-affinity antibodies (e.g., antibodies with a KD of 0.1 pM to 1 nM) with a KD less than 1 nM in the cell population after tracking increases by at least 40% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high-affinity antibodies (e.g., antibodies with a KD of 0.1 pM to 1 nM) with a KD less than 1 nM in the cell population after tracking increases by at least 50% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high-affinity antibodies (e.g., antibodies with a KD of 0.1 pM to 1 nM) with a KD less than 1 nM in the cell population after tracking increases by at least 75% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high-affinity antibodies with a KD of less than 1 nM (e.g., antibodies with a KD of 0.1 pM to 1 nM) in the cell population after tracking increases by at least 100% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high-affinity antibodies with a KD of less than 1 nM (e.g., antibodies with a KD of 0.1 pM to 1 nM) in the cell population after tracking increases by at least 200% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high-affinity antibodies with a KD of less than 0.1 nM (e.g., antibodies with a KD of 0.1 pM to 0.1 nM) in the cell population after tracking increases by at least 40% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting a high-affinity antibody (e.g., an antibody with a KD of 0.1 pM to 0.1 nM) with a KD less than 0.1 nM in the cell population after tracking increases by at least 50% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting a high-affinity antibody (e.g., an antibody with a KD of 0.1 pM to 0.1 nM) with a KD less than 0.1 nM in the cell population after tracking increases by at least 75% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting a high-affinity antibody (e.g., an antibody with a KD of 0.1 pM to 0.1 nM) with a KD less than 0.1 nM in the cell population after tracking increases by at least 100% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells secreting high affinity antibodies with a KD of less than 0.1 nM (eg, antibodies with a KD of 0.1 pM to 0.1 nM) in the cell population increases by at least 200% after chasing compared to the frequency in the cell population before or without chasing.

[0106] The term "binding affinity" as known in the art generally refers to the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule for its binding partner can generally be expressed in terms of the dissociation equilibrium constant (KD or K D ) indicates. D The (molar) value is inversely proportional to the binding affinity, so K D The smaller the value (M), the higher the affinity. Therefore, "higher affinity" refers to antibodies that generally bind to the antigen more strongly and / or more quickly and / or remain bound longer. Generally, because the antigen has a stronger binding interaction, a lower concentration (M) of antigen is required to achieve the desired effect.

[0107] The term "kd" (seconds-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction, or the dissociation rate constant of an antibody, Ig, antibody binding fragment, or molecule interaction. This value is also called k off value.

[0108] The term "ka" (M-1 x sec-1 or 1 / M) refers to the association rate constant for a particular antibody-antigen interaction, or the association rate constant for an antibody Ig, antibody binding fragment, or molecule interaction.

[0109] The term "KD" or "K D "(M)" refers to the equilibrium dissociation constant for a particular antibody-antigen interaction, or the equilibrium dissociation constant for an antibody, Ig, antibody binding fragment, or molecule interaction. The equilibrium dissociation constant is obtained by dividing ka by kd.

[0110] A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. As determined by surface plasmon resonance, the binding affinity obtained using this method is generally in the range of about 0.1 pM to about 25 nM. In some embodiments, the binding affinity is less than about 10 nM as determined by surface plasmon resonance.

[0111] The term "high affinity" antibody refers to an antibody having a binding affinity (expressed as KD) of 25 nM or less, for example, having a value of about 0.1 pM to about 25 nM. For this purpose, a high affinity antibody may have a binding affinity of about 25×10 -9 M (25nM) or less, about 10×10 -9M (10nM) or less, about 1×10 -9 M (1nM) or less, about 1×10 -10 M (0.1nM) or less, about 0.5×10 -10 M (0.05nM) or less, about 0.05×10 -10 The measured KD of an antibody may be about 5 pM (or less), about 1 pM or less, or about 0.5 pM or less. One skilled in the art will recognize that the KD value of an antibody may be expressed numerically as nE -z or n×10 -z , for example, 3.2E -12 Equivalent to 3.2×10 -12 And the indicated KD is 3.2 picomoles (pM). In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1pM to about 25nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1pM to about 20nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1pM to about 15nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1pM to about 10nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1pM to about 5nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1pM to about 1nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1pM to about 0.5nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1pM to about 0.1nM. In some embodiments, the high affinity antibody has a measured KD of less than about 20nM. In some embodiments, the high affinity antibody has a measured KD of less than about 15nM. In some embodiments, the high affinity antibody has a measured KD of less than about 10nM. In some embodiments, the high affinity antibody has a measured KD of less than about 5nM. In some embodiments, the high affinity antibody has a measured KD of less than about 1nM. In some embodiments, the high affinity antibody has a measured KD of less than about 0.1nM. In some embodiments, the high affinity antibody has a measured KD of less than about 0.5nM. In some embodiments, the high affinity antibody has a measured KD of less than about 0.01nM. In some embodiments, the high affinity antibody has a measured KD of less than about 0.001nM (or 1pM). In some embodiments, the high affinity antibody has a measured KD of less than about 0.5pM.

[0112] In some embodiments, the antigen is a protein that exists in monomeric form. Examples of proteins that exist in monomers include interleukin molecules, such as IL-13. In some embodiments, the antigen is a protein that exists in multimeric form, including homologs and heterologs. In some embodiments, the antigen is a protein that exists in both monomeric and multimeric forms, in which case a mixture of protein monomers and multimers can be used in the methods described herein.

[0113] Whether the antigen is a protein in the form of a monomer, a polymer or a mixture thereof, the antigen can be used in the methods described herein in a monovalent form or a multivalent form. The terms "monovalent" and "multivalent" are used to refer to the number of units of the antigen presented and to distinguish the antigen itself, which is a protein in the form of a monomer, a polymer or a mixture thereof. Therefore, the monovalent form of an antigen refers to a single unit form of an antigen, wherein the antigen itself can be a protein in the form of a monomer, a polymer or a mixture thereof. The multivalent form of an antigen refers to a plurality of antigen units presented by a multivalent molecule that is usually bound or connected to an antigen. The multivalent molecule can be a dimer, a trimer, a tetramer, a pentamer, a hexamer, etc., or a mixture thereof. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which can be complexed with biotin and then connected to the antigen, thereby providing a multivalent (e.g., tetravalent) antigen. In some embodiments, the streptavidin multimer includes a tetramer, and can additionally include a trimer and / or a dimer. In some embodiments, the streptavidin polymer is conjugated to a fluorophore, such as phycoerythrin. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment, to which an antigen can be attached to provide a divalent form of the antigen. In some embodiments, the multivalent molecule is a trimer of a trimeric molecule, such as a foldon, to which an antigen can be attached to provide a trivalent form of the antigen.

[0114] In some embodiments, the step of contacting a population of antibody-secreting cells with an antigen comprises: (a) contacting a population of antibody-secreting cells with a first labeled form of the antigen so that the antigen binds to antibodies including a target antibody captured on the cell surface, wherein the first labeled form of the antigen is conjugated to a first detectable label; (b) washing the cells to remove unbound antigen; (c) contacting the cells with: (i) an unlabeled form of the antigen, (ii) a second labeled form of the antigen, or (iii) an unlabeled form of the antigen and a second labeled form of the antigen; (d) washing the cells to remove unbound antigen; and (e) collecting a pool of antibody-secreting cells that remain bound to the first labeled form of the antigen, wherein the collected pool of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody, wherein the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex remains bound to the first labeled form of the antigen.

[0115] In some embodiments, the concentration of the first labeled form of the antigen is between 0.001nM and 1uM. In some embodiments, the concentration of the first labeled form of the antigen is between 0.01nM and 100nM. In some embodiments, the concentration of the first labeled form of the antigen is between 0.05nM and 10nM. In some embodiments, the concentration of the first labeled form of the antigen is between 0.05nM and 9nM, 0.05nM to 8nM, 0.05nM to 7nM, 0.05nM to 6nM, 0.05nM to 5nM, 0.05nM to 4nM, 0.05nM to 3nM, 0.05nM to 2nM or 0.05nM to 1nM. In some embodiments, the concentration of the first labeled form of the antigen is between 0.1nM and 7.5nM. In some embodiments, the concentration of the first labeled form of the antigen is between 0.1 nM and 7 nM, 0.1 nM and 6 nM, 0.1 nM and 5 nM, 0.1 nM and 4 nM, 0.1 nM and 3 nM, 0.1 nM and 2 nM, or 0.1 nM and 1 nM. In some embodiments, the concentration of the first labeled form of the antigen is between 0.2 nM and 7.5 nM. In some embodiments, the concentration of the first labeled form of the antigen is between 0.2nM and 7nM, 0.2nM and 6nM, 0.2nM and 5nM, 0.2nM and 4nM, 0.2nM and 3nM, 0.2nM and 2nM, 0.2nM and 1nM, 0.3nM and 7nM, 0.3nM and 6nM, 0.3nM and 5nM, 0.3nM and 4nM, 0.3nM and 3nM, 0.3nM and 2nM, 0.3nM and 1nM, 0.5nM and 7nM, 0.5nM and 6nM, 0.5nM and 5nM, 0.5nM and 4nM, 0.5nM and 3nM, 0.5nM and 2nM, 0.5nM and 1nM. In some embodiments, the concentration of the first labeled form of the antigen is between 1.0 nM and 10 nM, 1.0 nM and 9 nM, 1.0 nM and 8.0 nM, 1.0 nM and 7 nM, 1.0 nM and 6 nM, 1.0 nM and 5 nM, 1.0 nM and 4 nM, 1.0 nM and 3 nM, 1.0 nM and 2 nM, 2.0 nM and 10.0 nM or 5.0 nM and 10.0 nM. In a specific embodiment, the antibody secreting cell can be contacted with the first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 0.2 nM. In a specific embodiment, the antibody secreting cell can be contacted with the first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 5.0 nM. In a specific embodiment, the antibody secreting cell can be contacted with the first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 7.5 nM. In a specific embodiment, the antibody-secreting cells can be contacted with a first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 10 nM.In specific embodiments, the antibody-secreting cells can be contacted with a first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1.0 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, 4.5 nM, 5.0 nM, 5.5 nM, 6.0 nM, 6.5 nM, 7.0 nM, 8.0 nM, 8.5 nM, 9.0 nM, 9.5 nM or greater.

[0116] In some embodiments, contacting the antibody-secreting cells with the first labeled form of the antigen occurs for about 5 minutes to about 60 minutes, such as about 20 minutes, about 30 minutes, about 40 minutes, or about 50 minutes.

[0117] In some embodiments, the first labeled form of the antigen is a monovalent form of the antigen. In some embodiments, the first labeled form of the antigen is a multivalent form of the antigen. In some embodiments, the first labeled form of the antigen is a mixture of monovalent and multivalent forms of the antigen. Whether in monovalent form, multivalent form, or a mixture thereof, the antigen itself can be a protein that is a monomer, a multimer, or a mixture of monomers and multimers.

[0118] In some embodiments, the antigen in the first labeled form is an antigen conjugated to a first detectable label. Antigens can be labeled with small molecules, radioisotopes, enzyme proteins, and fluorescent dyes. In some embodiments, the detectable label is a small molecule. Detectable small molecule labels allow for easy labeling of proteins and can be used in many conventionally deployed detection assays known in the art.

[0119] In some embodiments, the detectable label is an enzyme reporter gene. Enzyme labels are larger than biotin, but they rarely disrupt the function of the antibody. Commonly used enzyme labels are horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and beta-galactosidase. In order to use enzyme-labeled antibodies, the sample is incubated with an enzyme-specific substrate, which is catalyzed by the enzyme to produce a colored product (chromogenic assay) or light (chemiluminescent assay). Each enzyme has a set of substrates and detection methods that can be used. For example, HRP can react with diaminobenzidine to produce a brown product, or with luminol to produce light. In contrast, AP can react with p-nitrophenyl phosphate (pNPP) to produce a yellow product detected by a spectrophotometer, or react with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) to produce a purple precipitate.

[0120] In some embodiments, the detectable label is a fluorescent label. The fluorescent label is directly conjugated to the antibody, and detection does not require enzyme / substrate or binding interactions. Therefore, the amount of fluorescent signal detected is proportional to the amount of target protein in the sample. The fluorescent label can be covalently attached to the antibody through a primary amine or thiol.

[0121] After the antibody secreting cell group is incubated with the antigen in the first labeled form (initial binding step), any unbound antigen can be removed from the cells producing the primary antibody. In some embodiments, unbound antigen is removed by washing. As known in the art, washing is a technique for removing unwanted components (including unbound antigen) using a wash buffer. Washing buffer is known in the art. In some embodiments, the wash buffer is a wash buffer based on phosphate buffered saline (PBS). In some embodiments, the wash buffer is a Tris buffered saline (TBS) wash buffer. In some embodiments, the wash buffer comprises a detergent. In some embodiments, the detergent is Tween-20. Wash the cells with the wash buffer for the duration of the allotment to remove unbound antigen. The amount of time allocated will be the amount of time sufficient to remove unbound antigen. In some embodiments, the allotment time can be about 10 minutes to about 60 minutes to remove unbound antigen; multiple washes of a total of 10 minutes to 60 minutes can be used, such as 3 washes of 10 minutes or a 30-minute wash; 2-4 washes of 5-15 minutes each, and the like. After washing and sucking out the supernatant containing wash buffer and unbound antigen, the precipitation containing the cells bound to the antigen can be used in subsequent steps. In some embodiments, the precipitation containing the bound antigen can be resuspended in a buffer and used in subsequent steps. In some embodiments, the buffer used to resuspend the precipitation can be the same wash buffer. In some embodiments, the buffer used to resuspend the precipitation can be a buffer different from the wash buffer.

[0122] After contacting the antibody secreting cell population with the first labeled form of the antigen (initial binding / contacting step), and once the unbound first labeled form of the antigen is removed, the cells are antigen-tracked, i.e., the cells are contacted with the antigen again, or "tracked" with the antigen, to selectively enrich for cells that secrete antibodies with high affinity for the antigen. This tracking can be performed using any of several forms of the antigen: (i) an unlabeled form of the antigen ("cold" tracking), (ii) a second labeled form of the antigen ("hot" tracking); or (iii) an unlabeled form of the antigen and a second labeled form of the antigen ("combined tracking"). Tracking allows the tracking antigen to bind to the antibody that was originally bound by the first labeled form of the antigen, thereby tracking the first labeled form of the antigen off the antibody, unless the antibody has a high affinity for the antigen and remains bound to the first labeled form of the antigen after tracking.

[0123] In some embodiments, the antigen in unlabeled form is used for tracking (cold tracking). In some embodiments, the antigen in unlabeled form is an antigen in monovalent form, that is, although the antigen itself can be a protein in a monomeric form, a polymeric form, or a mixture of monomeric and polymeric forms, the antigen in monovalent form is the antigen itself, without further secondary multimerization. In some embodiments, the antigen in unlabeled form is an antigen in multivalent form, wherein the antigen itself can be a protein in a monomeric form, a polymeric form, or a mixture of monomeric and polymeric forms. In some embodiments, the antigen in unlabeled form is a mixture of monovalent and multivalent forms of antigens. In embodiments using multivalent forms of antigens, this form can be provided by multivalent molecules that are bound or connected to the antigen. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which can be complexed with biotin and then connected to the antigen, thereby providing a multivalent (e.g., tetravalent) form of antigen. In some embodiments, in addition to tetramers, streptavidin multimers can include trimers and / or dimers. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment, to which an antigen can be attached to provide a divalent form of the antigen. In some embodiments, the multivalent molecule is a trimer of a trimeric molecule, such as a foldon, to which an antigen can be attached to provide a trivalent form of the antigen.

[0124] In some embodiments, a second labeled form of antigen is used for tracking (thermal tracking). In such embodiments, after the initial binding and washing steps, the antibody-secreting cells are tracked with a second labeled form of antigen. The second labeled form of antigen has a label that provides a detectable signal different from the label on the first labeled form of antigen. Suitable selections of labels are described herein, as long as the label on the second labeled form of antigen is different from the label on the first labeled form of antigen. These labels include small molecules, radioisotopes, enzyme proteins, and fluorescent dyes. In some embodiments, the first label is AlexaFluor647, and the second label is phycoerythrin.

[0125] In some embodiments, the antigen in the second labeled form is an antigen in a monovalent form. In some embodiments, the antigen in the second labeled form is an antigen in a multivalent form. In some embodiments, the antigen in the second labeled form comprises a mixture of monovalent and multivalent forms of antigens. In embodiments using multivalent forms of antigens, this form can be provided by a multivalent molecule that is bound or connected to the antigen. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which can be complexed with biotin and then connected to the antigen, thereby providing a multivalent (e.g., tetravalent) form of antigen. In some embodiments, in addition to tetramers, the streptavidin multimer can include trimers and / or dimers. In some embodiments, the streptavidin multimer is conjugated to a fluorophore (such as phycoerythrin). In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment, to which an antigen can be connected to provide a divalent form of the antigen. In some embodiments, the multivalent molecule is a trimer of a trimer molecule, such as a foldon, to which an antigen can be connected to provide a trivalent form of the antigen.

[0126] The first labeled form of the antigen and the second labeled form of the antigen can be the same or different valence forms of the antigen, but must have a label that emits a detectable signal that is different from each other. In some embodiments, the first labeled form of the antigen is a monovalent form of the antigen, and the second labeled form of the antigen is also a monovalent form. In some embodiments, the first labeled form of the antigen is a monovalent form of the antigen, and the second labeled form of the antigen is a multivalent form.

[0127] In some embodiments, tracking is performed with an unlabeled form of the antigen (cold) and a second labeled form of the antigen (hot), also referred to herein as combined tracking. In such embodiments, after the initial binding and washing steps, the antibody-secreting cells are tracked with an unlabeled form of the antigen and a second labeled form of the antigen. Two forms of tracking antigen: an unlabeled form of the antigen ("cold-tracked antigen") and a second labeled form of the antigen ("hot-tracked antigen") can be contacted with the cells simultaneously or sequentially (e.g., first adding the cold-tracked antigen, then adding the hot-tracked antigen, or vice versa). In some embodiments, the unlabeled form of the antigen is a monovalent form. In some embodiments, the unlabeled form of the antigen is a multivalent form. In some embodiments, the unlabeled form of the antigen is a mixture of a monovalent form and a multivalent form. In some embodiments, the second labeled form of the antigen is a monovalent form. In some embodiments, the second labeled form of the antigen is a multivalent form. In some embodiments, the second labeled form of the antigen includes a mixture of a monovalent form and a multivalent form. In embodiments where a multivalent form of the antigen is used in combined tracking, this form can be provided by a multivalent molecule that is bound or connected to the antigen. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which can be complexed with biotin and then attached to the antigen to provide a multivalent (e.g., tetravalent) form of the antigen. In some embodiments, in addition to tetramers, the streptavidin multimer can include trimers and / or dimers. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment, to which the antigen can be attached to provide a divalent form of the antigen. In some embodiments, the multivalent molecule is a trimer of a trimeric molecule, such as a foldon, to which the antigen can be attached to provide a trivalent form of the antigen.

[0128] For any form of tracking, the concentration of the tracking antigen used is excessive compared to the concentration of the first labeled form of the antigen, regardless of the form of the antigen used for tracking (i.e., unlabeled form, second labeled form, or unlabeled form and second labeled form). In the embodiment using the unlabeled form of the antigen (in cold tracking or combined tracking), the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen is at least 2 times, i.e., 2 times to up to, for example, 2500 times. In some embodiments, the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen is 2 times. In some embodiments, the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen is 3 times. In some embodiments, the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen is 4 times. In some embodiments, the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen is 5 times. In some embodiments, the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen is 6 times, 7 times, 8 times or 9 times. In some embodiments, the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen is 10 times, 15 times, 20 times or 25 times. In some embodiments, the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen is 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times or more. In some embodiments, depending on the concentration of the first labeled form of the antigen, the concentration of the unlabeled form of the antigen can be between 0.4 nM and 1 uM or between 10 nM and 600 nM. In some embodiments, depending on the concentration of the first labeled form of the antigen, the concentration of the unlabeled form of the antigen is between 10nM and 600nM, 10nM to 500nM, 10nM to 400nM, 10nM to 300nM, 10nM to 200nM, 10nM to 150nM, 10nM to 100nM, 10nM to 75nM, 10nM to 65nM, 10nM to 50nM, 10nM to 40nM, 10nM to 30nM, 10nM to 25nM or 10nM to 20nM. In embodiments using a second labeled form of the antigen (in heat tracking or combined tracking), the molar ratio of the second labeled form of the antigen relative to the first labeled form is at least 2 times, i.e., 2 times to as high as, for example, 2500 times. In some embodiments, the molar ratio of the second labeled form of the antigen relative to the first labeled form of the antigen is 2 times. In some embodiments, the molar ratio of the second labeled form of the antigen relative to the first labeled form of the antigen is 3 times. In some embodiments, the molar ratio of the second labeled form of the antigen relative to the first labeled form of the antigen is 4 times. In some embodiments, the molar ratio of the second labeled form of the antigen relative to the first labeled form of the antigen is 5 times.In some embodiments, the molar ratio of the second labeled form of the antigen relative to the first labeled form of the antigen is 6 times, 7 times, 8 times or 9 times. In some embodiments, the molar ratio of the second labeled form of the antigen relative to the first labeled form of the antigen is 10 times, 15 times, 20 times or 25 times. In some embodiments, the molar ratio of the second labeled form of the antigen relative to the first labeled form of the antigen is 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times or more. In some embodiments, depending on the concentration of the first labeled form of the antigen, the concentration of the second labeled form of the antigen can be between 0.4nM and 1μm or 10nM and 600nM. In some embodiments, the concentration of the second labeled form of the antigen can be between 10nM to 600nM, 10nM to 500nM, 10nM to 400nM, 10nM to 300nM, 10nM to 200nM, 10nM to 150nM, 10nM to 100nM, 10nM to 75nM, 10nM to 65nM, 10nM to 50nM, 10nM to 40nM, 10nM to 30nM, 10nM to 25nM, or 10nM to 20nM, depending on the concentration of the first labeled form of the antigen.

[0129] In some embodiments where combined tracing is performed, the cold tracing antigen and the hot tracing antigen can be at the same concentration or at different concentrations.

[0130] In some embodiments of combined tracing, cells are contacted sequentially with a cold tracing antigen and a hot tracing antigen; and in some such embodiments, a washing step may be included between the two tracing antigens. In such embodiments, cells are washed with a wash buffer for an allotted time sufficient to remove unbound antigen. In some embodiments, cells are washed for a time of about 10 minutes to about 60 minutes in one or more washes.

[0131] Tracking is performed for a period of time sufficient to track the antigen and antibody. In some embodiments, tracking is performed with an unlabeled form or a second labeled form of antigen for about 5 minutes to about 60 minutes, such as about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 45 minutes or about 50 minutes. In some embodiments of combined tracking, tracking is performed with an unlabeled form of antigen for about 5 minutes to about 60 minutes, and then incubated with the second labeled form of antigen for about 5 minutes to about 60 minutes. The incubation time with the unlabeled form of antigen and the second labeled form of antigen can be the same or different. As a non-limiting example, the unlabeled form of antigen can contact the cell for about 30 minutes, and the second labeled form of antigen can subsequently contact the cell for about 45 minutes, and vice versa. In some embodiments of combined tracking, tracking is performed with the second labeled form of antigen for about 5 minutes to about 60 minutes. In some embodiments of combined tracking, tracking is performed with the unlabeled form of antigen and the second labeled form of antigen simultaneously for about 5 minutes to about 60 minutes.

[0132] In some embodiments, tracing is performed more than once. In some embodiments, cold tracing is performed more than once. In some embodiments, hot tracing is performed more than once. In some embodiments, cold tracing is performed once, followed by more than one hot tracing. In some embodiments, cold tracing is performed more than once, followed by one hot tracing. In some embodiments, cold tracing is performed more than once, followed by more than one hot tracing. In some embodiments, combined tracing is performed more than once. In some embodiments, a washing step is included after each tracing step. In some embodiments, more than one washing step, such as 2 washes or 3 washes, is included after each tracing step.

[0133] After the chase is complete, unbound antigen is again removed by washing, and cells still bound to the first labeled form of the antigen are collected.

[0134] In some embodiments, wherein the first detectable label is a first fluorescent label, fluorescence activated cell sorting (FACS) can be used to collect cells that remain bound to the antigen in the first labeled form. In some embodiments, wherein the first detectable label is a first fluorescent label, and the second detectable label is a second fluorescent label different from the first fluorescent label, two-dimensional FACS is used to collect cells that remain bound to the antigen in the first labeled form. In specific embodiments, the first detectable label is A647, and the second detectable label is phycoerythrin.

[0135] Isolation of nucleic acid encoding the antibody

[0136] Antibody secreting cell pools can be sorted or separated into single cells. The protocol for separating single cells by flow cytometry is well known (Huang, J. et al., 2013, supra). Single cells can be sorted and collected by alternative methods known in the art, including but not limited to manual single cell picking, limiting dilution, microfluidics, laser capture microdissection and gel bead emulsions (GEMs), which are well known in the art. See, for example, Rolink et al., J Exp Med (1996) 183: 187-194; Lightwood, D. et al., J. Immunol. Methods (2006) 316 (1-2): 133-43; Gross et al., Int. J. Mol. Sci. (2015) 16: 16897-16919; and Zheng et al., Nature Communications (2017) 8: 14049. Gel bead emulsions (GEMs) are also commercially available (eg, 10X Chromium System from 10x Genomics, Pleasanton, CA).

[0137] Once obtained, cells secreting individual antibodies can be propagated by common cell culture techniques for subsequent DNA preparation. Alternatively, antibody genes can be amplified directly from individual antibody-producing cells and then cloned into DNA vectors.

[0138] Nucleic acids encoding antibodies or their fragments can be separated from single antibody secreting cells obtained herein. For example, genes or nucleic acids encoding immunoglobulin variable heavy chains and variable light chains (i.e., VH, VL, VL can be Vκ or Vλ) can be recovered using nucleic acids separated from antibody secreting cells using RT-PCR protocols. For example, nucleic acids encoding antibody fragments are first reverse transcribed (RT) into complementary DNA (cDNA). Subsequently, primers specific to antibody gene sequences (e.g., constant regions of antibody chains) are used to amplify cDNA in a PCR reaction. Chain-specific primers can be used to amplify all chains in multiple or separate ways. Then the amplicon is sequenced to obtain the nucleic acid sequence encoding the antibody fragment. These RT-PCR protocols are well-known conventional techniques, such as, for example, Wang et al., J. Immunol. Methods (2000) 244: described in 217-225 and described herein.

[0139] In some embodiments, nucleic acid encoding antibody fragments, such as variable domains, constant domains or combinations thereof. In certain embodiments, nucleic acid encoding antibody variable domains isolated from antibody production cells. In some embodiments, nucleic acid encoding antibody heavy chain or fragments thereof. In other embodiments, nucleic acid encoding antibody light chain or fragments thereof.

[0140] Once recovered, the gene or nucleic acid encoding the antibody can be cloned into an IgG heavy chain and light chain expression vector and expressed by the transfection of a host cell. For example, the gene or nucleic acid encoding the antibody can be inserted into a reproducible vector for further cloning (amplification of DNA) or for expression in a cell (stable or transient). Many vectors (particularly expression vectors) are available or can be engineered to contain the appropriate regulatory elements required for regulating the expression of antibody encoding genes or nucleic acids.

[0141] The expression vector in the context of the present disclosure can be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (containing a nucleic acid sequence of a suitable set of expression control elements) as described herein. Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors.

[0142] In some embodiments, the nucleic acid molecule is contained in a naked DNA or RNA vector, including, for example, a linear expression element (as described, for example, in Sykes and Johnston, Nat Biotech (1997) 12: 355-59), a compact nucleic acid vector (as described, for example, in US 6,077,835), or a plasmid vector such as pBR322 or pUC 19 / 18. Such nucleic acid vectors and their uses are well known in the art. See, for example, US 5,589,466 and US 5,973,972.

[0143] In certain embodiments, the expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as yeast alpha factor, alcohol oxidase, and PGH. See F. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987); and Grant et al., Methods in Enzymol 153, 516-544 (1987).

[0144] In some embodiments, an expression vector carrying a nucleic acid separated from an antibody secreting cell and encoding an antibody or its fragment is introduced into a host cell for expression of the antibody or its fragment. Host cells include, for example, mammalian cells, yeast cells, bacterial cells, or insect cells. In some embodiments, the host cell is cultured under conditions of expressing nucleic acid, and then an antibody or its portion can be produced, and separated for further use. In some embodiments, a host cell comprising one or more of the above-mentioned nucleic acids is cultured under conditions of expressing a full-length antibody, and then antibodies can be produced and separated for further use. In certain embodiments, the host cell comprises nucleic acids encoding the variable domains of an antibody, and the cell is cultured under conditions of expressing the variable domains. In other embodiments, the host cell comprises nucleic acids encoding the variable heavy chain (VH) domains of an antibody, and the cell is cultured under conditions of expressing the VH domains. In another embodiment, the host cell comprises nucleic acids encoding the variable light chain (VL) domains of an antibody, and the cell is cultured under conditions of expressing the VL domains. In a specific embodiment, the host cell comprises a nucleic acid encoding an antibody VH domain and a nucleic acid encoding an antibody VL domain, and the cell is cultured under conditions in which the VH domain and the VL domain are expressed.

[0145] In some embodiments, the host cell is a bacterial or yeast cell. In some embodiments, the host cell is a mammalian cell. In other embodiments, the host cell can be, for example, Chinese hamster ovary cells (CHO), such as CHO K1, DXB-11CHO, Veggie-CHO cells; COS (e.g., COS-7); stem cells; retinal cells; Vero cells; CV1 cells; kidney cells, such as, for example, HEK293, 293EBNA, MSR 293, MDCK, aHaK, BHK21 cells; HeLa cells; HepG2 cells; WI38; MRC 5; Colo25; HB 8065; HL-60; Jurkat or Daudi cells; A431 (epidermal) cells; CV-1, U937, 3T3 or L- cells; C127 cells, SP2 / 0, NS-0 or MMT cells, tumor cells and cell lines derived from any of the foregoing cells. In a specific embodiment, the host cell is a CHO cell. In a specific embodiment, the host cell is a CHO K1 cell.

[0146] The present disclosure also provides a method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by an antibody-secreting cell comprising a modified cell surface, and wherein the target antibody is subsequently captured on the modified cell surface, the method comprising: a) providing an antibody-secreting cell comprising a modified cell surface, wherein the cell secretes the target antibody, wherein the target antibody is subsequently captured on the modified cell surface, wherein the target antibody captured on the modified cell surface is bound to an antigen, wherein the antigen is linked to a barcode nucleic acid molecule, and wherein the antibody-secreting cell further comprises a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of the target antibody captured on the modified cell surface; b) subjecting the barcode nucleic acid molecule to a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of the target antibody captured on the modified cell surface; The invention relates to a method for preparing an antigen barcode nucleic acid molecule comprising a first amplicon library for gene expression in antibody-secreting cells, a second amplicon library for variable regions, diversity regions, and joining regions (VDJ) in antibody-secreting cells, and a third amplicon library for antigen barcode nucleic acid molecules; e) sequencing each of the three amplicon libraries; and f) identifying the region of the gene encoding the antigen binding fragment of the target antibody captured on the surface of the modified cell.

[0147] In the above method, regarding step b), a portion of the barcode nucleic acid molecule is hybridized with a portion of the first nucleic acid molecule attached to a solid surface, in a separator (e.g., a droplet), the separator (e.g., a bead) is dissolved and the cell barcode nucleic acid molecule / TSO oligomer floats freely.

[0148] In the above method, with respect to step c), a portion of a nucleic acid molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on a modified cell surface is hybridized with a portion of a second nucleic acid molecule attached to a solid surface, and a nucleic acid molecule (e.g., BCR mRNA) in a region of a gene encoding an antigen-binding fragment of a target antibody captured on a modified cell surface is not directly hybridized. Instead, it is first made into a first-strand cDNA, and then a reverse transcriptase adds CCC to the end of the cDNA. CCC is then hybridized with the rGrGrG portion of TSO. In addition, with respect to a second nucleic acid molecule attached to a solid surface, in a separator (e.g., a droplet), the separator (e.g., a bead) dissolves and the second nucleic acid molecule floats freely.

[0149] In some embodiments, the modification of the modified cell surface comprises avidin, streptavidin, avidin, biotin, jun protein or a portion thereof, fos protein or a portion thereof, mad protein or a portion thereof, max protein or a portion thereof, myc protein or a portion thereof, azide, alkyne and / or phosphine. In some embodiments, the modification of the modified cell surface comprises avidin. In some embodiments, the modification of the modified cell surface comprises streptavidin. In some embodiments, the modification of the modified cell surface comprises biotin. In some embodiments, the modification of the modified cell surface comprises jun protein or a portion thereof. In some embodiments, the modification of the modified cell surface comprises fos protein or a portion thereof. In some embodiments, the modification of the modified cell surface comprises mad protein or a portion thereof. In some embodiments, the modification of the modified cell surface comprises max protein or a portion thereof. In some embodiments, the modification of the modified cell surface comprises myc protein or a portion thereof. In some embodiments, the modification of the modified cell surface comprises azide. In some embodiments, the modification of the modified cell surface comprises alkyne. In some embodiments, the modification of the surface of the modified cell comprises a phosphine.

[0150] In some embodiments, a portion of the barcode nucleic acid molecule is complementary to a first template switch oligonucleotide (TSO). In some embodiments, a portion of the barcode nucleic acid molecule can be attached to a first TSO. In some embodiments, a 3' barcode system can be used. In this case, the antigen barcode will have polyA. VDJ enrichment and sequencing can be performed in different ways.

[0151] In some embodiments, the portion of the first nucleic acid molecule attached to the solid surface comprises a first template switch oligonucleotide (TSO). In some embodiments, the 3' end of the first nucleic acid molecule attached to the solid surface comprises a first sequence of three consecutive riboguanosine residues. In some embodiments, the first nucleic acid molecule attached to the solid surface further comprises a first unique molecular identifier (UMI). In some embodiments, the first nucleic acid molecule attached to the solid surface further comprises a first surface barcode. In some embodiments, the first nucleic acid molecule attached to the solid surface further comprises a first sequencing primer. In some embodiments, the first nucleic acid molecule attached to the solid surface comprises a first sequence of three riboguanosine residues, a first TSO, a first UMI, a first surface barcode, and a first sequencing primer from 3' to 5'. In some embodiments, the first nucleic acid molecule attached to the solid surface is attached via the 5' end of the first nucleic acid molecule attached to the solid surface.

[0152] In some embodiments, the first nucleic acid molecule attached to the solid surface comprises a first DNA molecule attached to the solid surface. In some embodiments, the first DNA molecule attached to the solid surface comprises a first single-stranded DNA molecule attached to the solid surface. In some embodiments, the portion of the first single-stranded DNA molecule attached to the solid surface comprises a first TSO, and the first single-stranded DNA molecule attached to the solid surface is reverse transcribed from the 3' end of the portion of the barcode nucleic acid molecule that is complementary to the first TSO. Reverse transcription also occurs in the other strand, where the antigen barcode nucleic acid extends from its 3' and the first nucleic acid molecule attached to the solid surface also extends from its 3'.

[0153] In some embodiments, the barcode nucleic acid molecule is a single-stranded DNA barcode nucleic acid molecule, wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first TSO, and the single-stranded DNA barcode nucleic acid molecule is reverse transcribed from the 3' end of the first TSO.

[0154] In some embodiments, the 3' end of a portion of a barcode nucleic acid molecule comprises three consecutive cytidine or ribocytidine residues.

[0155] In some embodiments, an mRNA molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on a modified cell surface is reverse transcribed, thereby generating a single-stranded DNA molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on a modified cell surface. The actual chemical reaction occurs in a solution in a separator (e.g., a droplet).

[0156] In some embodiments, the nucleotide sequence of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface is different from the complementary sequence of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface in that the 3' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface contains three additional continuous cytidine or ribocytidine residues. In some embodiments, the 3' end of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface contains multiple continuous adenine residues. In some embodiments, the 5' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface contains multiple continuous thymine residues. In some embodiments, the 5' end of the second nucleic acid molecule attached to the solid surface contains a second sequence of three continuous riboguanosine residues.

[0157] In some embodiments, the second nucleic acid molecule attached to the solid surface further comprises a second UMI. In some embodiments, the second nucleic acid molecule attached to the solid surface further comprises a second surface barcode. In some embodiments, the second surface barcode is the same surface (cell) barcode as the first nucleic acid molecule, so as to match the antigen with the BCR. In some embodiments, the second nucleic acid molecule attached to the solid surface further comprises a second sequencing primer. In some embodiments, the second nucleic acid molecule attached to the solid surface comprises a second sequence of three riboguanosine residues from 3' to 5', a second TSO, a second UMI, a second surface barcode, and a second sequencing primer. In some embodiments using the 10X Genomics system, for each nucleic acid molecule on the same surface, only the UMI is different. As long as the cell barcode is the same, other components (e.g., TSO and sequencing primers) may be different. In some embodiments, the second nucleic acid molecule attached to the solid surface is attached via the 5' end of the second nucleic acid molecule attached to the solid surface.

[0158] In some embodiments, the second nucleic acid molecule attached to the solid surface comprises a second DNA molecule attached to the solid surface. In some embodiments, the second DNA molecule attached to the solid surface comprises a second single-stranded DNA molecule attached to the solid surface. In some embodiments, the 3' end of the portion of the second single-stranded DNA molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues. In some embodiments, the 3' end of the portion of the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface comprises three consecutive cytidine residues. In some embodiments, the second single-stranded DNA molecule attached to the solid surface is reverse transcribed from the 3' end of the three consecutive cytidine residues.

[0159] In some embodiments, the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface comprises a second single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the antibody captured on the modified cell surface. In some embodiments, the 3' end of the portion of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface comprises three consecutive cytidine or ribocytidine residues, and the 5' end of the portion of the second single-stranded DNA molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues. In some embodiments, the second single-stranded DNA molecule attached to the solid surface is reverse transcribed from the 3' end of the single-stranded DNA molecule portion of the region encoding the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface. In some embodiments, the single-stranded DNA molecule of the region encoding the gene is reverse transcribed from the 3' end of the second single-stranded DNA molecule attached to the solid surface.

[0160] In some embodiments, the antibody secreting cell is placed in a separator with a solid surface. In some embodiments, the separator comprises a single antibody secreting cell. In some embodiments, the separator comprises a single plasma cell or plasmablast. In some embodiments, the solid surface comprises beads. In some embodiments, the antibody secreting cell in the separator is lysed. In some embodiments, the solid surface (e.g., beads) dissolves when separated, and the DNA molecules attached to its surface are released into the solution in the separator. In some embodiments, the separator is oil. In some embodiments, the separator is in the form of droplets.

[0161] In some embodiments, the barcoded nucleic acid molecule further comprises a third sequencing primer.

[0162] In some embodiments, each amplicon is generated from a first library of amplicons comprising a gene encoding an antigen-binding fragment of a target antibody captured on the surface of a modified cell.

[0163] In some embodiments, the first amplicon library is sample demultiplexed, aligned, filtered, and UMI counted. In some embodiments, sequencing of the first amplicon library comprises next generation sequencing.

[0164] In some embodiments, each amplicon of the first amplicon library is mapped to and aligned with a standard reference genome. In some embodiments, the standard reference genome comprises a human standard reference genome. In some embodiments, the human standard reference genome is GRCh38. In some embodiments, the standard reference genome comprises a mouse standard reference genome. In some embodiments, the mouse standard reference genome is mm10.

[0165] In some embodiments, each amplicon of the first amplicon library is mapped to a standard reference genome using single cell alignment software, such as STAR or kallisto (Bray et al., Nat. Biotechnol., 2016, 34, 525-527; Erratumin: Nat. Biotechnol., 2016, 34, 888), wherein each amplicon of the first amplicon library comprises a unique molecular identifier, wherein all amplicons of the first amplicon library mapped to annotated genes of the standard reference genome are classified, and wherein the classified amplicons are counted for each unique molecular identifier (UMI), thereby generating a single cell count matrix comprising a mapped count for each annotated gene and a count for each UMI.

[0166] In some implementations, the total number of annotated genes is calculated by dividing the log of the total number of UMIs counted. 10In some embodiments, cells with a gene to UMI ratio below about 0.1 are filtered out. In some embodiments, cells with an interquartile range of more than about four times the total number of UMIs counted are filtered out. In some embodiments, cells with more than about 80% of reads mapped to mitochondrial genes are filtered out. In some embodiments, cells with a gene to UMI ratio below about 0.1, cells with an interquartile range of more than about four times the total number of UMIs counted, and cells with more than about 80% of reads mapped to mitochondrial genes are filtered out. In some embodiments, any thresholds can be set for the gene to UMI ratio and the mitochondrial threshold. These values ​​are highly dependent on the tissue used. More stable tissues (such as blood and lymph nodes) require less stringent thresholds, while tissues with high mitochondrial contamination (such as intestine, lung, or skin) will have higher numbers of dead / dying cells.

[0167] In some implementations, the single cell count matrix is ​​normalized so that the total number of UMIs counted is about 1,000, 5,000, 10,000, 20,000, 50,000, or 100,000. In some implementations, the single cell count matrix is ​​normalized so that the total number of UMIs counted is about 10,000. In some implementations, the single cell count matrix is ​​normalized so that the total number of UMIs counted is about 5,000. In some implementations, the single cell count matrix is ​​normalized so that the total number of UMIs counted is about 20,000.

[0168] In some embodiments, the principal component embedding of the single cell count matrix is ​​calculated by principal component analysis (PCA), and used as input for calculating the unified manifold approximation and projection (UMAP). Alternatively, tSNE projections can also be calculated based on PCA embedding (van der Maaten et al., J.Mach.Learning Res., 2008, 9, 2579-2605). Additional iterative clustering can be performed on the main cell types to identify similarities between batches. In some embodiments, UMAP is generated using the method disclosed in Korsunsky et al., Nat.Methods, 2019, 16, 1289-1296.

[0169] In some embodiments, a linear adjustment function for each unit is determined using a cluster-specific centroid, the linear adjustment function for each unit is applied to correct for differences in the batch, thereby generating a batch-corrected embedding, and the batch-corrected embedding is used to generate a two-dimensional unified manifold approximation and projection (UMAP). This method is called harmony (Korsunsky et al., Nat. Methods, 2019, 16, 1289-1296), however, there are several alternatives, including the calculation of integration anchors from Seurat (Hao et al., Cell, 2021, 184, 3573-3587).

[0170] In some embodiments, a weighted k-neighbor graph is determined, and the Leiden algorithm is applied to a weighted k-neighbor graph with a resolution value of about 0.1, about 0.2, about 0.5, and about 1.0 to determine cell type clustering in an unsupervised manner. In some embodiments, a weighted k-neighbor graph is determined, and the Leiden algorithm is applied to a weighted k-neighbor graph with a resolution value of about 0.1 to determine cell type clustering in an unsupervised manner. In some embodiments, a weighted k-neighbor graph is determined, and the Leiden algorithm is applied to a weighted k-neighbor graph with a resolution value of about 0.2 to determine cell type clustering in an unsupervised manner. In some embodiments, a weighted k-neighbor graph is determined, and the Leiden algorithm is applied to a weighted k-neighbor graph with a resolution value of about 0.5 to determine cell type clustering in an unsupervised manner. In some embodiments, a weighted k-neighbor graph is determined, and the Leiden algorithm is applied to a weighted k-neighbor graph with a resolution value of about 1.0 to determine cell type clustering in an unsupervised manner. Leiden is a community detection algorithm that iteratively clusters nodes by splitting similar units to generate well-connected clusters (Traag et al., Scientific Reports, 2019, 9, 5233). Alternative algorithms include Louvain, K-means, and NMF clustering.

[0171] In some embodiments, a paired Wilcox test is performed on all cells in a cluster, and the results of the first paired Wilcox test are compared, and another paired Wilcox test is performed on all cells in every other cluster to quantify the p-value and fold change of each gene in each cluster. In some embodiments, when a gene has a low p-value and a high fold change, the cell is labeled with a common marker of antibody secreting cells, and the cell is labeled with a subtype of the cluster named by the top gene from the differential expression result. This can be repeated for cells with and without antigen specificity in each isotype (mainly IgE). In some embodiments, a test such as DESeq2 can be used for differential expression.

[0172] In some embodiments, sample demultiplexing is performed, de novo assembly of read pairs into contigs is completed, and contigs against germline segment V(D)J reference sequences are aligned and annotated for the second amplicon family.

[0173] In some embodiments, the alignment of the contigs against the germline segment V(D)J reference sequences comprises alignment against a human germline reference database or a mouse germline reference database using the software IgBlast (Ye et al., Nuc. Acids Res., 2013, 41, W34-W40). In some embodiments, the human germline reference database comprises the IMGT database of human germline immunoglobulin sequences (World Wide Web "imgt.org / IMGTrepertoire / LocusGenes / "). In some embodiments, the mouse germline reference database comprises the IMGT germline reference database.

[0174] In some embodiments, the VDJ sequence is compared to the sequence in the human germline reference database or the mouse germline reference database, thereby marking the VDJ sequence for quality comparison by checking the in-frame comparison, the length of CDR3 and / or the absence of the stop codon in the variable region sequence and the VDJ sequence filtered based on the tag. In some embodiments, the VDJ sequence is compared to the sequence in the human germline reference database or the mouse germline reference database, thereby marking the VDJ sequence for quality comparison by checking the in-frame comparison. In some embodiments, the VDJ sequence is compared to the sequence in the human germline reference database or the mouse germline reference database, thereby marking the VDJ sequence of the length of CDR3. In some embodiments, the VDJ sequence is compared to the sequence in the human germline reference database or the mouse germline reference database, thereby marking the VDJ sequence lacking the stop codon in the variable region sequence. Alternatively, this step can be omitted to retain all possible VDJ sequences, which can be compared with IgBlast to confirm the high-quality sequence in the human or mouse variable region frame.

[0175] Without a database of germline VDJ sequences, this step is impossible for less-studied organisms.

[0176] In some embodiments, the VDJ sequence is mapped to a reference database of immunoglobulin chains (e.g., IMGT).

[0177] In some embodiments, the accuracy of the immunoglobulin isotype, variable region map, connecting region map, diversity region map, full-length variable region of the heavy chain and light chain sequence of each cell is confirmed by comparison. In some embodiments, the immunoglobulin isotype is confirmed by comparison. In some embodiments, the variable region map is confirmed by comparison. In some embodiments, the connecting region map is confirmed by comparison. In some embodiments, the diversity region map is confirmed by comparison. In some embodiments, the accuracy of the full-length variable region of the heavy chain and light chain sequence of each cell is confirmed by comparison. In some embodiments, the VDJ sequence is mapped and annotated to the germline by IgBlast.

[0178] In some embodiments, the third amplicon library is sample demultiplexed, aligned, filtered, and UMI counted. In some embodiments, single cell alignment software such as STAR or kallisto can be used.

[0179] In some embodiments, the third amplicon library is mapped to a customized short read reference that includes a barcode nucleic acid molecule reference associated with each antigen. In some embodiments, for each cell in the barcoded antigen single cell matrix, the counts for each uniquely mapped barcode nucleic acid molecule are summed.

[0180] In some embodiments, the barcode nucleic acid molecules in all cells are quantified and normalized by taking the center logarithm ratio of each of the multiple barcode nucleic acid molecules captured for each sample. Alternatively, the center logarithm ratio or background denoising and scaling (DSB) can be used (Mulè et al., bioRxiv, 2020.02.24.963603).

[0181] In some embodiments, for each barcoded nucleic acid molecule in the plurality of barcoded nucleic acid molecules, background antigen signals are removed by DSBs. Alternatively, a central log ratio can be used.

[0182] In some embodiments, the antigen signal distribution is determined, wherein when there is a well-separated bimodal distribution in the antigen signal distribution, the z-transform value is used to calculate the antigen specificity, and wherein when there is no well-separated bimodal distribution in the antigen signal distribution, the quantile value is used. In some embodiments, a rank order can be used instead of a quantile value.

[0183] In some embodiments, analysis of the first amplicon library, the second amplicon library, and the third amplicon library are performed simultaneously to obtain at least one candidate sequence of an antigen-positive target antibody captured on the surface of a modified cell.

[0184] In some embodiments, the antibody secreting cells having effective antibody constant regions are grouped using analysis of the first amplicon library and analysis of the second amplicon library. In some embodiments, the antibody constant region comprises an IgE constant region. In some embodiments, the grouped antibody secreting cells comprise detectable levels of IgE heavy chain and CD79a, but lack detectable levels of Ms4a1 and CD19, wherein the grouped antibody secreting cells are IgE + Plasma cells or plasmablasts.

[0185] In some embodiments, IgE is assessed for IgA secreting cell isotypes, IgG secreting cell isotypes, and / or IgM secreting cell isotypes. + Differential expression of antibody secreting cells. In some embodiments, the assessment of differential expression further characterizes the IgE + Distinct transcriptional signatures of antibody-secreting cells.

[0186] In some embodiments, the IgE + Antigen specificity of antibody secreting cells. In some embodiments, the assessment includes the use of IgE + The antigen specificity of the antibody secreting cell is compared to a plurality of antigens and a control antigen, and wherein the plurality of antigens comprises a target antigen. In some embodiments, the comparison comprises calculating an empirical score for each of the plurality of antigens and the control antigen by subtracting the quantile value of the signal associated with the antigen (qT) from the quantile value of the signal associated with the control antigen (qC) with a penalty factor (x) to determine an antigen specificity score, wherein the antigen specificity score = qT - qC x In some embodiments, the penalty factor is the same for all antigens. In some embodiments, the penalty factor is different for different antibody isotypes. In some embodiments, the penalty factor is different when other antigens are used.

[0187] In some embodiments, the antigen specificity score for each of the plurality of antigens and a control antigen is determined. In some embodiments, the control antigen specificity score is calculated, wherein cells are selected when the antigen specificity score is high and the control antigen specificity score is low. In some embodiments, the VDJ region identifies antigen specific antibodies.

[0188] In some embodiments, paired V H :V L Antibody sequences select antibody candidates from a hierarchical list of antigenic signals.

[0189] In some embodiments, differential expression between isotypes, conditions, and / or antigen specificities is determined, wherein determining differential expression between isotypes comprises performing a paired Wilcox test on all cells in one isotype and performing a paired Wilcox test on all cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster; and wherein determining differential expression between antigen specificities comprises performing a paired Wilcox test on all cells in one isotype and performing a paired Wilcox test on all cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster.

[0190] There are many models / statistics for differential expression analysis. Some examples include: 1) "wilcox", which uses the Wilcoxon rank sum test (as described herein) to identify genes that are differentially expressed between two groups of cells; 2) "bimod", which is a likelihood ratio test of single cell gene expression, (McDavid et al., Bioinformatics, 2013); 3) "roc", which uses ROC analysis to identify "markers" of gene expression; for each gene, evaluate (using AUC) a classifier built based only on that gene to classify between the two groups of cells; an AUC value of 1 means that the expression value of the gene alone can classify the two groups very well (i.e., each of the cells in cell 1 shows a higher level than each of the cells in cell 2); an AUC value of 0 also means that there is a perfect classification, but in the opposite direction; a value of 0.5 means that the gene has no predictive power to classify the two groups; it returns the "predictive power" (abs(AUC - 0.5)*2) ranking matrix; 4) "t", which uses Student's t-test to identify differentially expressed genes between two groups of cells; 5) "negbinom", which uses a negative binomial generalized linear model to identify differentially expressed genes between two groups of cells; 6) "Poisson", which uses a Poisson generalized linear model to identify differentially expressed genes between two groups of cells; 7) "LR", which uses a logistic regression framework to determine differentially expressed genes; build a logistic regression model to predict group membership based on each feature separately and compare it with the null model using a likelihood ratio test; 8) "MAST", which uses a fence model customized for scRNA-seq data to identify differentially expressed genes between two groups of cells; run the DE test using the MAST package in R; 9) "DESeq2", which identifies differentially expressed genes between two groups of cells based on a model using DESeq2, which uses a negative binomial distribution (Love et al., Genome Biol., 2014); and 10) "Detection rate", which identifies differentially expressed genes between two groups of cells based on the percentage detection rate between cell groups.

[0191] In some embodiments, VDJ sequence similarities in antigen-positive target cells are clustered, wherein clustering the VDJ sequence similarities comprises grouping similar variable regions by amino acid sequence based on sequence alignment.

[0192] In some embodiments, the antigen-positive target cell is IgE + .

[0193] In some embodiments, redundant sequences are trimmed.

[0194] The present disclosure also provides a method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with a second component of a binding pair so that the second component of the binding pair binds to a cell surface, wherein the population of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody; b) contacting the population of antibody-secreting cells with an antibody capture complex, wherein the antibody capture complex comprises a first component of a binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to the second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cell; c) contacting the population of antibody-secreting cells with a second anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cell and captured by the antibody capture complex binds to the second anti-Ig antibody; d) contacting the population of antibody-secreting cells with an antigen comprising a barcode nucleic acid molecule, whereby the target antibody secreted by the antibody-secreting cell and captured by the antibody capture complex binds to the antigen; e) collecting the antibody-secreting a cell pool, wherein each cell in the pool secretes an antibody captured by an antibody capture complex and bound by a second anti-Ig antibody and an antigen comprising a barcode nucleic acid molecule, and wherein the antibody-secreting cell pool comprises antibody-secreting cells that secrete a target antibody; f) separating the antibody-secreting cell pool into single antibody-secreting cells, and for each single antibody-secreting cell: 1) hybridizing a portion of the barcode nucleic acid molecule with a portion of a first nucleic acid molecule attached to a solid surface, wherein the solid surface and the first nucleic acid molecule attached thereto are unique to each single antibody-secreting cell; 2) hybridizing a portion of a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of the target antibody with a portion of a second nucleic acid molecule attached to the solid surface; 3) preparing a first amplicon library of gene expression, a second amplicon library of variable regions, diversity regions, and joining regions (VDJ), and a third amplicon library of antigen barcode nucleic acid molecules; and 4) sequencing each of the three amplicon libraries; and g) identifying a region of a gene encoding an antigen-binding fragment of the target antibody.

[0195] In order to more effectively understand the subject matter disclosed herein, examples are provided below. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the claimed subject matter in any way. In all of these examples, molecular cloning reactions and other standard recombinant DNA techniques are performed according to the methods described in Maniatis et al., Molecular Cloning-A Laboratory Manual, 2nd edition, Cold Spring Harbor Press (1989), using commercially available reagents, unless otherwise stated.

[0196] Example

[0197] Materials and Methods

[0198] Reagents used

[0199] Mouse Fluorescent Antibody (Fluorescent Conjugated)

[0200]

[0201]

[0202] Human Fluid Antibody (Fluorescent Conjugated)

[0203]

[0204] Reagents and batch numbers used

[0205]

[0206]

[0207]

[0208] Biotinylation of cell surface

[0209] The cells were washed and resuspended in PBS containing 5% w / v BSA. The surface Fc was blocked with a 1:10 dilution of Fc blocker (eBioscience), while the surface BCR (for cell line determination) was detected at 4°C for 15 minutes. The cells were washed twice more with PBS, centrifuged at 500g for 10 minutes, resuspended in 1mL of freshly prepared NHS-biotin (Sigma, 0.5mg / mL in PBS) and incubated at 37°C for 15 minutes. The biotinylated cells were washed three times in cold PBS containing 5% w / v BSA, and the test tubes were changed for each washing step.

[0210] Preparation of Secretory IgE Capture Reagent

[0211] use The Streptavidin Conjugation Kit conjugates EctoFcεRIα to streptavidin to form a secretory IgE capture reagent. Briefly, a 0.5 mg / mL ectoFcεRIα solution containing 1 mg / mL streptavidin was prepared. One hundred microliters of modifier reagent was added to the solution under gentle mixing. The cap was removed from the vial of the streptavidin conjugated mixture and the mixture including the modifier reagent was added directly to the lyophilized material. The material was gently resuspended by withdrawing and redistributing the added suspension once or twice with a pipette. The cap was placed back on the vial and the vial was left to stand in the dark at 20-25°C for three hours. Thereafter, 100 μL of quencher was added under gentle mixing. Thus, a secretory IgE capture reagent was produced.

[0212] Incubation of cells with biotinylated cell surface and secreted IgE capture reagent

[0213] Washed biotinylated cells were plated at 1-3 × 10 6 Cells / mL were resuspended in 1 mL of PBS containing 5% w / v BSA and 30 g / mL of purified secretory IgE capture reagent and placed in a six-well plate. In some embodiments, a volume of up to 3 mL in a six-well plate can be used. The cells were incubated at 37°C on a rotating platform for one hour to capture secreted IgE. The cells were then washed twice in PBS containing 5% w / v BSA.

[0214] Lung cell preparation

[0215] Before obtaining lung samples, prepare the tubes and reagents. Add ten milliliters of sterile water to TH to give a final concentration of 26 collagenase Wunsch units / mL or 5.0 mg / mL collagenase. Aliquot one milliliter into individual tubes and store at -20°C. Prepare as needed Mixture (Digestion Buffer):

[0216]

[0217] For each mouse, 1.5 mL of Pipette the mixture into individual 5 mL flow cytometry tubes. Keep the tubes on ice or in a cold rack until needed for digestion.

[0218] For each mouse, the largest lobe of the right lung or the entire lung was placed in a separate 1 mL 2mL of the mixture In those tubes, the lung material is cut into small pieces, usually cubes of about 2-3 mm. The material is incubated at 37°C for twenty minutes. The digested material is added to the gentleMACS TM C tube and the digestion was terminated by adding 0.5 M EDTA to a final concentration of 10 mM. Two milliliters of Buffer. Tighten the lid and use the pre-loaded program m_lung_02_01 in gentleMACS TM Run the capped tubes on an Octo dissociator. Centrifuge the resulting cells at 400 g for four minutes and decant the supernatant. Lyse the erythrocytes in the pellet with 1 mL of erythrocyte lysis buffer for three minutes at ambient temperature. Thereafter, add 10 mL of DPBS to inactivate the lysis buffer. Centrifuge the cells at 400 g for four minutes, decant the supernatant and resuspend the pellet in 1 mL of DPBS. Pass the suspension through a 100 μm Plate filter into 2 mL deep well plate. Centrifuge cells at 400 g for four minutes and resuspend in 500 μL DPBS. Combine fifty microliters from each sample with 100 μL from the other sample and add 150 μL of the combined sample to a round bottom 96 well plate.

[0219] Draining lymph node-cell preparation

[0220] Add 1-2 mL of RPMI medium to a twelve-well 70 μm cell strainer plate. Collect drained lymph nodes and add to the plate. Prepare a PCR plate and punch each well with a 20G needle and place on a 2 mL deep well plate.

[0221] Using the back end of a 3 mL syringe, mash the draining lymph node in 2 mL of RPMI medium on a 74 μm cell strainer. Pass the preparation through a 100 μm Plate filter filtered into a 2 mL deep well plate and centrifuged filtered cells at 400 x g for four minutes. Resuspend the pellet in 500 μL DPBS and transfer to a 96-well deep bottom plate. Centrifuge the plate at 400 x g for four minutes. Resuspend the cells in 200 μL PBS. Add 200 μL of the suspension to a 96-well round bottom test tube. Centrifuge the plate at 400 x g for four minutes and flick off the supernatant. A typical cell count for mice administered 50 μg of HDM extract diluted in 20 μl saline intranasally three times a week for fifteen weeks was 230 × 10 6 cells.

[0222] Bone marrow cell preparation

[0223] 200 μL of RPMI medium was added to the 48-well plate. A PCR plate was prepared and punched in each well with a 20G needle and placed on a 2 mL deep well plate.

[0224] Extract the femur of the mouse and clean the bones. Cut off both ends of each bone and put it into a PCR plate with holes. Place the PCR plate on a 2mL deep well collection plate and centrifuge at 500x g for four minutes. If any BM remains in the bone, repeat the centrifugation step. Resuspend the precipitate in the added 500 μL red blood cell lysis buffer. Incubate the suspension at ambient temperature for three minutes, then add 1-2mL PBS to inactivate the lysis buffer. The resulting cells are centrifuged at 400x g for four minutes and the precipitate is resuspended in 1mL DPBS. The suspension is filtered into a 2mL deep well plate through a 100μm Millipore plate filter. Take out about 100-200 μL from each sample for fluorescence minus one (FMO), where the sample pool is used for FMO. The aliquoted merged sample is placed in twelve holes of FMO. The remaining sample is centrifuged at 400x g for four minutes.

[0225] The bone marrow samples were resuspended in 200 μL PBS. Half of the bone marrow samples were plated in a 96-well round-bottom plate. The plates were centrifuged at 400 x g for four minutes and the supernatant was flicked off. The typical cell count for mice administered 50 μg of HDM extract diluted in 20 μl of saline intranasally three times a week for fifteen weeks was 116 × 10 6 cells.

[0226] Splenocyte preparation

[0227] Spleens were extracted from mice and pre-loaded with TBA in gentleMACS TM Homogenize in 5mL DPBS in test tube. The spleen after homogenization is centrifuged at 400x g for four minutes and the supernatant is discarded. The spleen is then resuspended in 4mL red blood cell lysis buffer and incubated at room temperature for 5 minutes, and 10mL DPBS is added afterwards to neutralize the lysis buffer. The spleen is then centrifuged at 400x g for four minutes and the supernatant is discarded. The spleen is then resuspended in 10mL DPBS and the single cell suspension is filtered through a 70μM filter. Cell count and viability are measured, and then the spleen is subjected to antigen+B cell staining.

[0228] Pan-B cell isolation

[0229] use Mouse Pan-B Cell Isolation Kit was used to isolate B cells. Briefly, 1 × 10 8 Prepare the sample with 100 cells. Add 50 microliters of rat serum per milliliter of sample. Add the sample to a 14 mL polystyrene round-bottom test tube (Catalog No. 35205, Lot No. 00421123). Mix the cells and incubate at ambient temperature for five minutes. Add 50 microliters of vortexed 50 seconds per milliliter of sample. The mixture was incubated for 2.5 minutes. Phosphate buffered saline containing 5% w / v BSA was added to a volume of 5 mL and the tube was placed in a Big Easy Magnet (StemCell Cat. No. 18001) for 2.5 minutes. Pour the enriched cell suspension into a new tube and count. The procedure is outlined below:

[0230]

[0231] Example 1: Development of capture complex

[0232] To pair secreted antibodies with the ASCs that secrete them, immunoglobulin capture complexes were derived that can locally capture secreted antibodies on the surface of ASCs ( Figure 1 To this end, the cell surface was biotinylated using NHS-biotin, which has a hydrophilic reactive group that binds to primary amines (-NH2) on the cell surface. An affinity matrix was then assembled around the cells using streptavidin coupled to an Fc binding agent (αIgk for IgG and FcεRIα extracellular domain for IgE) to capture secreted immunoglobulins from ASCs ( Figure 1 To label PCs for antigen specificity, this method was combined with barcoded antigens and / or fluorescently labeled antigen staining ( Figure 1 ). Single-cell transcriptomes (scRNA-seq), barcoded antigen signals, and VH:VL antibody sequences were generated using 10X Genomics reagents.

[0233] To determine the functionality, sensitivity, and specificity of the IgG and IgE capture complexes, ARH77, a cell line secreting IgG (κ light chain), and U266, a myeloma cell line secreting IgE (λ light chain), were used in proof of principle experiments ( Figure 4A , Figure 4B , Figure 4D and Figure 4E ). It was first demonstrated that both cell lines lack BCR surface expression ( Fig.16A and Fig. 16B ARH77 and U266 cells were biotinylated using NHS-biotin, and the capture complex was assembled using streptavidin coupled to anti-human Igk for IgG (StAv-aIgk) and the extracellular domain of the high-affinity IgE receptor FcεRIα for IgE (StAv-FcεRIα). The sensitivity of capture was determined by staining for IgG and IgE ( Figure 4A and Figure 4B). Although ARH77 and U266, which lack any part of the affinity matrix, lack IgG and IgE on their cell surface, the successful assembly of all components of the secretion-capture complex allows the specific detection of secreted IgG and IgE on the surface of the cells that secrete them ( Figure 4A and Figure 4B To further determine the minimum concentrations of StAv-αIgk and StAv-FcεRIα required for binding to secreted antibodies, dose titration of the capture complex was performed and it was observed that StAv-αIgk saturated at 3.75 μg / mL, while StAv-FcεRIα saturated between 15-30 μg / mL ( Figure 4D and Figure 4E ).

[0234] To test the specificity of the capture complex, Ramos cells expressing IgM were incubated with U266 cells at a 1:1 ratio ( Figure 4F ) or 100:1( Fig. 16C ) were mixed, and the IgE secretion capture complex was assembled on the mixed population. No binding of secreted IgE was observed on the surface of Ramos cells, indicating minimal or no crosstalk with neighboring cells under the dilutions and culture conditions tested ( Figure 4F ; Fig. 16C ).

[0235] Example 2: Proof of concept for secretory IgE capture

[0236] Capture of secretory IgE: experimental procedure

[0237] A method for pairing a given secretory IgE antibody with a PC secreting that antibody was performed. Figure 2 (Step 1) shows a PC surface that has been biotinylated with NHS-biotin, because the N-hydroxysuccinimide group of NHS-biotin has a hydrophilic reactive group that can non-specifically react with primary amines (-NH2) on the cell surface. Step 2 shows a secretory IgE capture reagent bound to biotinylated PC. Figure 2 (Step 2) Also shown is ectoFceRIa, a secretory IgE capture reagent that captures PC-secreted IgE. Figure 2 (Step 3) Captured IgE bound to the barcoded antigen and bound by anti-IgE antibodies is shown.

[0238] Secretory IgE capture reagent successfully captured secretory IgE on the surface of U266 cells

[0239] The surface of IgE-secreting U266 cells was biotinylated as described herein, washed three times in cold PBS containing 5% w / v BSA, changing tubes for each wash step, and incubated with a secretory IgE capture reagent as described herein. Thereafter, the cells were washed and incubated with Brilliant TM Violet 711 labeled anti-IgE (BD) was stained for thirty minutes in PBS with 5% w / v BSA, 10 mg / mL dextran sulfate, and 1:50 Fc blocking agent. TM The cells were washed with Violet buffer. Thereafter, the cells were washed twice in PBS and fixed with BD Cytofix TM Fixation. Subsequently, flow cytometry data were acquired.

[0240] It was found that the secretory IgE capture reagent specifically captured the secretory IgE antibodies on the surface of U266 cells ( Figure 4B Importantly, the presence of biotinylated IgE in the absence of biotinylation, incubation with secreted IgE capture reagent, or incubation with Brilliant TM In the control cells exposed to Violet 711 labeled anti-IgE, no cell surface IgE antibodies were detected. This confirms that each step is necessary for the detection of captured secretory IgE. In addition, no IgE as BCR was detected on U266 cells. This confirms that captured secretory IgE antibodies, rather than IgE as part of the BCR, were detected on the surface of U266 cells. Figure 4C To test the specificity of the IgE secretion capture reagent, U266 cells were mixed with Ramos cells secreting human IgM at a 1:1 ratio. For U266 cells alone, the mixed cells were incubated with the secretory IgE capture reagent. The cells were then washed in PBS and incubated with Cytofix TM The flow cytometric data were obtained again. It was found that the secretory IgE capture reagent captured the secretory IgE antibodies on the surface of U266 cells, but did not capture the IgM secreted by Ramos cells. This indicates that the secretory IgE capture reagent has a specificity ( Figure 4C ).

[0241] Example 3: Capture of secretory IgE on the surface of primary plasma cells

[0242] IgE Venus and Blimp-1 mCherryReporter gene mice are used to determine whether secretory IgE capture reagents can capture secretory IgE on the surface of primary plasma cells that produce IgE. Specifically, the Venus reporter gene inserted downstream of the membrane IgE locus identifies cells that secrete IgE, while the mCherry reporter gene inserted downstream of Blimp-1 identifies PC (Asrat et al., Sci. Immunol., 2020, 10, eaav8402). Mice were challenged with 50 μg HDM extract diluted in 20 μl saline solution and administered intranasally three times a week for fifteen weeks. Mice were anesthetized and killed as described herein. BM cells were prepared as described herein and LDLN were prepared as described herein. BM from mice were merged together, and dLN from mice were merged together, respectively. Pan-B cells were separated and stained with Brilliant TM The cells were stained with a mixture of Violet 711-labeled anti-IgE (BD) antibodies to detect secretory IgE for thirty minutes at 4°C in PBS with 5% w / v BSA. The labeled cells were washed in PBS with 5% w / v BSA and incubated with Cytofix TM Fixed and used BD S6 flow cytometer sorting ( Fig. 7A and Figure 7B ). Plasma cells were gated in the following order: cells, single cells, live cells, DUMP - Cells, CD138 + Blimp-1 + cell( Fig. 7A and Figure 7B ). Dump is a collection of markers in the same channel, which includes CD3, CD11b, IgD, IgM, Ly6g, etc. Any cells expressing these markers are excluded. CD138 is detected using anti-CD138 antibody, and plasma cells are gated as Blimp1 + CD138 + PCs that secrete IgE are classified as IgE Venus+ cell( Figure 7C ).

[0243] Example 4: Preparation and sequencing of single cell libraries of antigen-specific IgE-expressing plasma cells

[0244] Fig. 7A A representative overview of the steps of Examples 3 to 5 is shown, and Fig. 9 The steps from barcoded antigen capture to secreted IgE on the PC surface combined with library selection and sequencing are shown.

[0245] Plasma cells were prepared as described in Example 3. Single PCs were suspended in PBS with 0.04% w / v BSA and loaded at 15,000 cells per lane. Controller device (10x Genomics). Form partitions of PCs in barcoded beads and lyse PCs. Perform reverse transcription and then resolve partitions. Use RNA-seq, signature barcode, and V(D)J libraries were prepared using the Next GEM Single Cell 5' Kit v.2 (10x Genomics). After library amplification, cDNA was separated into separate RNA-seq, signature barcode, and V(D)J aliquots. To enrich the V(D)J aliquot for BCR sequences, the Automated Single-Cell Mouse and Human BCR Amplification and Library Construction Kit (10xGenomics). 5' Signature Barcode Library Construction Kit (10x Genomics) is used to prepare a signature barcode library of DNA barcoded antigens or DNA barcoded cell surface proteins. Paired-end sequencing was performed on the RNA-seq library on the 6000 sequencing system, with read 1 of 26 bp for unique molecular identifiers (UMI) and cell barcodes, and read 2 of 80 bp for transcript reads, with 10 bp i7 and 10 bp i5 reads. Paired-end sequencing was also performed on the feature barcode library, with read 1 of 26 bp for UMI and cell barcodes, and read 2 of 35 bp for feature barcode reads, and with 10 bp i7 and 10 bp i5 reads. Paired-end sequencing was also performed on the V(D)J library, with read 1 of 150 bp, i7 of 10 bp, i5 of 10 bp, and read 2 of 150 bp. For RNA-seq and feature barcode libraries, sample demultiplexing, alignment, filtering, and UMI counting were performed using the Cell Ranger Single Cell Software Suite version 6.1.1 (10x Genomics). Human GRCh38 and mouse mm10 genome assemblies and RefSeq gene models for human and mouse were used for alignment, respectively. For V(D)J libraries, Cell Ranger software was used for sample demultiplexing, read pairs were reassembled into contigs, and contigs were aligned and annotated against all V(D)J germline reference sequences from the germline mouse IMGT reference database.

[0246] Example 5: Single Cell Analysis of Antigen-Specific IgE-Expressing Plasma Cells

[0247] Fig.10A representative flow chart of the steps described in this example is shown.After sequencing, the single cell gene expression library, the secreted antibody V(D)J region library and the barcoded antigen library are analyzed simultaneously to obtain candidate antigen-positive secreted IgE antibody sequences.

[0248] The single-cell gene expression library was mapped to the standard mouse reference genome Mm10 and aligned with it. Subsequently, the unique mapping count of each gene was determined by CellRanger software. The reads were mapped to the standard reference genome using the single-cell alignment software STAR, where each read contained a unique molecular identifier of an annotated gene mapped to the standard Mm10 reference genome. The mapped reads for each unique molecular identifier (UMI) were classified and counted to generate a single-cell count matrix including the mapping counts for each annotated gene and the counts for each UMI. The total number of annotated genes was then divided by the log of the total number of UMIs counted. 10 The single cell count matrix was filtered by the ratio of . Cells with a gene to UMI ratio below about 0.1 were filtered out. Cells with an interquartile range of more than four times the total number of UMIs were filtered out. Cells with more than about 80% of the reads mapped to mitochondrial genes were filtered out. High-quality cells with a gene to UMI ratio above about 0.1, an interquartile range of less than about four times the total number of UMIs counted, and less than 80% of the reads mapped to mitochondrial genes were retained. The count matrix from each individual sample capture was normalized to a total count of 10,000 and batch corrected using the Harmony algorithm to generate a combined unified manifold approximation and projection (UMAP). Unsupervised clustering using the Leiden algorithm was then used to determine cell type clusters, and the Wilcox test was used to identify specific cell types and cluster marker genes.

[0249] The V(D)J sequences were compared with the germline mouse IMGT reference database using the IgBlast sequence analysis tool (Ye et al., Nucleic Acids Res., 2013, 41, W34-W40). Valid V(D)J sequences were then filtered by valid in-frame alignments, full-length CDR3s, and the absence of stop codons in the sequence. The IgBlast tool also confirmed the immunoglobulin isotype, the gene mapping of the variable region, the difference region, and the joining region, as well as the accurate full-length variable region of the heavy and light chain sequences of each cell.

[0250] The barcoded antigen library was mapped to a customized short-read reference containing four barcoded antigens (i.e., Derp 1, Der p 2, Der f 1, and Ole e 1) by CellRanger software. These tag sequences were quantified in all cells and standardized by taking the center logarithm ratio (CLR) of each barcoded antigen in each sample capture. In addition, the background antigen signal of each barcoded antigen was removed by DSB (according to background denoising and calibration). Both CLR and DSB normalized values ​​were used to quantify the target antigen signal (i.e., the signal of Der p 1, Der p 2, and Der f 1) for the control signal (i.e., the signal of Ole e 1).

[0251] The antigen specific score (AgSS) of IgG was calculated by subtracting the quantile of the hIL-6Rα barcode UMI count from the quantile of the hIL-4Rα barcode UMI count multiplied by the penalty factor (x), AgSS = Q hIL-4Rα -Q hIL-6Rα x AgSS for IgE was calculated using a similar formula for mouse and human single cell data. For the IgE fraction, the target antigen was Der p 1, Der p 2, or Der f 1, while the control was always Ole e 1.

[0252] Ig BCR sequences were clustered using scirpy v0.10 with a Hamming distance of less than 4 using the function scirpy.pp.ir_dist based on amino acid sequence. Clustered BCRs were collected into clonotypes based on heavy chain CDR3 amino acid sequence using the function scirpy.tl.define_clonotypes based on the previous criterion of a Hamming distance of less than 4. Scirpy documentation can be found on the World Wide Web at "scverse.org / scirpy / latest / api.html".

[0253] After processing the three single cell libraries, gene expression and V (D) J region sequencing data were used to group PCs with effective IgE constant regions and detectable IgE and CD79a expression but lacking Ms4a1 and CD19 expression (usually found in non-PC B cells). The differential expression of PCs expressing IgE relative to other IgA, IgG and IgM plasma cell isotypes was then assessed to further characterize the unique transcriptional signature of PCs secreting IgE. The antigen specificity of PCs secreting IgE was then assessed by comparing the standardized levels of target antigens (i.e., Der p 1, Der p 2 or Der f 1) with the standardized levels of control antigens Ole e 1. This was done by calculating the empirical score for each target antigen and subtracting the quantile rank of the target antigen from the quantile rank of the control antigen with a penalty factor. The formula for doing so is as follows:

[0254] Antigen specificity = qT-qC x

[0255] where qT refers to the quantile of the target signal, qC refers to the quantile of the control signal, and x is the penalty factor.

[0256] For each of the three target antigens, the antigen specific score (AgSS) of each cell was determined and used to prioritize cells with the strongest target antigen signal while minimizing the signal of the control antigen. Using paired VH:VL antibody sequences from the cells, antibody candidates were selected from a ranked list of target antigen signals. Fig.11 Visualization of antigen specificity is shown.

[0257] Example 6: IgE Binding ELISA

[0258] After selection of allergen-specific antibodies, the relative binding of Der p 1, Der p 2 and Der f 1 was quantified by ELISA. Fig.12 A table of IgE candidates based on antigenic signaling is shown. 1,069 IgE + Specifically, binding microtiter plates were coated overnight at 4°C with 100 μL of 2 g / mL native Der p 1, Der p 2, Der f 1, and Ole e 1 in PBS. IgE antibodies were prepared at a concentration of 1 μg / mL and serially diluted three-fold in PBS with 0.5% w / v BSA. After washing with PBS at 20°C and blocking with 0.5% w / v BSA in PBS, 100 μL of diluted IgE antibody was transferred to the plate and incubated at ambient temperature for one hour. Subsequently, 100 μL of anti-mouse IgE antibody conjugated to horseradish peroxidase (HRP) at a dilution of 1:5000 was added. The plate was then incubated at ambient temperature for one hour in a PBS solution containing 0.05% v / v / The cells were washed in PBS at 20 °C. Subsequently, the HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added. The reaction was stopped (with 100 μL sulfuric acid 2.0 N) and the optical density was measured at 450 nm using a plate reader. IgE mAb1, mAb3, and mAb6 were found to bind effectively to Der p 1 and Der f 1 ( Fig.13A However, these antibodies did not bind to the negative control allergen Ole e 1 ( Fig. 13B ). The other IgE mAbs bound less efficiently to Der p 1 and Der f 1 than IgE mAb1, mAb4, and mAb6. Overall, these data demonstrate that the three IgE antibodies captured and identified by the secretory IgE capture reagent specifically bind to Der p 1 and Der f 1.

[0259] Example 7: In vivo capture and profiling of IgE plasma cells

[0260] To validate in vivo IgE capture, StAv-FcεRIα ectodomain was used to capture IgE from HDM cells exposed to extracts for 15 weeks and secreting IgE as previously described. Venus and Blimp-1 mCherry Reporter gene mice ( Fig. 7A ),like Figure 1 Cells were stained with a cocktail of antibodies containing PC markers, secretory IgE αIgE, and oligonucleotide barcoded HDM allergens including Der p 1, Der p 2, Der f 1, and Olive allergen (Ole e 1) as a negative control. PCs were gated using Blimp-1 signal ( Figure 7B and Figure 7C ), IgE PC and non-IgE PC ( Figure 7B and Figure 7C ) sort together ( Figure 7B and 7C ) for comparison. This is consistent with what was observed in U266 and Ramos ( Figure 4F ), secretory IgE was detected only on IgEPCs but not on other isotypes (non-IgE PCs), confirming the specificity of TRAP in mixed cell populations ( Figure 7B and Figure 7C ).

[0261] Example 8: Sequencing of IgE plasma cells in the bone marrow and lymph nodes of HDM-exposed mice

[0262] To elucidate the biology of IgE PCs within tissues, single-cell transcriptional profiles from lung dLN and BM were combined and cell clustering was performed to compare IgE and non-IgE PCs. 12,646 IgG+ cells, 17,524 IgA+ cells, 3,503 IgM+ cells, and 1,703 IgE+ cells ( Fig.7D ). From the single-cell transcriptome, the expression of immunoglobulins was confirmed and the specific gene expression programs of each isotype were determined. IgE PCs expressed high levels of Ighe, Slpi, Sdf2l1, and Pdia4. IgG-type PCs expressed Ighg1 and slightly higher levels of Cd74 and B2m, while IgA-type PCs expressed Igha and higher levels of Cd79a and Cd69. IgM PCs expressed Ighm and higher levels of Slc3a2, Ctss, and Ghg ( Fig. 7E ).

[0263] Using barcoded HDM allergens, IgE PCs can be directly mapped to their specificity for Der p 1, Der p 2, Der f 1 (HDM allergens) and Ole e 1 (olive allergen, negative control) and their AgSS compared, calculated as described in Example 4. The antigens were Der p 1, Der p 2 and Der f 1, and all controls were Ole e 1 (olive antigen) ( Figure 7F ).

[0264] Based on the reactivity profile (see Table 3), some allergen-specific IgE antibodies were selected and their relative binding to Der p 1, Der p 2 and Der f 1 as well as the olive allergen Ole e 1 was tested by ELISA. Two IgE antibodies (IgEmAb12_2 and mAb 3_1) bound to Der p 1 ( Fig. 6F , Fig.19A ), 3 IgEs bound to Der p 2 (IgE mAb12_2, mAb8_2, and mAb21_2), and 6 IgEs bound to Der f 1 (IgE mAb12_2, mAb1_2, mAb 1_1, mAb3_1, mAb4_1, and mAb6_1). In addition to Der p 1, Der p 2, and Der f 1, Ab12_2 also bound to Ole e 1 ( Figure 7G ), but did not show any binding to Fel d 1 ( Fig.19B ).

[0265] To determine whether the IgE showing HDM-allergen binding had allergic potential, a passive cutaneous anaphylaxis (PCA) assay was performed. The left ear of mice was inoculated with a mixture of Der p 1 or Der p 2-specific IgE ( Figure 7G and Fig.19A ) and in the right ear with irrelevant DNP-IgE as a negative control. After 24 hours, mice were challenged intravenously with Evans blue containing Der p 1 of group A or Der p 2 of group B ( Figure 7H Mast cell degranulation, as measured by Evans blue dye leakage, was observed only in ears sensitized with Der p 1 or Der p 2-specific IgE (left ears). Figure 7H ), thus demonstrating that the IgE antibody mixture used induced cross-linking of FcεRIα and mast cell degranulation.

[0266] Example 9: Human primary cells: Identification and profiling of IgE PCs in the bone marrow of allergic donors

[0267] To determine whether IgE secretion capture could be extended to human primary cells, we performed IgE secretion capture on BM from self-reported allergic donors. B lineage-enriched cells were coated with NHS-biotin, and secretory IgE was captured with StAv-FcεRIα ectodomain as previously described. Figure 1 PCs were gated as CD38 high / CD20 low ( Fig. 20A ), and the specificity and functionality of StAv-FcεRIα secretome capture were enhanced in non-allergic BM ( Fig. 8A ) and allergic BM lacking StAv-FcεRIα ( Fig. 8A ). To fully validate the capture of human IgE secretions, donors with confirmed cat, timothy, and HDM reactive IgE in serum were selected ( Figure 8B , Fig. 20B ), IgE secretion capture was performed, and all IgE PCs and non-IgE PCs from BM were sorted for comparison.

[0268] After sorting, scRNA-seq recovered complete transcriptomes and BCR sequences from 5,559 bone marrow PCs from individuals with cat, grass, and dust allergies. The specificity of PC capture was confirmed based on the abundant expression of various immunoglobulin genes and PC markers such as XBP1, SLAMF7, and CD74, but B cell markers such as MS4A1 and CD19 were not observed ( Figure 8CTen IgE PCs were identified by VH:VL sequencing and alignment with the human germline database of IgBlast. These IgE PCs were closely related to IGHE transcript expression and a small fraction of cells from IgA and IgG isotypes that also expressed IGHE, which may reflect sequential class switching between these isotypes in some IgA and IgG cells or sterile IGHE transcripts ( Figure 8C and Fig.8D ). This demonstrates that despite the rarity of IgE PCs in human bone marrow, the capture method of the present disclosure is able to isolate and profile IgE PCs.

[0269] To investigate clonal evolution, VH sequences from all 5,559 cells were clustered based on the amino acid sequence of the heavy chain CDR3 region, allowing for up to 3 amino acid mismatches, deletions, or insertions. The 10 IgE PCs were uniquely clustered into 5 clonotypes, which were defined as a group of PCs with highly similar heavy chain amino acid CDR3s (see Table 4). Two clonotypes, denoted 753 (n = 4 cells) and 696 (n = 3 cells), consisted of only IgE cells, and each clonotype displayed the same CDR3 sequence ( Fig. 8E The other two clonotypes, denoted as 1330 and 576, are single-cell clones derived from a single IgE cell ( Fig. 8E Interestingly, clonotype 21 contained 14 IgG cells and a single IgE ( Fig. 8E Closer inspection of the CDR3 nucleotide sequence of clonotype 21 revealed that individual IgE sequences contained three point mutations within the CDR3 region, whereas all IgG members of clonotype 21 carried the same CDR3 region ( Figure 8F ). In addition, the IgE sequence contained somatic hypermutations at the upstream V gene locus compared to the IgG clonotype members ( Figure 8F ), thus indicating additional rounds of affinity maturation.

[0270] After selection of IgE clones, we tested the binding of purified IgE and control (DNP-IgE) to relevant allergens to which the donor was allergic (Fel d 1 from cat, Der p 1 from dust, or Can e 1 from dog as a control) by ELISA ( Figure 8G One of the purified IgE mAbs bound to Fel d 1 but not to e1 or Der p 1 ( Figure 8G ), thus indicating that human IgE antibodies can be successfully isolated from allergic BM using the IgE capture method of the present disclosure.

[0271] Example 10: Proof of concept for secreted IgG capture

[0272] Preparation of Secreted IgG Capture Reagent

[0273] use The streptavidin conjugation kit conjugates anti-mouse IgK antibodies to streptavidin to form a secretory IgG capture reagent. Briefly, an anti-mouse IgK antibody solution containing 1 mg / mL streptavidin was prepared. One hundred microliters of modifier reagent was added to the solution under gentle mixing. The cap was removed from the vial of the streptavidin conjugated mixture and the mixture including the modifier was added directly to the lyophilized material. The material was gently resuspended by withdrawing and redistributing the added suspension once or twice with a pipette. The cap was placed back on the vial and the vial was left to stand in the dark at 20-25°C for three hours. Thereafter, 100 μL of quencher was added under gentle mixing. Thus, a secretory IgK capture reagent was produced.

[0274] Capture of secreted IgG: Experimental procedure

[0275] Figure 3 (Step 1) shows the surface of PC that has been biotinylated with NHS-biotin. Figure 3 (Step 2) shows secreted IgG capture reagent bound to biotinylated PC. Figure 3 (Step 2) Also shown is an anti-mouse IgK antibody for the secretory IgG capture reagent that captures PC-secreted IgG. Figure 3 (Step 3) Captured IgG bound to the barcoded antigen and bound by anti-IgG antibodies is shown.

[0276] Capture of secreted IgG on the surface of primary plasma cells

[0277] To detect antigen-specific IgG-secreting PCs, the footpads of VI3 / VelocImmune mice were immunized with the immunogen twice a week for four weeks. The mice were allowed to rest for a month and then boosted with the immunogen four days before anesthesia, sacrifice, and harvesting of BM, spleen, and draining lymph nodes. Cells from bone marrow, spleen, and draining lymph nodes were prepared as described herein. Single cell preparations from spleen and lymph nodes were combined and surface Fc receptors were blocked with a 1:10 dilution of Fc blocker for fifteen minutes at 4°C. The combined preparations were then stained with an antibody cocktail to screen for antigen-specific IgG + B cells ( Fig.14A). The antibody cocktail for spleen and lymph nodes included antibodies against mouse IgG1, IgG2a, CD19, IgM, IgD, and CD3. The antibody cocktail for BM included antibodies against mouse B220, CD138, TACI, CD98, TCRB, CD200R3, Ly6G, CD49b, CD11b, IgM, CD19, IgG1, IgG2a, and IgD. All antibodies are commercially available. The surface of PC was biotinylated as described herein, washed three times in cold PBS with 5% w / v BSA, changing tubes for each wash step, and incubated with secretory IgG capture reagent in the presence of secretory IgG capture reagent and in the absence of secretory IgE capture reagent as described herein. Cells were incubated for one hour at 37°C on a rotating platform to capture secretory Ig antibodies and stained for thirty minutes at 4°C with an antibody cocktail containing fluorescein isothiocyanate-labeled anti-IgG (BD Biosciences and Southern Biotech) to detect captured IgG and barcoded, phycoerythrin-labeled immunogens in PBS with 5% BSA. The labeled PCs were sorted on an S6 flow cytometer and the PCs that captured secreted IgG were identified ( Fig. 14B ). The control was used to detect antigen-specific populations of secreted IgG captured on the cell surface ( Fig. 14C ).

[0278] Example 11: Single-cell library preparation and sequencing of plasma cells secreting IgG captured on their surface

[0279] For PCs that have captured IgG secreting IgG, gene expression libraries, secreted IgG V(D)J regions, and barcoded antigens were prepared and sequenced as described in Example 3. Each library was also mapped, quantified, normalized, and processed as described in Example 4. The humanized nature of the mouse V(D)J region, the V(D)J library was mapped to the standard human germline IMGT reference. After processing the three single cell libraries, the gene expression and V(D)J region sequencing data were used to group PCs with effective IgG constant regions and detectable IgG and CD79a expression but lacking Ms4a1 and CD19 expression (usually found in non-PC B cells). To select antigen-specific IgG-expressing PCs, the antigen-specific score of the target immunogen against the control antigen (hIL-6Rα) was calculated as described in Example 4. The results are shown in Figure 15. The UMAP of the bone marrow is shown in Fig.15A and Fig. 15B and the spleen is shown in Fig. 15C and Fig.15D middle. Fig. 15B and Fig.15DThe x-axis in the graph represents the centered log ratio of the immunogen antigen signal, and the y-axis represents the centered log ratio α of the hIL-6R antigen signal. Fig.15D Only plasma cells identified by expression of CD79A, but lacking CD19 and MS4A1 are shown. Cells are colored according to plasma cell isotype as determined by VDJ library sequencing.

[0280] Example 12: IgG and Ag+ ELISA

[0281] In order to test the antigen specificity and IgG expression of the BCR sequence from the IgG secretion trap, the hIL-4Rα antigen or anti-human IgG (Jackson Labs) was plated at 1ug / mL or 2ug / mL, respectively, at +4°C overnight. The next day, the plate was washed three times in PBS supplemented with 0.05% Tween20 (PBS-T) and blocked with PBS with 0.5% BSA for 1 hour at room temperature. The plate was then washed three times with PBS-T, and the diluted supernatant from the Expi293F cells transfected with a single BCR from BMPC, PB / PC-like or spleen B cell part was plated at room temperature for 1 hour. The plate was then washed three times with PBS-T and incubated with anti-human IgG-HRP antibody (Jackson Labs) at room temperature for 1 hour at 1:10,000. The plate was washed three times in PBS-T, developed using TMB substrate, stopped using 1N sulfuric acid, and read at 450nm.

[0282] Example 13: Identification and isolation of antigen-specific IgG antibody-secreting cells from immunized mice

[0283] like Figure 1 As described in , αIgk antibodies were conjugated to streptavidin to capture secreted antibodies using kappa light chains. The StAv-αIgk capture complex was then used to isolate the secreted antibodies from mice immunized with human IL-4Rα (hIL-4Rα). Mouse NHS-biotin-coated ASCs. Secreted Ag was detected by staining cells with anti-mouse IgG and barcoded hIL-4Rα antigen. + / IgG + α barcoded hIL-6R was used as a negative control ( Figure 5A ).

[0284] As shown in Figures 5 and 17, in order to detect Ag + / IgG + ASC, mice were immunized with hIL-4Rα monomeric protein via footpad ( Figure 5A and Fig.17A ). Single cell suspensions of BM cells were generated and enriched for B lineage cells by negative selection ( Figure 5AAs described herein, cells were coated with NHS-biotin and secreted IgG captured with StAv-aIgk ( Figure 1 ). After assembling the biotin-StAv-aIgk complex on the surface, the enriched B cells / ASCs were stained with an antibody cocktail containing PC-specific markers, αIgG for detection of secreted IgG, barcoded hIL-4Rα for detection of Ag+ cells, and barcoded hIL-6Rα as a negative control. Spleens and draining lymph nodes (dLNs) from the same mice were combined and stained with a similar antibody cocktail for comparison of VH:VL sequences from Ag+ B cells and plasmablasts (PB) with BMPCs ( Fig. 17B and Fig. 17C ). Ag from BM was isolated using a staining control. + / IgG + PCs were sorted together with B cells expressing surface BCR (PB / PC) and ASCs from spleen / dLN ( Figure 5B as well as Fig. 17B , Fig. 17C and Fig.17D ).

[0285] scRNA-seq was performed on sorted BMPCs, spleen / dLN B cells, and PB / PCs to determine transcriptional differences, identify VH:VL repertoire changes, and quantify antigen specificity at the single-cell level. Ag was validated based on barcoded antigen counts of hIL-4Rα and the negative control antigen hIL-6Rα + / IgG + cell( Figure 5C ). To prioritize specificity and minimize polyreactivity, the antigen specificity score (AgSS) was determined to select cells with strong specificity for hIL-4Rα and minimal hIL-6Rα binding. Cells with high AgSS were preferentially expressed for hIL-4Rα and had minimal barcode UMI counts for the control antigen hIL-6Rα ( Figure 5C Combined transcriptional analysis and unbiased clustering indicated that plasma cells from spleen or BM clustered together and expressed typical PC markers such as Slamf7, Xbp1, Sdc1, Prdm1, and Slc3a2 ( Figure 5D , Fig.18A ). However, splenic B cells clustered very clearly with plasma cells and expressed typical B cell markers such as Ms4a1 and Cd79a ( Fig.18A In addition, Ag+ cells with high AgSS (>0.5) from BM and spleen were often clustered together ( Figure 5D ). Especially in BM PCs, cells with high AgSS clustered together at the bottom of the UMAP ( Figure 5D ).

[0286] Example 14: Affinity of Antigen-Specific IgG Plasma Cells Isolated from Bone Marrow and Spleen / dLN B Cells

[0287] scRNA-seq generated three distinct cell populations: bone marrow plasma cells (BMPCs), spleen / dLN plasmablasts / plasmacytoid cells (Sp / dLN PB / PCs), and spleen / dLN B cells (Sp / dLN B cells). From each of these populations, 20 unique VH:VL sequences were selected from cells with the highest hIL-4Rα barcode signal for cloning and expression analysis ( Fig.18B and Fig.18C ). 20 heavy and light chain variable sequences were successfully cloned from BMPC, 19 Sp / dLN B cells and 19 Sp / dLNPB / PC into vectors expressing human IgG4 and human Igk constants (see Table 1).

[0288] Consistent IgG expression was observed in all three populations, but anti-hIL-4Rα activity was observed in 80% (16 / 20) of BMPCs, compared with 32% (6 / 19) and 53% (10 / 19) of Sp / dLN PB / PCs and Sp / dLN B cells, respectively ( Figure 5E ; Table 1). BMPCs also had a higher median hIL-4Rα:total IgG binding ratio when considering different levels of IgG in the supernatant, indicating that these sequences will have higher affinity ( Figure 5E ). Biacore analysis of supernatants and purified antibodies confirmed this, with antibodies from BMPCs having the highest affinity as measured by the negative log10 of the binding Kd ( Fig. 5F and Fig.18D ).

[0289] Example 15: scRNA-seq of captured IgG cells

[0290] In verifying Ag + We then investigated the unique BCR sequences obtained by scRNA-seq of the VDJ region to compare Ag + BMPCs and B and PB / PCs from spleen / dLN. BCR sequences from all cells were mapped to specific V, D, and J gene segments by IgBlast and the extent of somatic hypermutation was measured by the percentage similarity to the germline V region. Ag in all three compartments + Cells have a lower percentage of similarity to the germline V region and therefore mutations are more frequent than non-Ag+ cells ( Fig. 6AThe BCR sequences of Ag+ cells were further investigated by identifying BCR clonotypes in cells that shared the same heavy and light chain V gene segments and had the same CDR3 amino acid sequences in the heavy and light chains (Table 2). Ten unique CDR3 sequence pairs were clonally shared in all three compartments, while the majority of each BCR repertoire was specific to each compartment (Table 2).

[0291] In addition to BCR sequence analysis, the transcriptomes of Ag+ and non-Ag+ cells in the three compartments were compared to identify gene signatures associated with hIL-4Rα specificity. In BMPCs, several gene expression programs differ based on hIL-4Ra specificity ( Figure 6C and Fig.6D ). Tmsbx4, Cd24a, Tmsb10, and Fkbp11 all showed significant upregulation in hIL-4Rα-specific BMPCs. Non-Ag+ cells showed an increase in Ly6d, Prg2, Tmem176a, and Tmem176b ( Figure 6C and Fig.6D ). Subclustering of BMPCs indicated that Cd24a, Ppib, Fkbp11, and Ssr2 transcripts were all highly expressed at the bottom of the UMAP projection ( Fig. 6E ), while the gene programs marked by the expression of Tmem176a, Tmem176b, Ly6d, and Cd74 were clustered in the middle and top regions of the UMAP projection ( Fig. 6E ). However, despite the significant transcriptional differences, BCR sequences cloned from either transcriptionally distinct region generated high-affinity hIL-4Rα-specific antibodies, as measured by the negative log10 of the binding Kd ( Fig. 6F ).

[0292] Example 16: Expanding Trap-N-Seq to human bone marrow to characterize short-term and long-term vaccine-specific IgG plasma cells.

[0293] In this example, TRAPnSeq was used to isolate vaccine-specific IgG+ plasma cells (PC) from human bone marrow. After enrichment of B cells and antibody-secreting cells (ABC), the cells were biotinylated with NHS-biotin, and an affinity matrix was assembled using streptavidin coupled to anti-human IgG (StAv-αIgk) to capture secreted IgG. After assembling the biotin-StAv-αIgk complex on the surface, the enriched B cells / ASC were stained with an antibody mixture containing PC-specific markers, αIgG for detecting secreted IgG, and fluorescently labeled antigens (listed in the table below) for detecting Ag+ cells.

[0294] After gating PCs as CD20-CD38++ ( Fig.21A), the use of secretion traps to isolate vaccine-specific IgG+PC from human bone marrow has been validated. Fig. 21B The necessary components for detecting secreted IgG on the surface of PC are shown in Fig. 21C The necessary components for detecting antigen-specific IgG are shown in Figure 2. Complete assembly of all components of the secretion trap allows detection of secreted IgG antibodies ( Fig.21D , left), and detection of vaccine-specific antibodies ( Fig.21D ,right).

[0295] Antigen List

[0296]

[0297] Example 17: Secreted Trap Combined with Antigen Tracking

[0298] Fig. 22 The workflow of the secretion trap combined with antigen tracking is shown.

[0299] The biotinylated human IL-4 receptor α (hIL-4Rα) extracellular domain was conjugated to PE-labeled streptavidin (“SA-PE”) tetramer overnight at 4° C. Separately, the trap antibody (mouse anti-human IgG (BD catalog number 555784, clone number G18-145)) was conjugated to streptavidin overnight using a commercially available kit (abcam catalog number ab102921).

[0300] A25 cells were suspended in NHS-biotin (Sigma catalog number 203112) in DPBS / FBS at a final concentration of 0.5 mg / mL at 37°C for 15 minutes. A25 cells were derived from the commercial A20 cell line by knocking out the endogenous surface BCR. Therefore, A25 cells are mouse B cells without surface or secreted BCR.

[0301] Following incubation, cells were washed 3 times in DPBS / FBS, transferring to new conical tubes after each wash to remove residual biotin.

[0302] Biotinylated cells were incubated with or without streptavidin-conjugated trap antibody (or "SA-anti-huIgG") (at a 1:100 dilution) in DPBS / FBS at 10 million cells / mL at 4°C for approximately 15 minutes. The 1:100 cell to trap antibody ratio ensured that the trap reagent bound to all biotinylated cells before incorporating the antibody in the next step. After incubation, cells were washed twice in DPBS / FBS and then resuspended in DPBS / FBS.

[0303] The following four antibodies of different affinities were used. Each single antibody was spiked into the cell suspension at a concentration of approximately 1 mg antibody per 2 million cells. After incubation, the cells were washed twice in DPBS / FBS.

[0304]

[0305] Next, cells were incubated with 5 nM hIL-4Rα labeled with AlexaFluor 647 ("AF647") in DPBS / FBS for 30 minutes at room temperature (RT). After incubation, cells were washed twice in DPBS / FBS.

[0306] Afterwards, the cells were incubated twice with pre-conjugated hIL-4Rα biotin-StAv-PE in DPBS / FBS at room temperature for 45 minutes each time (or 2×45 minutes). The concentration of the antigen hIL-4Rα was 20 nM. After incubation, the cells were washed twice in DPBS / FBS and analyzed by flow cytometry. Fig.23C As shown in , the four antibodies show clear separation by affinity.

[0307] As a control, after incubation with the tracking antigen (hIL-4Rα-biotin-StAv-PE), the cells were washed twice in DPBS / FBS and then stained with anti-huIgK-PE-Cy7 antibody to detect the antibody on the surface of the cells. The Ab 4 / negative control did not stain for the antigen but stained positive for IgK, indicating that the antibody was still captured on the surface of the cells.

[0308] Table 1: From

[0309] Antigen-specific IgG plasma cell sequences isolated from bone marrow plasma cells and spleen plasma cells and B cells

[0310]

[0311]

[0312]

[0313] Table 1 (continued)

[0314]

[0315]

[0316]

[0317] Table 1 (continued)

[0318]

[0319]

[0320]

[0321] Table 1 (continued)

[0322]

[0323]

[0324]

[0325] Table 1 (continued)

[0326]

[0327]

[0328]

[0329] Table 1 (continued)

[0330]

[0331]

[0332]

[0333] Table 1 (continued)

[0334]

[0335]

[0336]

[0337] Table 1 (continued)

[0338]

[0339]

[0340]

[0341] Table 2: hIL-Ra IgG-specific BCR sequences shared among bone marrow plasma cells, splenic B cells, and plasma cells

[0342]

[0343]

[0344]

[0345] Table 2 (continued)

[0346]

[0347]

[0348]

[0349] Table 2 (continued)

[0350]

[0351]

[0352]

[0353] Table 2 (continued)

[0354]

[0355]

[0356]

[0357]

[0358]

[0359] Table 2 (continued)

[0360]

[0361]

[0362]

[0363]

[0364] Table 2 (continued)

[0365]

[0366]

[0367]

[0368] Table 3: IgE HDM-specific BCR sequence metadata

[0369]

[0370] Table 3 (continued)

[0371]

[0372]

[0373] Table 3 (continued)

[0374]

[0375] Table 3 (continued)

[0376]

[0377] Table 3 (continued)

[0378]

[0379]

[0380] Table 3 (continued)

[0381]

[0382] Table 3 (continued)

[0383]

[0384] Table 3 (continued)

[0385]

[0386]

[0387] Table 4: Clustered BCR sequences containing HDM-specific IgE clones from myeloid

[0388]

[0389]

[0390] Table 4 (continued)

[0391]

[0392] Table 4 (continued)

[0393]

[0394]

[0395] Table 4 (continued)

[0396]

[0397]

[0398] Table 4 (continued)

[0399]

[0400]

[0401] Table 4 (continued)

[0402]

[0403]

[0404] The present disclosure provides the following illustrative embodiments:

[0405] 1. An antibody capture complex comprising a first component of a binding pair linked to an antibody capture molecule;

[0406] wherein the first member of the binding pair is capable of binding to the second member of the binding pair;

[0407] wherein the antibody capture molecule is capable of binding to a target antibody, wherein the target antibody is secreted by an antibody-secreting cell;

[0408] Wherein when the second component of the binding pair is biotin and the first component of the binding pair is streptavidin, the antibody capture molecule does not bind to the kappa light chain of the target antibody.

[0409] 2. The antibody capture complex of embodiment 1, wherein:

[0410] The first component of the binding pair comprises avidin, streptavidin or avidin, and the second component of the binding pair comprises biotin;

[0411] The first component of the binding pair comprises one of jun and fos, and the second component of the binding pair comprises the other of jun and fos;

[0412] The first component of the binding pair comprises one of mad and max, and the second component of the binding pair comprises the other of mad and max;

[0413] The first component of the binding pair comprises one of myc and max, and the second component of the binding pair comprises the other of myc and max;

[0414] The first member of the binding pair comprises one of an azide and an alkyne, and the second member of the binding pair comprises the other of an azide and an alkyne; or

[0415] The first component of the binding pair comprises one of an azide and a phosphine, and the second component of the binding pair comprises the other of an azide and a phosphine.

[0416] 3. The antibody capture complex of embodiment 1, wherein the first component of the binding pair comprises avidin or streptavidin, and the second component of the binding pair comprises biotin.

[0417] 4. An antibody capture complex as described in embodiment 1, wherein the second component of the binding pair comprises a surface marker of the antibody-secreting cell, and the first component of the binding pair comprises an antibody that binds to the surface marker.

[0418] 5. The antibody capture complex of embodiment 4, wherein the cell surface marker comprises CD27, CD38, CD45, CD138, CD98, CD78, CD319, CXCR4, BCMA, GPRC5D, FCRL5, CD19, Ly6D, CD52 or transmembrane activator calcium regulator and cyclophilin ligand interactor (TACI).

[0419] 6. An antibody capture complex as described in embodiment 1, wherein the second component of the binding pair comprises an interleukin-6 receptor (IL-6R), and the first component of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6).

[0420] 7. The antibody capture complex of embodiment 1, wherein the second component of the binding pair comprises CD27, and the first component of the binding pair comprises an anti-CD27 antibody or CD70.

[0421] 8. The antibody capture complex of embodiment 1, wherein:

[0422] The second component of the binding pair comprises fluorescein isothiocyanate (FITC), and the first component of the binding pair comprises an anti-FITC antibody;

[0423] The second component of the binding pair comprises phycoerythrin (PE), and the first component of the binding pair comprises an anti-PE antibody;

[0424] The second component of the binding pair comprises an APC, and the first component of the binding pair comprises an anti-APC antibody;

[0425] The second component of the binding pair comprises BV421, and the first component of the binding pair comprises an anti-BV421 antibody;

[0426] The second component of the binding pair comprises BV510, and the first component of the binding pair comprises an anti-BV510 antibody;

[0427] The second component of the binding pair comprises BV605, and the first component of the binding pair comprises an anti-BV605 antibody;

[0428] The second component of the binding pair comprises BV650, and the first component of the binding pair comprises an anti-BV650 antibody;

[0429] The second component of the binding pair comprises BV711, and the first component of the binding pair comprises an anti-BV711 antibody; or

[0430] The second component of the binding pair comprises BV786, and the first component of the binding pair comprises an anti-BV786 antibody.

[0431] 9. The antibody capture complex of any one of embodiments 1 to 8, wherein the antibody capture molecule comprises a capture antibody.

[0432] 10. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain κ antibody, or an anti-light chain λ antibody.

[0433] 11. The antibody capture complex of embodiment 10, wherein the anti-Fc antibody comprises an anti-Fcγ antibody, an anti-Fcα antibody or an anti-Fcε antibody.

[0434] 12. The antibody capture complex of embodiment 11, wherein the anti-Fcγ antibody comprises an anti-FcγRI antibody, an anti-FcγRIIA antibody, an anti-FcγRIIB antibody, an anti-FcγRIIB1 antibody, an anti-FcγRIIB2 antibody, an anti-FcγRIIIA antibody, or an anti-FcγRIIIB antibody.

[0435] 13. The antibody capture complex of embodiment 11, wherein the anti-Fcα antibody comprises an anti-FcαRI antibody.

[0436] 14. The antibody capture complex of embodiment 11, wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody.

[0437] 15. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-IgM antibody.

[0438] 16. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-IgG antibody.

[0439] 17. The antibody capture complex of embodiment 16, wherein the anti-IgG antibody comprises an anti-IgG1 antibody, an anti-IgG2 antibody, an anti-IgG2a antibody, an anti-IgG2b antibody, an anti-IgG3 antibody or an anti-IgG4 antibody.

[0440] 18. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-IgA antibody.

[0441] 19. The antibody capture complex of embodiment 18, wherein the anti-IgA antibody comprises an anti-IgA1 antibody, an anti-IgA2 antibody, an anti-secretory IgA antibody, or a polymeric anti-IgA antibody.

[0442] 20. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-IgE antibody.

[0443] 21. The antibody capture complex of any one of embodiments 1 to 8, wherein the antibody capture molecule comprises an Fc receptor or an extracellular domain of the Fc receptor.

[0444] 22. The antibody capture complex of embodiment 21, wherein the Fc receptor comprises an Fcγ receptor, an Fcα receptor or an Fcε receptor.

[0445] 23. The antibody capture complex of embodiment 22, wherein the Fcγ receptor comprises an FcγRI receptor, an FcγRIIA receptor, an FcγRIIB receptor, an FcγRIIB1 receptor, an FcγRIIB2 receptor, an FcγRIIIA receptor, an FcγRIIIB receptor or an FcRn receptor.

[0446] 24. The antibody capture complex of embodiment 22, wherein the Fcα receptor comprises an FcαRI receptor or an Fcα / μR receptor.

[0447] 25. The antibody capture complex of embodiment 22, wherein the Fcε receptor comprises an FcεRI receptor or an FcεRII receptor.

[0448] 26. The antibody capture complex of any one of embodiments 1 to 8, wherein the antibody capture molecule comprises protein A or protein G.

[0449] 27. An antibody capture complex as described in any one of embodiments 1 to 8, wherein the antibody capture molecule comprises protein L.

[0450] 28. The antibody capture complex of embodiment 1, wherein the antibody capture complex comprises avidin or streptavidin linked to the extracellular domain of the high affinity IgE receptor (FcεRIa).

[0451] 29. A method for capturing a target antibody secreted by an antibody-secreting cell, the method comprising:

[0452] contacting a population of antibody-secreting cells with a second component of a binding pair such that the second component of the binding pair binds to a cell surface of the population of antibody-secreting cells, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody;

[0453] contacting the population of antibody-secreting cells with an antibody capture complex, wherein the antibody capture complex comprises a first component of the binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to the second component of the binding pair on the cell surface of the antibody-secreting cells, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; and

[0454] The antibody-secreting cell population is contacted with an antigen, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the antigen.

[0455] 30. The method of embodiment 29, wherein:

[0456] The first component of the binding pair comprises avidin, streptavidin or avidin, and the second component of the binding pair comprises biotin;

[0457] The first component of the binding pair comprises one of jun and fos, and the second component of the binding pair comprises the other of jun and fos;

[0458] The first component of the binding pair comprises one of mad and max, and the second component of the binding pair comprises the other of mad and max;

[0459] The first component of the binding pair comprises one of myc and max, and the second component of the binding pair comprises the other of myc and max;

[0460] The first member of the binding pair comprises one of an azide and an alkyne, and the second member of the binding pair comprises the other of an azide and an alkyne; or

[0461] The first component of the binding pair comprises one of an azide and a phosphine, and the second component of the binding pair comprises the other of an azide and a phosphine.

[0462] 31. The method of embodiment 29, wherein the first component of the binding pair comprises avidin or streptavidin, and the second component of the binding pair comprises biotin.

[0463] 32. The method of embodiment 29, wherein:

[0464] The second component of the binding pair comprises fluorescein isothiocyanate (FITC), and the first component of the binding pair comprises an anti-FITC antibody;

[0465] The second component of the binding pair comprises phycoerythrin (PE), and the first component of the binding pair comprises an anti-PE antibody;

[0466] The second component of the binding pair comprises an APC, and the first component of the binding pair comprises an anti-APC antibody;

[0467] The second component of the binding pair comprises BV421, and the first component of the binding pair comprises an anti-BV421 antibody;

[0468] The second component of the binding pair comprises BV510, and the first component of the binding pair comprises an anti-BV510 antibody;

[0469] The second component of the binding pair comprises BV605, and the first component of the binding pair comprises an anti-BV605 antibody;

[0470] The second component of the binding pair comprises BV650, and the first component of the binding pair comprises an anti-BV650 antibody;

[0471] The second component of the binding pair comprises BV711, and the first component of the binding pair comprises an anti-BV711 antibody; or

[0472] The second component of the binding pair comprises BV786, and the first component of the binding pair comprises an anti-BV786 antibody.

[0473] 33. A method for capturing a target antibody secreted by an antibody-secreting cell, the method comprising:

[0474] contacting a population of antibody-secreting cells, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody, with an antibody capture complex, wherein the antibody capture complex comprises a first component of a binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to a second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; and

[0475] The antibody-secreting cell population is contacted with an antigen, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the antigen.

[0476] 34. A method as described in embodiment 33, wherein the second component of the binding pair comprises a surface marker of the antibody-secreting cell population, and the first component of the binding pair is an antibody that binds to the surface marker.

[0477] 35. The method of embodiment 34, wherein the surface markers comprise CD27, CD38, CD45, CD138, CD98, CD78, CD319, CXCR4, BCMA, GPRC5D, FCRL5, CD19, Ly6D, CD52, or transmembrane activator calcium regulator and cyclophilin ligand interactor (TACI).

[0478] 36. A method as described in embodiment 33, wherein the second component of the binding pair comprises an interleukin-6 receptor (IL-6R), and the first component of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6).

[0479] 37. The method of embodiment 33, wherein the second component of the binding pair comprises CD27 and the first component of the binding pair comprises an anti-CD27 antibody or CD70.

[0480] 38. The method of any one of embodiments 29 to 37, wherein the antibody capture molecule comprises a capture antibody.

[0481] 39. The method of embodiment 38, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain κ antibody, or an anti-light chain λ antibody.

[0482] 40. The method of embodiment 39, wherein the anti-Fc antibody comprises an anti-Fcγ antibody, an anti-Fcα antibody, or an anti-Fcε antibody.

[0483] 41. The method of embodiment 40, wherein the anti-Fcγ antibody comprises an anti-FcγRI antibody, an anti-FcγRIIA antibody, an anti-FcγRIIB antibody, an anti-FcγRIIB1 antibody, an anti-FcγRIIB2 antibody, an anti-FcγRIIIA antibody, or an anti-FcγRIIIB antibody.

[0484] 42. The method of embodiment 40, wherein the anti-Fcα antibody comprises an anti-FcαRI antibody.

[0485] 43. The method of embodiment 40, wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody.

[0486] 44. The method of embodiment 38, wherein the capture antibody comprises an anti-IgM antibody.

[0487] 45. The method of embodiment 38, wherein the capture antibody comprises an anti-IgG antibody.

[0488] 46. ​​The method of embodiment 45, wherein the anti-IgG antibody comprises an anti-IgG1 antibody, an anti-IgG2 antibody, an anti-IgG2a antibody, an anti-IgG2b antibody, an anti-IgG3 antibody, or an anti-IgG4 antibody.

[0489] 47. The method of embodiment 38, wherein the capture antibody comprises an anti-IgA antibody.

[0490] 48. The method of embodiment 47, wherein the anti-IgA antibody comprises an anti-IgA1 antibody, an anti-IgA2 antibody, an anti-secretory IgA antibody, or a polymeric anti-IgA antibody.

[0491] 49. The method of embodiment 38, wherein the capture antibody comprises an anti-IgE antibody.

[0492] 50. The method of any one of embodiments 29 to 37, wherein the antibody capture molecule comprises an Fc receptor or an extracellular domain of the Fc receptor.

[0493] 51. The method of embodiment 50, wherein the Fc receptor comprises an Fcγ receptor, an Fcα receptor, or an Fcε receptor.

[0494] 52. A method as described in embodiment 51, wherein the Fcγ receptor comprises an FcγRI receptor, an FcγRIIA receptor, an FcγRIIB receptor, an FcγRIIB1 receptor, an FcγRIIB2 receptor, an FcγRIIIA receptor, an FcγRIIIB receptor or an FcRn receptor.

[0495] 53. The method of embodiment 51, wherein the Fcα receptor comprises an FcαRI receptor or an Fcα / μR receptor.

[0496] 54. A method as described in embodiment 51, wherein the Fcε receptor comprises an FcεRI receptor or an FcεRII receptor.

[0497] 55. The method of any one of embodiments 29 to 37, wherein the antibody capture molecule comprises protein A or protein G.

[0498] 56. A method as described in any one of embodiments 29 to 37, wherein the antibody capture molecule comprises protein L.

[0499] 57. The method of any one of embodiments 29 to 56, further comprising contacting the antibody-secreting cell population with a second anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the second anti-Ig antibody.

[0500] 58. The method of embodiment 57, wherein the second anti-Ig antibody is anti-IgE or anti-IgG.

[0501] 59. The method of embodiment 57 or 58, wherein the second anti-Ig antibody is detectably labeled.

[0502] 60. The method of embodiment 59, wherein the second anti-Ig antibody is labeled with:

[0503] Radioactive compounds, fluorescent compounds, or enzymes.

[0504] 61. The method of embodiment 60, wherein the radioactive compound comprises 3 H. 14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 Tc.

[0505] 62. The method of embodiment 60, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or fluorescent protein.

[0506] 63. The method of embodiment 60, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase.

[0507] 64. The method of any one of embodiments 29 to 63, wherein in the step of contacting the antibody-secreting cell population with an antigen, the antigen is a barcoded antigen.

[0508] 65. The method of any one of embodiments 29 to 64, wherein in the step of contacting the antibody-secreting cell population with an antigen, the antibody-secreting cell population is contacted with a plurality of barcoded antigens, wherein each barcoded antigen in the plurality of barcoded antigens comprises a unique antigen linked to a unique nucleic acid molecule.

[0509] 66. The method of embodiment 65, wherein the unique nucleic acid molecule comprises DNA.

[0510] 67. The method of embodiment 65, wherein the unique nucleic acid molecule comprises RNA.

[0511] 68. The method of any one of embodiments 65 to 67, wherein each unique nucleic acid molecule comprises about 10 nucleotides to about 500 nucleotides.

[0512] 69. The method of any one of embodiments 65 to 68, wherein each barcoded antigen is detectably labeled.

[0513] 70. The method of embodiment 69, wherein each barcoded antigen is labeled with:

[0514] Radioactive compounds, fluorescent compounds, or enzymes.

[0515] 71. The method of embodiment 70, wherein the radioactive compound comprises 3 H. 14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 Tc.

[0516] 72. The method of embodiment 70, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or fluorescent protein.

[0517] 73. The method of embodiment 70, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase.

[0518] 74. The method of any one of embodiments 64 to 73, wherein the barcoded antigen comprises an allergen.

[0519] 75. The method of any one of embodiments 29 to 74, wherein the antibody-secreting cell is a plasma cell or a plasmablast.

[0520] 76. The method of any one of embodiments 29 to 75, wherein the antibody-secreting cell population is obtained by enriching a cell population obtained from a human being for immune cells.

[0521] 77. A method as described in embodiment 76, wherein the cell population obtained from the human is enriched in plasma cells or plasmablasts.

[0522] 78. A method as described in embodiment 77, wherein pan-B cell enrichment is used to enrich the immune cell population for plasma cells or plasmablasts.

[0523] 79. The method of any one of embodiments 29 to 78, wherein the antibody-secreting cell population is obtained from lymph nodes, lungs, bone marrow, or blood of a human.

[0524] 80. The method of any one of embodiments 29 to 79, further comprising detecting an antigen bound by the target antibody.

[0525] 81. A method as described in any one of embodiments 29 to 80, which further includes sorting the antibody-secreting cell population to collect an antibody-secreting cell pool, wherein the antibody-secreting cells in the pool each secrete an antibody captured by the antibody capture molecule and bound by the antigen, wherein the collected antibody-secreting cell pool contains the antibody-secreting cells that secrete the target antibody.

[0526] 82. A method as described in embodiment 81, wherein the collected antibody-secreting cell pool is separated into single cells and the antibody-secreting cells that secrete the target antibody are separated.

[0527] 83. The method of any one of embodiments 57 to 80, further comprising detecting the second anti-Ig antibody.

[0528] 84. A method as described in embodiment 83, which also includes sorting the antibody-secreting cell population to collect an antibody-secreting cell pool, wherein the antibody-secreting cells in the pool each secrete an antibody captured by the antibody capture molecule and bound by the antigen and the second anti-Ig antibody, wherein the collected antibody-secreting cell pool contains the antibody-secreting cells that secrete the target antibody bound by the antigen and the second anti-Ig antibody.

[0529] 85. The method as described in embodiment 84 further includes separating the collected antibody-secreting cell pool into single cells and isolating the antibody-secreting cells that secrete the target antibody.

[0530] 86. The method of any one of embodiments 29 to 85, further comprising contacting the antibody-secreting cell population with an Fc blocker prior to contacting the antibody-secreting cell population with the second component of the binding pair.

[0531] 87. A method for capturing IgE antibodies secreted by antibody-secreting cells, wherein the IgE antibodies are directed against allergens, the method comprising:

[0532] contacting a population of antibody-secreting cells with NHS-biotin to bind biotin to the cell surface, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the IgE antibody;

[0533] contacting the antibody-secreting cell population with an antibody capture complex, wherein the antibody capture complex comprises streptavidin linked to the extracellular domain of a high affinity IgE receptor (FcεRIa), whereby the antibody capture molecule binds to the IgE antibodies secreted by the antibody-secreting cells; and

[0534] The antibody-secreting cell population is contacted with an antigen, wherein the antigen is an antigenic portion or a mixture of multiple antigenic portions of the allergen, whereby the IgE antibodies captured by the antibody-capture complex bind to the antigen.

[0535] 88. The method of embodiment 87, wherein the antigen comprises a plurality of barcoded antigens, wherein each of the plurality of barcoded antigens is a different antigenic portion of the allergen.

[0536] 89. A method as described in embodiment 87 or 88, which also includes sorting the antibody-secreting cell population to collect an antibody-secreting cell pool, wherein the antibody-secreting cells in the pool each secrete an IgE antibody captured by the antibody capture complex and bound by the antigen, wherein the collected antibody-secreting cell pool contains the antibody-secreting cells that secrete the IgE antibody.

[0537] 90. The method of embodiment 87 or 88, further comprising contacting the antibody-secreting cell population with an anti-IgE antibody so that the IgE antibodies secreted by the antibody-secreting cells and captured by the antibody capture complex bind to the anti-IgE antibody.

[0538] 91. A method as described in embodiment 90, which also includes sorting the antibody-secreting cell population to collect an antibody-secreting cell pool, wherein the antibody-secreting cells in the pool each secrete an IgE antibody captured by the antibody capture complex and bound by the antigen and the anti-IgE antibody, wherein the collected antibody-secreting cell pool contains the antibody-secreting cells that secrete the IgE antibody.

[0539] 92. The method of embodiment 89 or 91, further comprising separating the collected antibody-secreting cell pool into single cells and isolating the antibody-secreting cells that secrete the IgE antibody.

[0540] 93. The method of any one of embodiments 29 to 92, wherein the step of contacting the population of antibody-secreting cells with an antigen comprises:

[0541] (a) contacting the antibody-secreting cell population with a first labeled form of the antigen so that the antigen binds to antibodies including the target antibody captured on the cell surface of the antibody-secreting cell population, wherein the first labeled form of the antigen is conjugated to a first detectable label;

[0542] (b) washing the antibody-secreting cell population to remove unbound antigen;

[0543] (c) contacting the antibody-secreting cell population with:

[0544] (i) said antigen in unlabelled form,

[0545] (ii) a second labelled form of said antigen, or

[0546] (iii) said unlabeled form of said antigen and said second labeled form of said antigen;

[0547] (d) washing the antibody secreting cell population to remove unbound antigen, and

[0548] (e) collecting a pool of antibody-secreting cells that remain bound to the antigen in the first labeled form, wherein the collected pool of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody, wherein the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex remains bound to the antigen in the first labeled form.

[0549] 94. A method as described in embodiment 93, wherein the concentration of the antigen in the first labeled form is between 0.001 nM and 1 uM.

[0550] 95. A method as described in embodiment 93, wherein the concentration of the first labeled form of the antigen is between 0.1 nM and 7.5 nM

[0551] 96. The method of any one of embodiments 93 to 95, wherein the first detectable marker is a first fluorescent marker.

[0552] 97. The method of any one of embodiments 93 to 96, wherein the antigen is a protein in monomeric form.

[0553] 98. The method of any one of embodiments 93 to 96, wherein the antigen is a protein in a multimeric form.

[0554] 99. The method of any one of embodiments 93 to 96, wherein the antigen is a protein that exists in both monomeric and multimeric forms.

[0555] 100. The method of any one of embodiments 97 to 99, wherein the first labeled form of the antigen is a monovalent form of the antigen.

[0556] 101. The method of any one of embodiments 97 to 100, wherein the unlabeled form of the antigen is a monovalent form of the antigen.

[0557] 102. The method of any one of embodiments 97 to 100, wherein the unlabeled form of the antigen is a multivalent form of the antigen.

[0558] 103. A method as described in embodiment 102, wherein the multivalent form of the antigen is provided by a multivalent molecule to which the antigen binds.

[0559] 104. A method as described in embodiment 103, wherein the multivalent molecule is selected from a streptavidin multimer such as a tetramer, a dimer of an immunoglobulin Fc fragment, or a trimer of a trimerized molecule such as a foldon.

[0560] 105. The method of any one of embodiments 97 to 104, wherein the second labeled form of the antigen is a monovalent form of the antigen.

[0561] 106. The method of any one of embodiments 99 to 104, wherein the second labeled form of the antigen is a multivalent form of the antigen.

[0562] 107. The method of embodiment 106, wherein the multivalent form of the antigen is provided by a multivalent molecule to which the antigen is bound or linked.

[0563] 108. A method as described in embodiment 107, wherein the multivalent molecule is a streptavidin multimer such as a tetramer, a dimer of an immunoglobulin Fc fragment, or a trimer of a trimerized molecule such as a foldon.

[0564] 109. The method of any one of embodiments 106 to 108, wherein the multivalent form of the antigen is labeled with a second detectable marker.

[0565] 110. The method of embodiment 109, wherein the second detectable marker is a second fluorescent marker.

[0566] 111. The method of embodiment 100, wherein in step (c), the antibody-secreting cell population is contacted with the antigen in an unlabeled form.

[0567] 112. The method of embodiment 111, wherein the unlabeled form of the antigen is a monovalent form of the antigen.

[0568] 113. A method as described in embodiment 111 or 112, wherein the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen used in step (a) is at least 2-fold to 4-fold.

[0569] 114. A method as described in embodiment 100, wherein in step (c), the antibody-secreting cell population is contacted with the antigen in the second labeled form.

[0570] 115. The method of embodiment 114, wherein the second labeled form of the antigen is a multivalent form of the antigen.

[0571] 116. A method as described in embodiment 114 or 115, wherein the molar ratio of the second labeled form of the antigen relative to the first labeled form of the antigen used in step (a) is at least 2 to 4 times.

[0572] 117. A method as described in embodiments 111 to 116, wherein the contacting in step (c) and the washing in step (d) are repeated at least once before collecting the cells in step (e).

[0573] 118. A method as described in embodiment 100, wherein in step (c), the antibody-secreting cell population is contacted with an unlabeled form of the antigen and a second labeled form of the antigen.

[0574] 119. The method of embodiment 118, wherein the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a multivalent form of the antigen.

[0575] 120. A method as described in embodiment 118, wherein the antigen is a monomeric protein, the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a multivalent form of the antigen.

[0576] 121. A method as described in embodiment 118, wherein the antigen is a multimeric protein, the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a multivalent form of the antigen.

[0577] 122. A method as described in embodiment 118, wherein the antigen is a protein that exists in both monomeric form and multimeric form, the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a multivalent form of the antigen.

[0578] 123. The method of any one of embodiments 118 to 122, wherein the cell is contacted simultaneously with the unlabeled form of the antigen and the second labeled form of the antigen.

[0579] 124. The method of any one of embodiments 118 to 123, wherein the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen used in step (a) is at least 2-fold to 4-fold.

[0580] 125. The method of any one of embodiments 93 to 124, wherein the first detectable marker is a fluorescent marker, and wherein fluorescence activated cell sorting is used to collect cells that remain bound to the antigen in the first labeled form.

[0581] 126. A method as described in any one of embodiments 111 to 124, wherein the first detectable marker is a first fluorescent marker, and the second detectable marker is a second fluorescent marker different from the first fluorescent marker, wherein two-dimensional fluorescence-activated cell sorting is performed to collect a pool of antibody-secreting cells that are still bound to the antigen in the first labeled form, and wherein the collected pool of antibody-secreting cells contains the antibody-secreting cells that secrete the target antibody that is still bound to the antigen in the first labeled form.

[0582] 127. The method of any one of embodiments 93 to 126, wherein the target antibody has an affinity in the range of about 0.1 pM to about 25 nM (KD).

[0583] 128. The method of embodiment 127, wherein the affinity is less than about 10 nM (KD).

[0584] 129. The method of any one of embodiments 93 to 128, further comprising separating the pool of antibody-secreting cells collected in step (e) into single cells and isolating the antibody-secreting cells that secrete the target antibody.

[0585] 130. The method of embodiments 82, 85, 92 or 129, further comprising isolating nucleic acid encoding the antibody from the isolated antibody-secreting cells that secrete the target antibody.

[0586] 131. A method of identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by a cell comprising a modified cell surface, and wherein the target antibody is subsequently captured on the modified cell surface, the method comprising:

[0587] a) providing an antibody-secreting cell comprising a modified cell surface, wherein the antibody-secreting cell secretes a target antibody, wherein the target antibody is subsequently captured on the surface of the modified cell, wherein the target antibody captured on the surface of the modified cell is bound to an antigen, wherein the antigen is linked to a barcode nucleic acid molecule, and wherein the antibody-secreting cell further comprises a nucleic acid molecule comprising the region of the gene encoding the antigen-binding fragment of the target antibody captured on the surface of the modified cell;

[0588] b) hybridizing a portion of the barcode nucleic acid molecule to a portion of a first nucleic acid molecule attached to a solid surface;

[0589] c) hybridizing a portion of the nucleic acid molecule encoding the region of the gene of the antigen-binding fragment of the target antibody captured on the surface of the modified cell with a portion of a second nucleic acid molecule attached to the solid surface;

[0590] d) preparing a first amplicon library of gene expression in the antibody-secreting cells, a second amplicon library of variable regions, diversity regions, and joining regions (VDJ) in the antibody-secreting cells, and a third amplicon library of antigen barcode nucleic acid molecules;

[0591] e) sequencing each of the three amplicon libraries; and

[0592] f) identifying said region of said gene encoding said antigen-binding fragment of said target antibody captured on the surface of said modified cell.

[0593] 132. The method of embodiment 131, wherein the portion of the barcode nucleic acid molecule is complementary to a first template switch oligonucleotide (TSO), wherein the portion of the barcode nucleic acid molecule terminates at the 3' end of the barcode nucleic acid molecule.

[0594] 133. The method of embodiment 131 or embodiment 132, wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first template switch oligonucleotide (TSO).

[0595] 134. The method of any one of embodiments 131 to 133, wherein the 5' end of the first nucleic acid molecule attached to the solid surface comprises a first sequence of three consecutive riboguanosine residues.

[0596] 135. The method of any one of embodiments 131 to 134, wherein the first nucleic acid molecule attached to the solid surface further comprises a first unique molecular identifier (UMI).

[0597] 136. The method of any one of embodiments 131 to 135, wherein the first nucleic acid molecule attached to the solid surface further comprises a first surface barcode.

[0598] 137. The method of any one of embodiments 131 to 136, wherein the first nucleic acid molecule attached to the solid surface further comprises a first sequencing primer.

[0599] 138. The method of any one of embodiments 131 to 137, wherein the first nucleic acid molecule attached to the solid surface comprises, from 5′ to 3′, the first sequence of three riboguanosine residues, the first TSO, the first UMI, the first surface barcode, and the first sequencing primer.

[0600] 139. The method of any one of embodiments 131 to 138, wherein the first nucleic acid molecule attached to the solid surface is attached via the 3' end of the first nucleic acid molecule attached to the solid surface.

[0601] 140. The method of any one of embodiments 131 to 139, wherein the first nucleic acid molecule attached to the solid surface comprises a first DNA molecule attached to the solid surface.

[0602] 141. A method as described in embodiment 140, wherein the first DNA molecule attached to the solid surface comprises a first single-stranded DNA molecule attached to the solid surface.

[0603] 142. A method as described in embodiment 141, wherein the portion of the first single-stranded DNA molecule attached to the solid surface comprises a first TSO, wherein the portion of the barcode nucleic acid molecule hybridized to the first single-stranded DNA molecule is complementary to the first TSO, and wherein the method further comprises reverse transcribing the first single-stranded DNA molecule attached to the solid surface starting from the 3' end of the portion of the barcode nucleic acid molecule complementary to the first TSO.

[0604] 143. A method as described in any one of embodiments 131 to 142, wherein the barcode nucleic acid molecule is a single-stranded DNA barcode nucleic acid molecule, and wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first TSO, and the method further comprises reverse transcribing the single-stranded DNA barcode nucleic acid molecule starting from the 5' end of the first TSO.

[0605] 144. The method of any one of embodiments 131 to 143, wherein the 3' end of the portion of the barcode nucleic acid molecule comprises three consecutive cytidine or ribocytidine residues.

[0606] 145. A method as described in embodiment 144, which also includes reverse transcribing an mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the surface of the modified cell, thereby generating a single-stranded cDNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the surface of the modified cell, wherein the nucleotide sequence of the single-stranded cDNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the surface of the modified cell differs from the DNA complementary sequence of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the surface of the modified cell in that the 3' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the surface of the modified cell contains three additional, consecutive cytidine or ribocytidine residues.

[0607] 146. A method as described in embodiment 145, wherein the 3' end of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface contains a plurality of consecutive adenine residues.

[0608] 147. A method as described in embodiment 145 or embodiment 146, wherein the 5' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface contains a plurality of consecutive thymine residues.

[0609] 148. The method of any one of embodiments 131 to 147, wherein the 5' end of the second nucleic acid molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues.

[0610] 149. The method of any one of embodiments 131 to 148, wherein the second nucleic acid molecule attached to the solid surface further comprises a second UMI.

[0611] 150. The method of any one of embodiments 131 to 149, wherein the second nucleic acid molecule attached to the solid surface further comprises a second surface barcode, wherein the nucleotide sequences of the first surface barcode nucleic acid molecule and the second surface barcode nucleic acid molecule are identical.

[0612] 151. The method of any one of embodiments 131 to 150, wherein the second nucleic acid molecule attached to the solid surface further comprises a second sequencing primer.

[0613] 152. The method of any one of embodiments 131 to 151, wherein the second nucleic acid molecule attached to the solid surface comprises, from 5' to 3', the second sequence of three riboguanosine residues, the second TSO, the second UMI, the second surface barcode, and the second sequencing primer, wherein the nucleotide sequences of the first surface barcode nucleic acid molecule and the second surface barcode nucleic acid molecule are identical.

[0614] 153. The method of any one of embodiments 131 to 152, wherein the second nucleic acid molecule attached to the solid surface is attached via the 3' end of the second nucleic acid molecule attached to the solid surface.

[0615] 154. The method of any one of embodiments 131 to 153, wherein the second nucleic acid molecule attached to the solid surface comprises a second DNA molecule attached to the solid surface.

[0616] 155. A method as described in embodiment 154, wherein the second DNA molecule attached to the solid surface comprises a second single-stranded DNA molecule attached to the solid surface.

[0617] 156. A method as described in embodiment 155, wherein the 5' end of the portion of the second single-stranded DNA molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues, and wherein the 3' end of the portion of the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface comprises three consecutive cytidine or ribocytidine residues, and wherein the method further comprises reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting from the 3' end of the three consecutive cytidine or ribocytidine residues.

[0618] 157. The method of any one of embodiments 131 to 156, wherein the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the surface of the modified cell comprises a second single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the surface of the modified cell.

[0619] 158. A method as described in embodiment 156, wherein the 3' end of the portion of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface contains three consecutive cytidine or ribocytidine residues, and wherein the 5' end of the portion of the second single-stranded DNA molecule attached to the solid surface contains a second sequence of three consecutive riboguanosine residues, wherein the method also includes reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting from the 3' end of the portion of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface.

[0620] 159. The method of any one of embodiments 131 to 158, wherein the antibody-secreting cells are disposed within a separator having the solid surface.

[0621] 160. The method of any one of embodiments 131 to 159, wherein the solid surface comprises beads.

[0622] 161. The method of embodiment 159 or 160, further comprising lysing the antibody-secreting cells within the separator.

[0623] 162. The method of any one of embodiments 131 to 161, wherein the barcoded nucleic acid molecule further comprises a third sequencing primer, wherein the third sequencing primer begins at the 5′ end of the barcoded nucleic acid molecule.

[0624] 163. The method of any one of embodiments 131 to 162, wherein each amplicon of the first amplicon library comprises a gene, wherein each amplicon does not comprise a nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface.

[0625] 164. The method of embodiment 163, further comprising performing sample demultiplexing, alignment, filtering, and UMI counting on the first amplicon library.

[0626] 165. The method of any one of embodiments 131 to 164, wherein sequencing of the first amplicon library comprises next generation sequencing.

[0627] 166. The method of any one of embodiments 131 to 165, further comprising mapping each amplicon of the first amplicon library to a standard reference genome and aligning them.

[0628] 167. A method as described in embodiment 166, wherein the standard reference genome includes a human standard reference genome.

[0629] 168. A method as described in embodiment 167, wherein the human standard reference genome is GRCh38.

[0630] 169. A method as described in embodiment 166, wherein the standard reference genome comprises a mouse standard reference genome.

[0631] 170. A method as described in embodiment 169, wherein the mouse standard reference genome is mm10.

[0632] 171. The method of any one of embodiments 166 to 170, wherein the mapping and aligning comprises mapping each amplicon of the first amplicon library to the standard reference genome with a splicing-aware aligner (STAR), wherein each amplicon of the first amplicon library comprises a unique molecular identifier, wherein all amplicons of the first amplicon library mapped to annotated genes of the standard reference genome are classified, and wherein the classified amplicons are counted for each unique molecular identifier (UMI), thereby generating a single cell count matrix comprising mapped counts for each annotated gene and counts for each UMI.

[0633] 172. A method as described in embodiment 171, further comprising calculating the total number of annotated genes divided by the log of the total number of UMIs counted. 10 The single cell count matrices for high-quality cells and broadly distributed genes were filtered using a ratio of , wherein cells with a ratio below about 0.1 were removed, wherein cells with an interquartile range of more than about four times the total number of UMIs counted were removed, and cells with more than about 80% of reads mapped to mitochondrial genes were removed.

[0634] 173. The method of embodiment 172, further comprising normalizing the single cell counting matrix so that the total number of UMIs counted is approximately 10,000.

[0635] 174. A method as described in embodiment 172 or 173, wherein the method also includes calculating a PCA embedding of the single cell count matrix and iteratively clustering the major cell types to identify similarities between batches, thereby generating a unified manifold approximation and projection (UMAP).

[0636] 175. The method of any one of embodiments 172 to 174, further comprising determining a linear adjustment function for each unit using a cluster-specific centroid, and applying the linear adjustment function for each unit to correct for differences in a batch, thereby generating a batch-corrected embedding, and using the batch-corrected embedding to generate a two-dimensional unified manifold approximation and projection (UMAP).

[0637] 176. A method as described in embodiment 174 or 175, wherein the method also includes determining a weighted k-nearest neighbor graph and applying the Leiden algorithm to the weighted k-nearest neighbor graph, wherein the weighted k-nearest neighbor graph has resolution values ​​of approximately 0.1, approximately 0.2, approximately 0.5, and approximately 1.0 to determine cell type clusters in an unsupervised manner.

[0638] 177. A method as described in embodiment 176, wherein the method also includes performing a paired Wilcox test on all cells in a cluster and comparing the results of the first paired Wilcox test, and performing another paired Wilcox test on all cells in every other cluster to quantify the p-value and fold change of each gene in each cluster, wherein when a gene has a low p-value and a high fold change, the cell is labeled with a common marker of antibody-secreting cells, and the cell is labeled with a subtype of the cluster named by the top gene from the differential expression results.

[0639] 178. The method of any one of embodiments 131 to 177, further comprising performing sample demultiplexing, reassembling read pairs into contigs, and aligning and annotating the contigs against a germline segment V(D)J reference sequence of a second amplicon family.

[0640] 179. A method as described in embodiment 178, wherein the alignment of the contig to the germline segment V(D)J reference sequence includes alignment relative to a human germline reference database or a mouse germline reference database.

[0641] 180. The method of embodiment 179, wherein the human germline reference database comprises the IMGT database of human germline immunoglobulin sequences.

[0642] 181. The method of embodiment 179, wherein the mouse germline reference database comprises the IMGT germline reference database.

[0643] 182. The method of any one of embodiments 179 to 181, further comprising comparing the VDJ sequence against sequences in the human germline reference database or the mouse germline reference database, thereby marking the VDJ sequence for quality alignment by checking in-frame alignment, length of CDR3, and absence of stop codons in the variable region sequence and filtering the VDJ sequence based on the markings.

[0644] 183. The method of embodiment 182, further comprising mapping the VDJ sequence to a reference database of immunoglobulin chains.

[0645] 184. A method as described in embodiment 179, wherein the accuracy of the immunoglobulin isotypes, variable region maps, joining region maps, diversity region maps, and full-length variable regions of the heavy chain and light chain sequences of each cell are confirmed by the alignment.

[0646] 185. The method of any one of embodiments 131 to 184, further comprising performing sample demultiplexing, alignment, filtering, and UMI counting on the third amplicon library.

[0647] 186. The method of any one of embodiments 131 to 185, further comprising mapping the third amplicon library to a custom short read reference comprising the barcode nucleic acid molecule reference associated with each antigen, wherein the counts of each uniquely mapped barcode nucleic acid molecule are summed for each cell in the barcoded antigen single cell matrix.

[0648] 187. The method of embodiment 186 further comprises quantifying the barcode nucleic acid molecules in all cells and normalizing the quantification by taking the center log ratio of each barcode nucleic acid molecule in the multiple barcode nucleic acid molecules in each sample capture.

[0649] 188. The method of embodiment 187 further comprises removing background antigen signals by performing background denoising and scaling (DSB) on each of the plurality of barcoded nucleic acid molecules.

[0650] 189. A method as described in embodiment 188, wherein the method also includes determining an antigen signal distribution, wherein when a well-separated bimodal distribution exists in the antigen signal distribution, a z-transform value is used to calculate the antigen specificity, and wherein when a well-separated bimodal distribution does not exist in the antigen signal distribution, a quantile value is used.

[0651] 190. The method of embodiment 189 further comprises performing statistical modeling on the target antigen and negative control antigen distribution.

[0652] 191. The method of any one of embodiments 131 to 190, wherein the first amplicon library, the second amplicon library, and the third amplicon library are analyzed simultaneously to obtain at least one candidate sequence of the antigen-positive target antibody captured on the surface of the modified cell.

[0653] 192. The method of embodiment 191, further comprising grouping antibody secreting cells using effective antibody constant regions using analysis of the first amplicon library and analysis of the second amplicon library.

[0654] 193. The method of embodiment 192, wherein the antibody constant region comprises an IgE constant region.

[0655] 194. A method as described in embodiment 192 or 193, wherein the grouped antibody-secreting cells contain detectable levels of IgE heavy chain and CD79a, but lack detectable levels of Ms4a1 and CD19, wherein the grouped antibody-secreting cells are IgE + Antibody-secreting cells.

[0656] 195. The method of embodiment 194, further comprising evaluating the IgE + Differential expression of antibody secreting cells relative to IgA secreting cell isotypes, IgG secreting cell isotypes, and / or IgM secreting cell isotypes.

[0657] 196. A method as described in embodiment 195, wherein the evaluation of differential expression further characterizes the IgE + Distinct transcriptional signatures of antibody-secreting cells.

[0658] 197. The method of embodiment 196, further comprising evaluating the IgE + Antigen specificity of antibody-secreting cells.

[0659] 198. The method of embodiment 197, wherein the assessment comprises converting the IgE + The antigen specificity of the antibody secreting cells is compared relative to a plurality of antigens and a control antigen, and wherein the plurality of antigens comprises the target antigen.

[0660] 199. A method as described in embodiment 198, wherein the comparison includes calculating an empirical score for each antigen in the plurality of antigens and the control antigen by subtracting the quantile value of the signal associated with the antigen (qT) from the quantile value of the signal associated with the control antigen (qC) with a penalty factor (x) to determine an antigen-specific score, wherein the antigen-specific score=qT-qC x .

[0661] 200. The method of embodiment 199, wherein the penalty factor is the same for all antigens.

[0662] 201. The method of embodiment 199, wherein the penalty factor is different for different antibody isotypes.

[0663] 202. The method of embodiment 199, wherein the penalty factor is different when other antigens are used.

[0664] 203. The method of embodiment 199, further comprising determining the antigen-specific score for each of the plurality of antigens and the control antigen.

[0665] 204. The method as described in embodiment 203 also includes calculating a control antigen-specific score, wherein the cell is selected when the antigen-specific score is high and the control antigen-specific score is low.

[0666] 205. The method of embodiment 204, wherein the VDJ region identifies antigen-specific antibodies.

[0667] 206. The method of embodiment 204, further comprising using a paired V H :V L Antibody sequences select antibody candidates from a hierarchical list of antigenic signals.

[0668] 207. The method of any one of embodiments 191 to 206, further comprising determining differential expression between isotypes, conditions, and / or antigen specificities, wherein determining differential expression between isotypes comprises performing a paired Wilcox test on all cells in one isotype and performing a paired Wilcox test on all cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster; and wherein determining differential expression between antigen specificities comprises performing a paired Wilcox test on all cells in one isotype and performing a paired Wilcox test on all cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster.

[0669] 208. The method of any one of embodiments 191 to 207, further comprising clustering VDJ sequence similarities in antigen-positive target cells, wherein clustering VDJ sequence similarities comprises grouping similar variable regions by amino acid sequence based on sequence alignment.

[0670] 209. The method of any one of embodiments 191 to 208, wherein the antigen-positive target cells are IgE + .

[0671] 210. The method of any one of embodiments 191 to 209, further comprising trimming redundant sequences.

[0672] 211. A method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by an antibody-secreting cell, the method comprising:

[0673] a) contacting a population of antibody-secreting cells with a second component of a binding pair, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody, whereby the second component of the binding pair binds to the cell surface of the population of antibody-secreting cells;

[0674] b) contacting the antibody-secreting cell population with an antibody capture complex, wherein the antibody capture complex comprises a first component of the binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to the second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells;

[0675] c) contacting the antibody-secreting cell population with a second anti-Ig antibody so that the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the second anti-Ig antibody;

[0676] d) contacting the antibody-secreting cell population with an antigen comprising a barcode nucleic acid molecule so that the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the antigen;

[0677] e) collecting a pool of antibody-secreting cells, wherein each cell in the pool secretes an antibody captured by the antibody-capture complex and bound by the second anti-Ig antibody and the antigen comprising the barcode nucleic acid molecule, and wherein the pool of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody;

[0678] f) separating the antibody-secreting cell pool into single antibody-secreting cells, and for each single antibody-secreting cell:

[0679] 1) hybridizing a portion of the barcode nucleic acid molecule to a portion of a first nucleic acid molecule attached to a solid surface, wherein the solid surface and the first nucleic acid molecule attached thereto are unique to each single antibody-secreting cell;

[0680] 2) hybridizing a portion of the nucleic acid molecule comprising the region of the gene encoding the antigen-binding fragment of the target antibody with a portion of a second nucleic acid molecule attached to the solid surface;

[0681] 3) preparing a first amplicon library of gene expression, a second amplicon library of variable region, diversity region and joining region (VDJ), and a third amplicon library of antigen barcode nucleic acid molecules; and

[0682] 4) sequencing each of the three amplicon libraries; and

[0683] g) identifying said region of said gene encoding said antigen-binding fragment of said target antibody.

[0684] 212. The method of embodiment 211, wherein:

[0685] The first component of the binding pair comprises avidin, streptavidin or avidin, and the second component of the binding pair comprises biotin;

[0686] The first component of the binding pair comprises one of jun and fos, and the second component of the binding pair comprises the other of jun and fos;

[0687] The first component of the binding pair comprises one of mad and max, and the second component of the binding pair comprises the other of mad and max;

[0688] The first component of the binding pair comprises one of myc and max, and the second component of the binding pair comprises the other of myc and max;

[0689] The first member of the binding pair comprises one of an azide and an alkyne, and the second member of the binding pair comprises the other of an azide and an alkyne; or

[0690] The first component of the binding pair comprises one of an azide and a phosphine, and the second component of the binding pair comprises the other of an azide and a phosphine.

[0691] 213. A method as described in embodiment 211, wherein the first component of the binding pair comprises avidin or streptavidin, and the second component of the binding pair comprises biotin.

[0692] 214. The method of embodiment 211, wherein:

[0693] The second component of the binding pair comprises fluorescein isothiocyanate (FITC), and the first component of the binding pair comprises an anti-FITC antibody;

[0694] The second component of the binding pair comprises phycoerythrin (PE), and the first component of the binding pair comprises an anti-PE antibody;

[0695] The second component of the binding pair comprises an APC, and the first component of the binding pair comprises an anti-APC antibody;

[0696] The second component of the binding pair comprises BV421, and the first component of the binding pair comprises an anti-BV421 antibody;

[0697] The second component of the binding pair comprises BV510, and the first component of the binding pair comprises an anti-BV510 antibody;

[0698] The second component of the binding pair comprises BV605, and the first component of the binding pair comprises an anti-BV605 antibody;

[0699] The second component of the binding pair comprises BV650, and the first component of the binding pair comprises an anti-BV650 antibody;

[0700] The second component of the binding pair comprises BV711, and the first component of the binding pair comprises an anti-BV711 antibody; or

[0701] The second component of the binding pair comprises BV786, and the first component of the binding pair comprises an anti-BV786 antibody.

[0702] 215. A method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by an antibody-secreting cell, the method comprising:

[0703] a) contacting a population of antibody-secreting cells, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody, with an antibody capture complex, wherein the antibody capture complex comprises a first component of a binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to a second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells;

[0704] b) contacting the antibody-secreting cell population with a second anti-Ig antibody so that the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the second anti-Ig antibody;

[0705] c) contacting the antibody-secreting cell population with an antigen comprising a barcode nucleic acid molecule, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the antigen;

[0706] d) collecting a pool of antibody-secreting cells, wherein each cell in the pool secretes an antibody captured by the antibody-capture complex and bound by the second anti-Ig antibody and the antigen comprising the barcode nucleic acid molecule, and wherein the pool of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody;

[0707] e) separating the antibody-secreting cell pool into single antibody-secreting cells, and for each single antibody-secreting cell:

[0708] 1) hybridizing a portion of the barcode nucleic acid molecule to a portion of a first nucleic acid molecule attached to a solid surface, wherein the solid surface and the first nucleic acid molecule attached thereto are unique to each single antibody-secreting cell;

[0709] 2) hybridizing a portion of the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody with a portion of a second nucleic acid molecule attached to the solid surface;

[0710] 3) preparing a first amplicon library of gene expression in the antibody-secreting cells, a second amplicon library of variable regions, diversity regions, and joining regions (VDJ) in the antibody-secreting cells, and a third amplicon library of antigen barcode nucleic acid molecules; and

[0711] 4) sequencing each of the three amplicon libraries; and

[0712] f) identifying said region of said gene encoding said antigen-binding fragment of said target antibody.

[0713] 216. A method as described in embodiment 215, wherein the second component of the binding pair comprises a surface marker of the antibody-secreting cell, and the first component of the binding pair is an antibody that binds to the surface marker.

[0714] 217. A method as described in embodiment 216, wherein the surface markers comprise CD27, CD38, CD45, CD138, CD98, CD78, CD319, CXCR4, BCMA, GPRC5D, FCRL5, CD19, Ly6D, CD52 or transmembrane activator calcium regulator and cyclophilin ligand interactor (TACI).

[0715] 218. A method as described in embodiment 215, wherein the second component of the binding pair comprises an interleukin-6 receptor (IL-6R), and the first component of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6).

[0716] 219. A method as described in embodiment 215, wherein the second component of the binding pair comprises CD27, and the first component of the binding pair comprises an anti-CD27 antibody or CD70.

[0717] 220. The method of any one of embodiments 211 to 219, wherein the antibody capture molecule comprises a capture antibody.

[0718] 221. The method of embodiment 220, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain κ antibody, or an anti-light chain λ antibody.

[0719] 222. The method of embodiment 221, wherein the anti-Fc antibody comprises an anti-Fcγ antibody, an anti-Fcα antibody, or an anti-Fcε antibody.

[0720] 223. A method as described in embodiment 222, wherein the anti-Fcγ antibody comprises an anti-FcγRI antibody, an anti-FcγRIIA antibody, an anti-FcγRIIB antibody, an anti-FcγRIIB1 antibody, an anti-FcγRIIB2 antibody, an anti-FcγRIIIA antibody, or an anti-FcγRIIIB antibody.

[0721] 224. The method of embodiment 222, wherein the anti-Fcα antibody comprises an anti-FcαRI antibody.

[0722] 225. The method of embodiment 222, wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody.

[0723] 226. The method of embodiment 220, wherein the capture antibody comprises an anti-IgM antibody.

[0724] 227. The method of embodiment 220, wherein the capture antibody comprises an anti-IgG antibody.

[0725] 228. The method of embodiment 227, wherein the anti-IgG antibody comprises an anti-IgG1 antibody, an anti-IgG2 antibody, an anti-IgG2a antibody, an anti-IgG2b antibody, an anti-IgG3 antibody, or an anti-IgG4 antibody.

[0726] 229. The method of embodiment 220, wherein the capture antibody comprises an anti-IgA antibody.

[0727] 230. The method of embodiment 229, wherein the anti-IgA antibody comprises an anti-IgA1 antibody, an anti-IgA2 antibody, an anti-secretory IgA antibody, or a polymeric anti-IgA antibody.

[0728] 231. The method of embodiment 220, wherein the capture antibody comprises an anti-IgE antibody.

[0729] 232. The method of any one of embodiments 211 to 219, wherein the antibody capture molecule comprises an Fc receptor or an extracellular domain of the Fc receptor.

[0730] 233. A method as described in embodiment 232, wherein the Fc receptor comprises an Fcγ receptor, an Fcα receptor or an Fcε receptor.

[0731] 234. A method as described in embodiment 233, wherein the Fcγ receptor comprises an FcγRI receptor, an FcγRIIA receptor, an FcγRIIB receptor, an FcγRIIB1 receptor, an FcγRIIB2 receptor, an FcγRIIIA receptor, an FcγRIIIB receptor or an FcRn receptor.

[0732] 235. The method of embodiment 233, wherein the Fcα receptor comprises an FcαRI receptor or an Fcα / μR receptor.

[0733] 236. A method as described in embodiment 233, wherein the Fcε receptor comprises an FcεRI receptor or an FcεRII receptor.

[0734] 237. A method as described in any one of embodiments 211 to 219, wherein the antibody capture molecule comprises protein A or protein G.

[0735] 238. A method as described in any one of embodiments 211 to 219, wherein the antibody capture molecule comprises protein L.

[0736] 239. The method of any one of embodiments 211 to 238, wherein the second anti-Ig antibody is anti-IgE or anti-IgG.

[0737] 240. The method of any one of embodiments 211 to 239, wherein the second anti-Ig antibody is detectably labeled.

[0738] 241. The method of embodiment 240, wherein the second anti-Ig antibody is labeled with: a radioactive compound, a fluorescent compound, or an enzyme.

[0739] 242. The method of embodiment 241, wherein the radioactive compound comprises 3 H.14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 Tc.

[0740] 243. A method as described in embodiment 241, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin or fluorescent protein.

[0741] 244. The method of embodiment 241, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase.

[0742] 245. The method of any one of embodiments 211 to 244, wherein the antibody-secreting cells are contacted with a plurality of barcoded antigens, wherein each barcoded antigen in the plurality of barcoded antigens comprises a unique antigen linked to a unique nucleic acid molecule.

[0743] 246. A method as described in embodiment 245, wherein the unique nucleic acid molecule comprises DNA.

[0744] 247. The method of embodiment 245, wherein the unique nucleic acid molecule comprises RNA.

[0745] 248. The method of any one of embodiments 245 to 247, wherein each unique nucleic acid molecule comprises about 10 nucleotides to about 500 nucleotides.

[0746] 249. The method of any one of embodiments 245 to 248, wherein each barcoded antigen is detectably labeled.

[0747] 250. The method of embodiment 249, wherein each barcoded antigen is labeled with:

[0748] Radioactive compounds, fluorescent compounds, or enzymes.

[0749] 251. The method of embodiment 250, wherein the radioactive compound comprises 3 H. 14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 Tc.

[0750] 252. A method as described in embodiment 250, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin or fluorescent protein.

[0751] 253. The method of embodiment 250, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase.

[0752] 254. The method of any one of embodiments 211 to 253, wherein the barcoded antigen comprises an allergen.

[0753] 255. The method of any one of embodiments 211 to 254, wherein the antibody-secreting cell is a plasma cell or a plasmablast.

[0754] 256. The method of any one of embodiments 211 to 255, further comprising enriching a cell population obtained from humans for immune cells prior to contacting the surface of the antibody-secreting cell population with the second component of a binding pair, or prior to contacting the surface of the antibody-secreting cell population with the antibody capture complex.

[0755] 257. The method of embodiment 256, wherein the cell population obtained from the human is enriched for plasma cells or plasmablasts.

[0756] 258. A method as described in embodiment 257, wherein pan-B cell enrichment is used to enrich the immune cell population for plasma cells or plasmablasts.

[0757] 259. The method of any one of embodiments 211 to 258, wherein the antibody-secreting cell population is obtained from human lymph nodes, lungs, bone marrow, or blood.

[0758] 260. The method of any one of embodiments 211 to 259, further comprising detecting one or more of the second anti-Ig antibody and / or the barcoded antigen.

[0759] 261. The method of any one of embodiments 211 to 260, further comprising sorting the antibody-secreting cell population to obtain a pool of antibody-secreting cells bound by a second anti-Ig antibody and / or one or more of the barcoded antigens.

[0760] 262. The method of any one of embodiments 211 to 261, further comprising contacting the antibody-secreting cell population with an Fc blocker prior to contacting the antibody-secreting cell population with the second component of the binding pair.

[0761] 263. The method of any one of embodiments 211 to 262, wherein the portion of the barcode nucleic acid molecule is complementary to a first template switch oligonucleotide (TSO).

[0762] 264. The method of any one of embodiments 211 to 263, wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first template switch oligonucleotide (TSO).

[0763] 265. The method of any one of embodiments 211 to 264, wherein the 5' end of the first nucleic acid molecule attached to the solid surface comprises a first sequence of three consecutive riboguanosine residues.

[0764] 266. The method of any one of embodiments 211 to 265, wherein the first nucleic acid molecule attached to the solid surface further comprises a first unique molecular identifier (UMI).

[0765] 267. The method of any one of embodiments 211 to 266, wherein the first nucleic acid molecule attached to the solid surface further comprises a first surface barcode.

[0766] 268. The method of any one of embodiments 211 to 267, wherein the first nucleic acid molecule attached to the solid surface further comprises a first sequencing primer.

[0767] 269. The method of any one of embodiments 211 to 268, wherein the first nucleic acid molecule attached to the solid surface comprises, from 5′ to 3′, the first sequence of three riboguanosine residues, the first TSO, the first UMI, the first surface barcode, and the first sequencing primer.

[0768] 270. The method of any one of embodiments 211 to 269, wherein the first nucleic acid molecule attached to the solid surface is attached via the 3' end of the first nucleic acid molecule attached to the solid surface.

[0769] 271. The method of any one of embodiments 211 to 270, wherein the first nucleic acid molecule attached to the solid surface comprises a first DNA molecule attached to the solid surface.

[0770] 272. A method as described in embodiment 271, wherein the first DNA molecule attached to the solid surface comprises a first single-stranded DNA molecule attached to the solid surface.

[0771] 273. A method as described in embodiment 272, wherein the portion of the first single-stranded DNA molecule attached to the solid surface comprises a first TSO, and the method further comprises reverse transcribing the first single-stranded DNA molecule attached to the solid surface starting from the 3' end of the portion of the barcode nucleic acid molecule that is complementary to the first TSO.

[0772] 274. The method of any one of embodiments 211 to 273, wherein the barcode nucleic acid molecule is a single-stranded DNA barcode nucleic acid molecule, and wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first TSO, and the method further comprises reverse transcribing the single-stranded DNA barcode nucleic acid molecule from the 3' end of the first TSO.

[0773] 275. The method of any one of embodiments 211 to 274, wherein the 3' end of the portion of the barcode nucleic acid molecule comprises three consecutive cytidine residues.

[0774] 276. A method as described in embodiment 275, which also includes reverse transcribing an mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody, thereby producing a single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody, and wherein the nucleotide sequence of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody differs from the complementary sequence of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody in that the 3' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody contains three additional consecutive cytidine residues.

[0775] 277. A method as described in embodiment 276, wherein the 3' end of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody contains a plurality of consecutive adenine residues.

[0776] 278. A method as described in embodiment 276 or embodiment 277, wherein the 5' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody contains a plurality of consecutive thymine residues.

[0777] 279. The method of any one of embodiments 211 to 278, wherein the 5' end of the second nucleic acid molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues.

[0778] 280. The method of any one of embodiments 211 to 279, wherein the second nucleic acid molecule attached to the solid surface further comprises a second UMI.

[0779] 281. The method of any one of embodiments 211 to 280, wherein the second nucleic acid molecule attached to the solid surface further comprises a second surface barcode.

[0780] 282. The method of any one of embodiments 211 to 281, wherein the second nucleic acid molecule attached to the solid surface further comprises a second sequencing primer.

[0781] 283. The method of any one of embodiments 211 to 282, wherein the second nucleic acid molecule attached to the solid surface comprises, from 5′ to 3′, the second sequence of three riboguanosine residues, the second TSO, the second UMI, the second surface barcode, and the second sequencing primer.

[0782] 284. The method of any one of embodiments 211 to 283, wherein the second nucleic acid molecule attached to the solid surface is attached via the 3' end of the second nucleic acid molecule attached to the solid surface.

[0783] 285. The method of any one of embodiments 211 to 284, wherein the second nucleic acid molecule attached to the solid surface comprises a second DNA molecule attached to the solid surface.

[0784] 286. A method as described in embodiment 285, wherein the second DNA molecule attached to the solid surface comprises a second single-stranded DNA molecule attached to the solid surface.

[0785] 287. A method as described in embodiment 286, wherein the 5' end of the portion of the second single-stranded DNA molecule attached to the solid surface contains a second sequence of three consecutive riboguanosine residues, and wherein the 3' end of the portion of the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody contains three consecutive cytidine residues, and wherein the method further includes reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting from the 3' end of the three consecutive cytidine residues.

[0786] 288. The method of any one of embodiments 211 to 287, wherein the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody comprises a second single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody.

[0787] 289. A method as described in embodiment 287, wherein the 3' end of the portion of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody contains three consecutive cytidine residues, and wherein the 5' end of the portion of the second single-stranded DNA molecule attached to the solid surface contains a second sequence of three consecutive riboguanosine residues, wherein the method also includes reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting from the 3' end of the portion of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody.

[0788] 290. The method of any one of embodiments 211 to 289, wherein each of the individual antibody-secreting cells is disposed within a separator having a solid surface.

[0789] 291. The method of any one of embodiments 211 to 290, wherein the solid surface comprises beads.

[0790] 292. The method of embodiment 290 or embodiment 291, further comprising lysing the single antibody-secreting cell within the separator.

[0791] 293. The method of any one of embodiments 211 to 292, wherein the barcoded nucleic acid molecule further comprises a third sequencing primer.

[0792] 294. The method of any one of embodiments 211 to 293, wherein each amplicon of the first amplicon library comprises a gene, wherein each amplicon does not comprise a nucleic acid molecule encoding the antigen-binding fragment of the target antibody comprising the region of the gene.

[0793] 295. The method of embodiment 294, further comprising performing sample demultiplexing, alignment, filtering, and UMI counting on the first amplicon library.

[0794] 296. The method of any one of embodiments 211 to 295, wherein sequencing of the first amplicon library comprises next generation sequencing.

[0795] 297. The method of any one of embodiments 211 to 296, further comprising mapping each amplicon of the third amplicon library to a standard reference genome and aligning them.

[0796] 298. A method as described in embodiment 297, wherein the standard reference genome includes a human standard reference genome.

[0797] 299. A method as described in embodiment 298, wherein the human standard reference genome is GRCh38.

[0798] 300. A method as described in embodiment 297, wherein the standard reference genome comprises a mouse standard reference genome.

[0799] 301. A method as described in embodiment 300, wherein the mouse standard reference genome is mm10.

[0800] 302. The method of any one of embodiments 297 to 301, wherein the mapping and aligning comprises mapping each amplicon of the first amplicon library to the standard reference genome with a splicing-aware aligner (STAR), wherein each amplicon of the first amplicon library comprises a unique molecular identifier, wherein all amplicons of the first amplicon library mapped to annotated genes of the standard reference genome are classified, and wherein the classified amplicons are counted for each unique molecular identifier (UMI), thereby generating a single cell count matrix comprising mapped counts for each annotated gene and counts for each UMI.

[0801] 303. A method as described in embodiment 302, the method also includes calculating the total number of annotated genes divided by the log of the total number of UMIs counted. 10 The single cell count matrices of high-quality single antibody-secreting cells and widely distributed genes were filtered using a ratio of , wherein single antibody-secreting cells with a ratio lower than about 0.1 were filtered out, wherein single antibody-secreting cells with an interquartile range of more than about four times the total number of UMIs counted were filtered out, and single antibody-secreting cells with more than about 80% of the reads mapped to mitochondrial genes were filtered out.

[0802] 304. The method of embodiment 303, further comprising normalizing the single cell counting matrix so that the total number of UMIs counted is approximately 10,000.

[0803] 305. The method of embodiment 303 or embodiment 304, further comprising calculating a PCA embedding of the single cell count matrix, iteratively clustering major cell types to identify similarities between batches, thereby generating a unified manifold approximation and projection (UMAP).

[0804] 306. The method of any one of embodiments 303 to 305, further comprising determining a linear adjustment function for each unit using a cluster-specific centroid, applying the linear adjustment function for each unit to correct for differences in a batch, thereby generating a batch-corrected embedding, and using the batch-corrected embedding to generate a two-dimensional unified manifold approximation and projection (UMAP).

[0805] 307. The method of embodiment 305 or embodiment 306 further comprises determining a weighted k-nearest neighbor graph and applying the Leiden algorithm to the weighted k-nearest neighbor graph having resolution values ​​of 0.1, 0.2, 0.5, and 1.0 to determine cell type clusters in an unsupervised manner.

[0806] 308. A method as described in embodiment 305, wherein the method also includes performing a paired Wilcox test on all single antibody-secreting cells in a cluster and comparing the results of the first paired Wilcox test, and performing another paired Wilcox test on all single antibody-secreting cells in every other cluster to quantify the p-value and fold change of each gene in each cluster, wherein when a gene has a low p-value and a high fold change, the single antibody-secreting cell is marked by a common marker of plasma cells, plasmablasts, or B cells, and the single antibody-secreting cell is marked by a subtype of the cluster named by the top gene from the differential expression results.

[0807] 309. The method of any one of embodiments 211 to 308, further comprising performing sample demultiplexing, reassembling the read pairs into contigs, and aligning and annotating the contigs against a germline segment V(D)J reference sequence of a second amplicon family.

[0808] 310. A method as described in embodiment 309, wherein the alignment of the contig to the germline segment V(D)J reference sequence includes alignment relative to a human germline reference database or a mouse germline reference database.

[0809] 311. A method as described in embodiment 310, wherein the human germline reference database comprises the IMGT database of human germline immunoglobulin sequences.

[0810] 312. A method as described in embodiment 310, wherein the mouse germline reference database comprises the IMGT germline reference database.

[0811] 313. The method of any one of embodiments 310 to 312, further comprising comparing the VDJ sequence against sequences in the human germline reference database or the mouse germline reference database, thereby marking the VDJ sequence for quality alignment by checking in-frame alignment, length of CDR3, and absence of stop codons in the variable region sequence and filtering the VDJ sequence based on the markings.

[0812] 314. The method of embodiment 313, further comprising mapping the VDJ sequence to a reference database of immunoglobulin chains.

[0813] 315. A method as described in embodiment 310, wherein the accuracy of the immunoglobulin isotypes, variable region maps, joining region maps, diversity region maps, and full-length variable regions of the heavy chain and light chain sequences of each cell are confirmed by the alignment.

[0814] 316. The method of any one of embodiments 211 to 315, further comprising performing sample demultiplexing, alignment, filtering, and UMI counting on the third amplicon library.

[0815] 317. The method of any one of embodiments 211 to 316, further comprising mapping the third amplicon library to a custom short read reference comprising the barcode nucleic acid molecule reference associated with each antigen, wherein the counts of each uniquely mapped barcode nucleic acid molecule are summed for each cell in the barcoded antigen single cell matrix.

[0816] 318. The method of embodiment 317 further comprises quantifying the barcode nucleic acid molecules in all individual antibody-secreting cells, and normalizing the quantification by taking the central logarithm ratio of each barcode nucleic acid molecule in the multiple barcode nucleic acid molecules in each sample capture.

[0817] 319. The method of embodiment 318, further comprising removing background antigen signals by performing background denoising and scaling (DSB) on each of the plurality of barcoded nucleic acid molecules.

[0818] 320. A method as described in embodiment 319, wherein the method also includes determining an antigen signal distribution, wherein when a well-separated bimodal distribution exists in the antigen signal distribution, a z-transform value is used to calculate the antigen specificity, and wherein when a well-separated bimodal distribution does not exist in the antigen signal distribution, a quantile value is used.

[0819] 321. The method of embodiment 320, further comprising performing statistical modeling on the target antigen and negative control antigen distribution.

[0820] 322. The method of any one of embodiments 211 to 321, wherein the first amplicon library, the second amplicon library, and the third amplicon library are analyzed simultaneously to obtain at least one candidate sequence of an antigen-positive target antibody.

[0821] 323. The method of embodiment 322, further comprising grouping plasma cells or plasmablasts using an analysis of the first amplicon library and an analysis of the second amplicon library using an effective antibody constant region.

[0822] 324. The method of embodiment 323, wherein the antibody constant region comprises an IgE constant region.

[0823] 325. A method as described in embodiment 323 or embodiment 324, wherein the grouped plasma cells or plasmablasts contain detectable levels of IgE heavy chain and CD79a, but lack detectable levels of Ms4a1 and CD19, wherein the grouped plasma cells or plasmablasts are IgE + Plasma cells or plasmablasts.

[0824] 326. The method as described in embodiment 325, further comprising evaluating the IgE + Differential expression of plasma cells or plasmablasts relative to IgA secreting plasma cells or plasmablast isotypes, IgG secreting plasma cells or plasmablast isotypes, and / or IgM secreting plasma cells or plasmablast isotypes.

[0825] 327. A method as described in embodiment 326, wherein the evaluation of differential expression further characterizes the IgE + Distinct transcriptional signature of plasma cells or plasmablasts.

[0826] 328. The method of embodiment 327, further comprising evaluating the IgE + Antigen specificity of plasma cells or plasmablasts.

[0827] 329. The method of embodiment 328, wherein the assessment comprises converting the IgE + The antigen specificity of plasma cells or plasmablasts relative to a plurality of antigens and a control antigen is compared, and wherein the plurality of antigens comprises the target antigen.

[0828] 330. A method as described in embodiment 329, wherein the comparison includes calculating an empirical score for each antigen in the plurality of antigens and the control antigen by subtracting the quantile value of the signal associated with the antigen (qT) from the quantile value of the signal associated with the control antigen (qC) with a penalty factor (x) to determine an antigen-specific score, wherein the antigen-specific score=qT-qC x .

[0829] 331. A method as in embodiment 330, wherein the penalty factor is the same for all antigens.

[0830] 332. A method as in embodiment 330, wherein the penalty factor is different for different antibody isotypes.

[0831] 333. A method as in embodiment 330, wherein the penalty factor is different when other antigens are used.

[0832] 334. The method of embodiment 331, further comprising determining the antigen-specific score for each of the plurality of antigens and the control antigen.

[0833] 335. The method of embodiment 334 further comprises calculating a control antigen-specific score, wherein the cell is selected when the antigen-specific score is high and the control antigen-specific score is low.

[0834] 336. A method as described in embodiment 335, wherein the VDJ region identifies antigen-specific antibodies.

[0835] 337. The method of embodiment 335, further comprising using a paired V H :V L Antibody sequences select antibody candidates from a hierarchical list of antigenic signals.

[0836] 338. The method of any one of embodiments 322 to 337, further comprising determining differential expression between isotypes, conditions, and / or antigen specificities, wherein determining differential expression between isotypes comprises performing a paired Wilcox test on all single antibody-secreting cells in one isotype and performing a paired Wilcox test on all single antibody-secreting cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster; and wherein determining differential expression between antigen specificities comprises performing a paired Wilcox test on all single antibody-secreting cells in one isotype and performing a paired Wilcox test on all single antibody-secreting cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster.

[0837] 339. The method of any one of embodiments 320 to 338, further comprising clustering VDJ sequence similarities in antigen-positive target cells, wherein clustering VDJ sequence similarities comprises grouping similar variable regions by amino acid sequence based on sequence alignment.

[0838] 340. The method of any one of embodiments 320 to 339, wherein the antigen-positive target cells are IgE + .

[0839] 341. The method of any one of embodiments 320 to 340, further comprising trimming redundant sequences.

[0840] Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims

1. An antibody capture complex comprising a first component of a binding pair linked to an antibody capture molecule; wherein the first member of the binding pair is capable of binding to the second member of the binding pair; wherein the antibody capture molecule is capable of binding to a target antibody, wherein the target antibody is secreted by an antibody-secreting cell; Wherein when the second component of the binding pair is biotin and the first component of the binding pair is streptavidin, the antibody capture molecule does not bind to the kappa light chain of the target antibody.

2. The antibody capture complex of claim 1, wherein: The first component of the binding pair comprises avidin, streptavidin or avidin, and the second component of the binding pair comprises biotin; The first component of the binding pair comprises one of jun and fos, and the second component of the binding pair comprises the other of jun and fos; The first component of the binding pair comprises one of mad and max, and the second component of the binding pair comprises the other of mad and max; The first component of the binding pair comprises one of myc and max, and the second component of the binding pair comprises the other of myc and max; The first member of the binding pair comprises one of an azide and an alkyne, and the second member of the binding pair comprises the other of an azide and an alkyne; or The first component of the binding pair comprises one of an azide and a phosphine, and the second component of the binding pair comprises the other of an azide and a phosphine.

3. The antibody capture complex of claim 1, wherein the first component of the binding pair comprises avidin or streptavidin and the second component of the binding pair comprises biotin.

4. The antibody capture complex of claim 1, wherein the second component of the binding pair comprises a surface marker of the antibody-secreting cell, and the first component of the binding pair comprises an antibody that binds to the surface marker.

5. The antibody capture complex of claim 4, wherein the cell surface marker comprises CD27, CD38, CD45, CD138, CD98, CD78, CD319, CXCR4, BCMA, GPRC5D, FCRL5, CD19, Ly6D, CD52 or transmembrane activator calcium regulator and cyclophilin ligand interactor (TACI).

6. The antibody capture complex of claim 1, wherein the second component of the binding pair comprises an interleukin-6 receptor (IL-6R), and the first component of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6).

7. The antibody capture complex of claim 1, wherein the second component of the binding pair comprises CD27 and the first component of the binding pair comprises an anti-CD27 antibody or CD70.

8. The antibody capture complex of claim 1, wherein: The second component of the binding pair comprises fluorescein isothiocyanate (FITC), and the first component of the binding pair comprises an anti-FITC antibody; The second component of the binding pair comprises phycoerythrin (PE), and the first component of the binding pair comprises an anti-PE antibody; The second component of the binding pair comprises an APC, and the first component of the binding pair comprises an anti-APC antibody; The second component of the binding pair comprises BV421, and the first component of the binding pair comprises an anti-BV421 antibody; The second component of the binding pair comprises BV510, and the first component of the binding pair comprises an anti-BV510 antibody; The second component of the binding pair comprises BV605, and the first component of the binding pair comprises an anti-BV605 antibody; The second component of the binding pair comprises BV650, and the first component of the binding pair comprises an anti-BV650 antibody; The second component of the binding pair comprises BV711, and the first component of the binding pair comprises an anti-BV711 antibody; or The second component of the binding pair comprises BV786, and the first component of the binding pair comprises an anti-BV786 antibody.

9. The antibody capture complex of any one of claims 1 to 8, wherein the antibody capture molecule comprises a capture antibody.

10. The antibody capture complex of claim 9, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain κ antibody, or an anti-light chain λ antibody. The antibody capture complex of claim 10 , wherein the anti-Fc antibody comprises an anti-Fcγ antibody, an anti-Fcα antibody or an anti-Fcε antibody.

12. The antibody capture complex of claim 11, wherein the anti-Fcγ antibody comprises an anti-FcγRI antibody, an anti-FcγRIIA antibody, an anti-FcγRIIB antibody, an anti-FcγRIIB1 antibody, an anti-FcγRIIB2 antibody, an anti-FcγRIIIA antibody, or an anti-FcγRIIIB antibody.

13. The antibody capture complex of claim 11, wherein the anti-Fcα antibody comprises an anti-FcαRI antibody. The antibody capture complex of claim 11 , wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody.

15. The antibody capture complex of claim 9, wherein the capture antibody comprises an anti-IgM antibody.

16. The antibody capture complex of claim 9, wherein the capture antibody comprises an anti-IgG antibody. 17 . The antibody capture complex of claim 16 , wherein the anti-IgG antibody comprises an anti-IgG1 antibody, an anti-IgG2 antibody, an anti-IgG2a antibody, an anti-IgG2b antibody, an anti-IgG3 antibody or an anti-IgG4 antibody.

18. The antibody capture complex of claim 9, wherein the capture antibody comprises an anti-IgA antibody. 19 . The antibody capture complex of claim 18 , wherein the anti-IgA antibody comprises an anti-IgA1 antibody, an anti-IgA2 antibody, an anti-secretory IgA antibody or a polymeric anti-IgA antibody.

20. The antibody capture complex of claim 9, wherein the capture antibody comprises an anti-IgE antibody.

21. The antibody capture complex of any one of claims 1 to 8, wherein the antibody capture molecule comprises an Fc receptor or an extracellular domain of the Fc receptor.

22. The antibody capture complex of claim 21, wherein the Fc receptor comprises an Fcγ receptor, an Fcα receptor, or an Fcε receptor.

23. The antibody capture complex of claim 22, wherein the Fcγ receptor comprises an FcγRI receptor, an FcγRIIA receptor, an FcγRIIB receptor, an FcγRIIB1 receptor, an FcγRIIB2 receptor, an FcγRIIIA receptor, an FcγRIIIB receptor, or an FcRn receptor.

24. The antibody capture complex of claim 22, wherein the Fcα receptor comprises an FcαRI receptor or an Fcα / μR receptor.

25. The antibody capture complex of claim 22, wherein the Fcε receptor comprises an FcεRI receptor or an FcεRII receptor.

26. The antibody capture complex of any one of claims 1 to 8, wherein the antibody capture molecule comprises protein A or protein G.

27. The antibody capture complex of any one of claims 1 to 8, wherein the antibody capture molecule comprises protein L.

28. The antibody capture complex of claim 1, wherein the antibody capture complex comprises avidin or streptavidin linked to the extracellular domain of the high affinity IgE receptor (FcεRIa).

29. A method for capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting a population of antibody-secreting cells with a second component of a binding pair such that the second component of the binding pair binds to a cell surface of the population of antibody-secreting cells, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody; contacting the population of antibody-secreting cells with an antibody capture complex, wherein the antibody capture complex comprises a first component of the binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to the second component of the binding pair on the cell surface of the antibody-secreting cells, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; as well as The antibody-secreting cell population is contacted with an antigen, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the antigen.

30. The method of claim 29, wherein: The first component of the binding pair comprises avidin, streptavidin or avidin, and the second component of the binding pair comprises biotin; The first component of the binding pair comprises one of jun and fos, and the second component of the binding pair comprises the other of jun and fos; The first component of the binding pair comprises one of mad and max, and the second component of the binding pair comprises the other of mad and max; The first component of the binding pair comprises one of myc and max, and the second component of the binding pair comprises the other of myc and max; The first member of the binding pair comprises one of an azide and an alkyne, and the second member of the binding pair comprises the other of an azide and an alkyne; or The first component of the binding pair comprises one of an azide and a phosphine, and the second component of the binding pair comprises the other of an azide and a phosphine.

31. The method of claim 29, wherein the first component of the binding pair comprises avidin or streptavidin and the second component of the binding pair comprises biotin.

32. The method of claim 29, wherein: The second component of the binding pair comprises fluorescein isothiocyanate (FITC), and the first component of the binding pair comprises an anti-FITC antibody; The second component of the binding pair comprises phycoerythrin (PE), and the first component of the binding pair comprises an anti-PE antibody; The second component of the binding pair comprises an APC, and the first component of the binding pair comprises an anti-APC antibody; The second component of the binding pair comprises BV421, and the first component of the binding pair comprises an anti-BV421 antibody; The second component of the binding pair comprises BV510, and the first component of the binding pair comprises an anti-BV510 antibody; The second component of the binding pair comprises BV605, and the first component of the binding pair comprises an anti-BV605 antibody; The second component of the binding pair comprises BV650, and the first component of the binding pair comprises an anti-BV650 antibody; The second component of the binding pair comprises BV711, and the first component of the binding pair comprises an anti-BV711 antibody; or The second component of the binding pair comprises BV786, and the first component of the binding pair comprises an anti-BV786 antibody.

33. A method for capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting a population of antibody-secreting cells, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody, with an antibody capture complex, wherein the antibody capture complex comprises a first component of a binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to a second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; as well as The antibody-secreting cell population is contacted with an antigen, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the antigen.

34. The method of claim 33, wherein the second component of the binding pair comprises a surface marker of the antibody-secreting cell population and the first component of the binding pair is an antibody that binds to the surface marker.

35. The method of claim 34, wherein the surface marker comprises CD27, CD38, CD45, CD138, CD98, CD78, CD319, CXCR4, BCMA, GPRC5D, FCRL5, CD19, Ly6D, CD52, or transmembrane activator calcium regulator and cyclophilin ligand interactor (TACI).

36. The method of claim 33, wherein the second component of the binding pair comprises an interleukin-6 receptor (IL-6R) and the first component of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6).

37. The method of claim 33, wherein the second component of the binding pair comprises CD27 and the first component of the binding pair comprises an anti-CD27 antibody or CD70.

38. The method of any one of claims 29 to 37, wherein the antibody capture molecule comprises a capture antibody.

39. The method of claim 38, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain kappa antibody, or an anti-light chain lambda antibody.

40. The method of claim 39, wherein the anti-Fc antibody comprises an anti-Fcγ antibody, an anti-Fcα antibody, or an anti-Fcε antibody.

41. The method of claim 40, wherein the anti-Fcγ antibody comprises an anti-FcγRI antibody, an anti-FcγRIIA antibody, an anti-FcγRIIB antibody, an anti-FcγRIIB1 antibody, an anti-FcγRIIB2 antibody, an anti-FcγRIIIA antibody, or an anti-FcγRIIIB antibody.

42. The method of claim 40, wherein the anti-Fca antibody comprises an anti-FcaRI antibody.

43. The method of claim 40, wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody.

44. The method of claim 38, wherein the capture antibody comprises an anti-IgM antibody.

45. The method of claim 38, wherein the capture antibody comprises an anti-IgG antibody.

46. ​​The method of claim 45, wherein the anti-IgG antibody comprises an anti-IgG1 antibody, an anti-IgG2 antibody, an anti-IgG2a antibody, an anti-IgG2b antibody, an anti-IgG3 antibody, or an anti-IgG4 antibody.

47. The method of claim 38, wherein the capture antibody comprises an anti-IgA antibody.

48. The method of claim 47, wherein the anti-IgA antibody comprises an anti-IgA1 antibody, an anti-IgA2 antibody, an anti-secretory IgA antibody, or a polymeric anti-IgA antibody.

49. The method of claim 38, wherein the capture antibody comprises an anti-IgE antibody.

50. The method of any one of claims 29 to 37, wherein the antibody capture molecule comprises an Fc receptor or an extracellular domain of the Fc receptor.

51. The method of claim 50, wherein the Fc receptor comprises an Fcγ receptor, an Fcα receptor, or an Fcε receptor.

52. The method of claim 51, wherein the Fcγ receptor comprises an FcγRI receptor, an FcγRIIA receptor, an FcγRIIB receptor, an FcγRIIB1 receptor, an FcγRIIB2 receptor, an FcγRIIIA receptor, an FcγRIIIB receptor, or an FcRn receptor.

53. The method of claim 51, wherein the Fcα receptor comprises an FcαRI receptor or an Fcα / μR receptor.

54. The method of claim 51, wherein the Fcε receptor comprises an FcεRI receptor or an FcεRII receptor.

55. The method of any one of claims 29 to 37, wherein the antibody capture molecule comprises protein A or protein G.

56. The method of any one of claims 29 to 37, wherein the antibody capture molecule comprises protein L.

57. The method of any one of claims 29 to 56, further comprising contacting the population of antibody-secreting cells with a second anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the second anti-Ig antibody.

58. The method of claim 57, wherein the second anti-Ig antibody is anti-IgE or anti-IgG.

59. The method of claim 57 or 58, wherein the second anti-Ig antibody is detectably labeled.

60. The method of claim 59, wherein the second anti-Ig antibody is labeled with: Radioactive compounds, fluorescent compounds, or enzymes.

61. The method of claim 60, wherein the radioactive compound comprises 3 H. 14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 Tc.

62. The method of claim 60, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or fluorescent protein.

63. The method of claim 60, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase.

64. The method of any one of claims 29 to 63, wherein in the step of contacting the antibody-secreting cell population with an antigen, the antigen is a barcoded antigen.

65. The method of any one of claims 29 to 64, wherein in the step of contacting the antibody-secreting cell population with an antigen, the antibody-secreting cell population is contacted with a plurality of barcoded antigens, wherein each barcoded antigen in the plurality of barcoded antigens comprises a unique antigen linked to a unique nucleic acid molecule.

66. The method of claim 65, wherein the unique nucleic acid molecule comprises DNA.

67. The method of claim 65, wherein the unique nucleic acid molecule comprises RNA.

68. The method of any one of claims 65 to 67, wherein each unique nucleic acid molecule comprises about 10 nucleotides to about 500 nucleotides.

69. The method of any one of claims 65 to 68, wherein each barcoded antigen is detectably labeled.

70. The method of claim 69, wherein each barcoded antigen is labeled with: Radioactive compounds, fluorescent compounds, or enzymes.

71. The method of claim 70, wherein the radioactive compound comprises 3 H. 14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 Tc.

72. The method of claim 70, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or fluorescent protein.

73. The method of claim 70, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase.

74. The method of any one of claims 64 to 73, wherein the barcoded antigen comprises an allergen.

75. The method of any one of claims 29 to 74, wherein the antibody secreting cell is a plasma cell or a plasmablast.

76. The method of any one of claims 29 to 75, wherein the antibody secreting cell population is obtained by enriching a cell population obtained from a human being for immune cells.

77. The method of claim 76, wherein the cell population obtained from the human is enriched for plasma cells or plasmablasts.

78. The method of claim 77, wherein the enriched immune cell population is enriched for plasma cells or plasmablasts using pan-B cell enrichment.

79. The method of any one of claims 29 to 78, wherein the antibody secreting cell population is obtained from human lymph nodes, lungs, bone marrow or blood.

80. The method of any one of claims 29 to 79, further comprising detecting an antigen bound by the target antibody.

81. methods as described in any one in claim 29 to 80, it also includes sorting the described antibody-secreting cell group to collect antibody-secreting cell pool, wherein the described antibody-secreting cells in the pool each secrete the antibody that is captured by the described antibody capture molecule and bound by the described antigen, and wherein the collected antibody-secreting cell pool comprises the described antibody-secreting cells that secrete the described target antibody.

82. The method of claim 81, wherein the collected antibody-secreting cell pool is separated into single cells and the antibody-secreting cells secreting the target antibody are isolated.

83. The method of any one of claims 57 to 80, further comprising detecting the second anti-Ig antibody.

84. The method of claim 83, further comprising sorting the antibody-secreting cell population to collect an antibody-secreting cell pool, wherein the antibody-secreting cells in the pool each secrete an antibody that is captured by the antibody capture molecule and bound by the antigen and the second anti-Ig antibody, wherein the collected antibody-secreting cell pool contains the antibody-secreting cells that secrete the target antibody bound by the antigen and the second anti-Ig antibody.

85. The method of claim 84, further comprising separating the collected antibody-secreting cell pool into single cells and isolating the antibody-secreting cells that secrete the target antibody.

86. The method of any one of claims 29 to 85, further comprising contacting the antibody-secreting cell population with an Fc blocker prior to contacting the antibody-secreting cell population with the second component of the binding pair.

87. A method for capturing IgE antibodies secreted by antibody-secreting cells, wherein the IgE antibodies are directed against allergens, the method comprising: contacting a population of antibody-secreting cells with NHS-biotin to bind biotin to the cell surface, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the IgE antibody; contacting the antibody-secreting cell population with an antibody capture complex, wherein the antibody capture complex comprises streptavidin linked to the extracellular domain of a high affinity IgE receptor (FcεRIa), whereby the antibody capture molecule binds to the IgE antibodies secreted by the antibody-secreting cells; and The antibody-secreting cell population is contacted with an antigen, wherein the antigen is an antigenic portion or a mixture of multiple antigenic portions of the allergen, whereby the IgE antibodies captured by the antibody-capture complex bind to the antigen.

88. The method of claim 87, wherein the antigen comprises a plurality of barcoded antigens, wherein each of the plurality of barcoded antigens is a different antigenic portion of the allergen.

89. A method as described in claim 87 or 88, further comprising sorting the antibody-secreting cell population to collect an antibody-secreting cell pool, wherein the antibody-secreting cells in the pool each secrete an IgE antibody captured by the antibody capture complex and bound by the antigen, wherein the collected antibody-secreting cell pool contains the antibody-secreting cells that secrete the IgE antibody.

90. The method of claim 87 or 88, further comprising contacting the antibody-secreting cell population with an anti-IgE antibody to allow the IgE antibodies secreted by the antibody-secreting cells and captured by the antibody capture complex to bind to the anti-IgE antibody.

91. The method of claim 90, further comprising sorting the antibody-secreting cell population to collect an antibody-secreting cell pool, wherein the antibody-secreting cells in the pool each secrete an IgE antibody captured by the antibody capture complex and bound by the antigen and the anti-IgE antibody, wherein the collected antibody-secreting cell pool contains the antibody-secreting cells that secrete the IgE antibody.

92. The method of claim 89 or 91, further comprising separating the collected antibody-secreting cell pool into single cells and isolating the antibody-secreting cells that secrete the IgE antibody.

93. The method of any one of claims 29 to 92, wherein the step of contacting the antibody secreting cell population with an antigen comprises: (a) contacting the antibody-secreting cell population with a first labeled form of the antigen so that the antigen binds to antibodies including the target antibody captured on the cell surface of the antibody-secreting cell population, wherein the first labeled form of the antigen is conjugated to a first detectable label; (b) washing the antibody-secreting cell population to remove unbound antigen; (c) contacting the antibody-secreting cell population with: (i) said antigen in unlabelled form, (ii) a second labelled form of said antigen, or (iii) said unlabeled form of said antigen and said second labeled form of said antigen Original; (d) washing the antibody secreting cell population to remove unbound antigen, and (e) collecting a pool of antibody-secreting cells that remain bound to the antigen in the first labeled form, wherein the collected pool of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody, wherein the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex remains bound to the antigen in the first labeled form.

94. The method of claim 93, wherein the concentration of the antigen in the first labeled form is between 0.001 nM and 1 uM.

95. The method of claim 93, wherein the concentration of the antigen in the first labeled form is between 0.1 nM and 7.5 nM.

96. The method of any one of claims 93 to 95, wherein the first detectable label is a first fluorescent label.

97. The method of any one of claims 93 to 96, wherein the antigen is a protein in monomeric form.

98. The method of any one of claims 93 to 96, wherein the antigen is a protein in a multimeric form.

99. The method of any one of claims 93 to 96, wherein the antigen is a protein that exists in both monomeric and multimeric forms.

100. The method of any one of claims 97 to 99, wherein the first labeled form of the antigen is a monovalent form of the antigen.

101. The method of any one of claims 97 to 100, wherein the unlabeled form of the antigen is a monovalent form of the antigen.

102. The method of any one of claims 97 to 100, wherein the unlabeled form of the antigen is a multivalent form of the antigen.

103. The method of claim 102, wherein the multivalent form of the antigen is provided by a multivalent molecule to which the antigen binds.

104. The method of claim 103, wherein the multivalent molecule is selected from a streptavidin multimer such as a tetramer, a dimer of an immunoglobulin Fc fragment, or a trimer of a trimerized molecule such as a foldon.

105. The method of any one of claims 97 to 104, wherein the second labeled form of the antigen is a monovalent form of the antigen.

106. The method of any one of claims 99-104, wherein the second labeled form of the antigen is a multivalent form of the antigen.

107. The method of claim 106, wherein the multivalent form of the antigen is provided by a multivalent molecule to which the antigen is bound or linked.

108. The method of claim 107, wherein the multivalent molecule is a streptavidin multimer such as a tetramer, a dimer of an immunoglobulin Fc fragment, or a trimer of a trimerized molecule such as a foldon.

109. The method of any one of claims 106 to 108, wherein the multivalent form of the antigen is labeled with a second detectable marker.

110. The method of claim 109, wherein the second detectable label is a second fluorescent label.

111. The method of claim 100, wherein in step (c), the antibody-secreting cell population is contacted with the antigen in an unlabeled form.

112. The method of claim 111, wherein the unlabeled form of the antigen is a monovalent form of the antigen.

113. The method of claim 111 or 112, wherein the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen used in step (a) is at least 2-fold to 4-fold.

114. The method of claim 100, wherein in step (c), the antibody-secreting cell population is contacted with the antigen in the second labeled form.

115. The method of claim 114, wherein the second labeled form of the antigen is a multivalent form of the antigen.

116. The method of claim 114 or 115, wherein the molar ratio of the second labeled form of the antigen relative to the first labeled form of the antigen used in step (a) is at least 2-fold to 4-fold.

117. The method of claims 111 to 116, wherein the contacting in step (c) and the washing in step (d) are repeated at least once prior to collecting the cells in step (e).

118. The method of claim 100, wherein in step (c), the population of antibody-secreting cells is contacted with an unlabeled form of the antigen and a second labeled form of the antigen.

119. The method of claim 118, wherein the unlabeled form is a monovalent form of the antigen and the second labeled form of the antigen is a multivalent form of the antigen.

120. The method of claim 118, wherein the antigen is a monomeric protein, the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a multivalent form of the antigen.

121. The method of claim 118, wherein the antigen is a multimeric protein, the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a multivalent form of the antigen.

122. The method of claim 118, wherein the antigen is a protein that exists in both monomeric and multimeric forms, the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a multivalent form of the antigen.

123. The method of any one of claims 118 to 122, wherein the cell is contacted simultaneously with the unlabeled form of the antigen and the second labeled form of the antigen.

124. The method of any one of claims 118 to 123, wherein the molar ratio of the unlabeled form of the antigen relative to the first labeled form of the antigen used in step (a) is at least 2-fold to 4-fold.

125. The method of any one of claims 93 to 124, wherein the first detectable label is a fluorescent label, and wherein fluorescence activated cell sorting is used to collect cells that retain binding to the antigen in the first labeled form.

126. A method as described in any one of claims 111 to 124, wherein the first detectable marker is a first fluorescent marker, and the second detectable marker is a second fluorescent marker different from the first fluorescent marker, wherein two-dimensional fluorescence-activated cell sorting is performed to collect a pool of antibody-secreting cells that are still bound to the antigen in the first labeled form, and wherein the collected pool of antibody-secreting cells contains the antibody-secreting cells that secrete the target antibody that is still bound to the antigen in the first labeled form.

127. The method of any one of claims 93 to 126, wherein the target antibody has an affinity in the range of about 0.1 pM to about 25 nM (KD).

128. The method of claim 127, wherein the affinity is less than about 10 nM (KD).

129. The method of any one of claims 93 to 128, further comprising separating the pool of antibody-secreting cells collected in step (e) into single cells and isolating the antibody-secreting cells that secrete the target antibody.

130. The method of claim 82, 85, 92 or 129, further comprising isolating nucleic acid encoding the antibody from the isolated antibody-secreting cells that secrete the target antibody.

131. A method of identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by a cell comprising a modified cell surface, and wherein the target antibody is subsequently captured on the modified cell surface, the method comprising: a) providing an antibody-secreting cell comprising a modified cell surface, wherein the antibody-secreting cell secretes a target antibody, wherein the target antibody is subsequently captured on the surface of the modified cell, wherein the target antibody captured on the surface of the modified cell is bound to an antigen, wherein the antigen is linked to a barcode nucleic acid molecule, and wherein the antibody-secreting cell further comprises a nucleic acid molecule comprising the region of the gene encoding the antigen-binding fragment of the target antibody captured on the surface of the modified cell; b) hybridizing a portion of the barcode nucleic acid molecule to a portion of a first nucleic acid molecule attached to a solid surface; c) hybridizing a portion of the nucleic acid molecule encoding the region of the gene of the antigen-binding fragment of the target antibody captured on the surface of the modified cell with a portion of a second nucleic acid molecule attached to the solid surface; d) preparing a first amplicon library of gene expression in the antibody-secreting cells, a second amplicon library of variable regions, diversity regions, and joining regions (VDJ) in the antibody-secreting cells, and a third amplicon library of antigen barcode nucleic acid molecules; e) sequencing each of the three amplicon libraries; and f) identifying said region of said gene encoding said antigen-binding fragment of said target antibody captured on the surface of said modified cell.

132. The method of claim 131, wherein the portion of the barcode nucleic acid molecule is complementary to a first template switch oligonucleotide (TSO), wherein the portion of the barcode nucleic acid molecule terminates at the 3' end of the barcode nucleic acid molecule.

133. The method of claim 131 or claim 132, wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first template switch oligonucleotide (TSO).

134. The method of any one of claims 131 to 133, wherein the 5' end of the first nucleic acid molecule attached to the solid surface comprises a first sequence of three consecutive riboguanosine residues.

135. The method of any one of claims 131 to 134, wherein the first nucleic acid molecule attached to the solid surface further comprises a first unique molecular identifier (UMI).

136. The method of any one of claims 131 to 135, wherein the first nucleic acid molecule attached to the solid surface further comprises a first surface barcode.

137. The method of any one of claims 131 to 136, wherein the first nucleic acid molecule attached to the solid surface further comprises a first sequencing primer.

138. The method of any one of claims 131 to 137, wherein the first nucleic acid molecule attached to the solid surface comprises, from 5' to 3', the first sequence of three riboguanosine residues, the first TSO, the first UMI, the first surface barcode, and the first sequencing primer.

139. The method of any one of claims 131 to 138, wherein the first nucleic acid molecule attached to the solid surface is attached via the 3' end of the first nucleic acid molecule attached to the solid surface.

140. The method of any one of claims 131 to 139, wherein the first nucleic acid molecule attached to the solid surface comprises a first DNA molecule attached to the solid surface.

141. The method of claim 140, wherein the first DNA molecule attached to the solid surface comprises a first single-stranded DNA molecule attached to the solid surface.

142. A method as claimed in claim 141, wherein the portion of the first single-stranded DNA molecule attached to the solid surface comprises a first TSO, wherein the portion of the barcode nucleic acid molecule hybridized to the first single-stranded DNA molecule is complementary to the first TSO, and wherein the method further comprises reverse transcribing the first single-stranded DNA molecule attached to the solid surface starting from the 3' end of the portion of the barcode nucleic acid molecule complementary to the first TSO.

143. The method of any one of claims 131 to 142, wherein the barcode nucleic acid molecule is a single-stranded DNA barcode nucleic acid molecule, and wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first TSO, and the method further comprises reverse transcribing the single-stranded DNA barcode nucleic acid molecule starting from the 5' end of the first TSO.

144. The method of any one of claims 131 to 143, wherein the 3' end of the portion of the barcode nucleic acid molecule comprises three consecutive cytidine or ribocytidine residues.

145. A method as described in claim 144, further comprising reverse transcribing an mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface, thereby producing a single-stranded cDNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface, wherein the nucleotide sequence of the single-stranded cDNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface differs from the DNA complementary sequence of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface in that the 3' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface contains three additional, consecutive cytidine or ribocytidine residues.

146. The method of claim 145, wherein the 3' end of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface comprises a plurality of consecutive adenine residues.

147. The method of claim 145 or claim 146, wherein the 5' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface comprises a plurality of consecutive thymine residues.

148. The method of any one of claims 131 to 147, wherein the 5' end of the second nucleic acid molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues.

149. The method of any one of claims 131 to 148, wherein the second nucleic acid molecule attached to the solid surface further comprises a second UMI.

150. The method of any one of claims 131 to 149, wherein the second nucleic acid molecule attached to the solid surface further comprises a second surface barcode, wherein the nucleotide sequences of the first surface barcode nucleic acid molecule and the second surface barcode nucleic acid molecule are identical.

151. The method of any one of claims 131 to 150, wherein the second nucleic acid molecule attached to the solid surface further comprises a second sequencing primer.

152. The method of any one of claims 131 to 151, wherein the second nucleic acid molecule attached to the solid surface comprises from 5' to 3' the second sequence of three riboguanosine residues, the second TSO, the second UMI, the second surface barcode, and the second sequencing primer, wherein the nucleotide sequences of the first surface barcode nucleic acid molecule and the second surface barcode nucleic acid molecule are identical.

153. The method of any one of claims 131 to 152, wherein the second nucleic acid molecule attached to the solid surface is attached via the 3' end of the second nucleic acid molecule attached to the solid surface.

154. The method of any one of claims 131 to 153, wherein the second nucleic acid molecule attached to the solid surface comprises a second DNA molecule attached to the solid surface.

155. The method of claim 154, wherein the second DNA molecule attached to the solid surface comprises a second single-stranded DNA molecule attached to the solid surface.

156. A method as claimed in claim 155, wherein the 5' end of the portion of the second single-stranded DNA molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues, and wherein the 3' end of the portion of the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface comprises three consecutive cytidine or ribocytidine residues, and wherein the method further comprises reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting from the 3' end of the three consecutive cytidine or ribocytidine residues.

157. The method of any one of claims 131 to 156, wherein the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface comprises a second single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface.

158. A method as claimed in claim 156, wherein the 3' end of the portion of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface contains three consecutive cytidine or ribocytidine residues, and wherein the 5' end of the portion of the second single-stranded DNA molecule attached to the solid surface contains a second sequence of three consecutive riboguanosine residues, wherein the method further comprises reverse transcribing the second single-stranded DNA molecule attached to the solid surface from the 3' end of the portion of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface.

159. The method of any one of claims 131 to 158, wherein the antibody-secreting cells are disposed within a separator having the solid surface.

160. The method of any one of claims 131 to 159, wherein the solid surface comprises beads.

161. The method of claim 159 or 160, further comprising lysing the antibody-secreting cells within the compartment.

162. The method of any one of claims 131 to 161, wherein the barcoded nucleic acid molecule further comprises a third sequencing primer, wherein the third sequencing primer begins at the 5' end of the barcoded nucleic acid molecule.

163. methods as described in any one in claim 131 to 162, each amplicon of wherein said first amplicon library comprises a gene, and wherein each amplicon does not comprise a nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface.

164. The method of claim 163, further comprising performing sample demultiplexing, alignment, filtering, and UMI counting on the first amplicon library.

165. The method of any one of claims 131 to 164, wherein sequencing of the first amplicon library comprises next generation sequencing.

166. The method of any one of claims 131 to 165, further comprising mapping each amplicon of the first amplicon library to a standard reference genome and aligning them.

167. The method of claim 166, wherein the standard reference genome comprises a human standard reference genome.

168. The method of claim 167, wherein the human standard reference genome is GRCh38.

169. The method of claim 166, wherein the standard reference genome comprises a mouse standard reference genome.

170. The method of claim 169, wherein the mouse standard reference genome is mm10.

171. The method of any one of claims 166 to 170, wherein the mapping and aligning comprises mapping each amplicon of the first amplicon library to the standard reference genome with a splicing-aware aligner (STAR), wherein each amplicon of the first amplicon library comprises a unique molecular identifier, wherein all amplicons of the first amplicon library mapped to annotated genes of the standard reference genome are classified, and wherein the classified amplicons are counted for each unique molecular identifier (UMI), thereby generating a single cell count matrix comprising mapped counts for each annotated gene and counts for each UMI.

172. The method of claim 171, further comprising calculating the total number of annotated genes divided by the log of the total number of UMIs counted. 10 The single cell count matrices for high-quality cells and broadly distributed genes were filtered using a ratio of , wherein cells with a ratio below about 0.1 were removed, wherein cells with an interquartile range of more than about four times the total number of UMIs counted were removed, and cells with more than about 80% of reads mapped to mitochondrial genes were removed.

173. The method of claim 172, further comprising normalizing the single cell count matrix so that a total number of UMIs counted is approximately 10,000.

174. The method of claim 172 or 173, further comprising computing a PCA embedding of the single cell count matrix and iteratively clustering major cell types to identify similarities between batches, thereby generating a unified manifold approximation and projection (UMAP).

175. The method of any one of claims 172 to 174, further comprising determining a linear adjustment function for each unit using the cluster-specific centroids, and applying the linear adjustment function for each unit to correct for differences in the batch, thereby generating a batch-corrected embedding, and using the batch-corrected embedding to generate a two-dimensional unified manifold approximation and projection (UMAP).

176. A method as described in claim 174 or 175, further comprising determining a weighted k-nearest neighbor graph and applying a Leiden algorithm to the weighted k-nearest neighbor graph, wherein the weighted k-nearest neighbor graph has resolution values ​​of approximately 0.1, approximately 0.2, approximately 0.5 and approximately 1.0 to determine cell type clusters in an unsupervised manner.

177. The method of claim 176, further comprising performing a paired Wilcox test on all cells in a cluster and comparing the results of the first paired Wilcox test, and performing another paired Wilcox test on all cells in every other cluster to quantify the p-value and fold change of each gene in each cluster, wherein when a gene has a low p-value and a high fold change, the cell is labeled with a common marker of antibody-secreting cells, and the cell is labeled with a subtype of the cluster named by the top gene from the differential expression results.

178. The method of any one of claims 131 to 177, further comprising performing sample demultiplexing, reassembling read pairs into contigs, and aligning and annotating the contigs against a germline segment V(D)J reference sequence of a second amplicon family.

179. The method of claim 178, wherein the alignment of the contigs to a germline segment V(D)J reference sequence comprises alignment relative to a human germline reference database or a mouse germline reference database.

180. The method of claim 179, wherein the human germline reference database comprises the IMGT database of human germline immunoglobulin sequences.

181. The method of claim 179, wherein the mouse germline reference database comprises the IMGT germline reference database.

182. The method of any one of claims 179 to 181, further comprising comparing the VDJ sequence against sequences in the human germline reference database or the mouse germline reference database, thereby marking the VDJ sequence for quality alignment by checking in-frame alignment, length of CDR3, and absence of stop codons in the variable region sequence and filtering the VDJ sequence based on the markings.

183. The method of claim 182, further comprising mapping the VDJ sequence to a reference database of immunoglobulin chains.

184. The method of claim 179, wherein the accuracy of the immunoglobulin isotypes, variable region maps, joining region maps, diversity region maps, and full-length variable regions of the heavy and light chain sequences of each cell are confirmed by the alignment.

185. The method of any one of claims 131 to 184, further comprising sample demultiplexing, aligning, filtering, and UMI counting of the third amplicon library.

186. The method of any one of claims 131 to 185, further comprising mapping the third amplicon library to a custom short read reference comprising the barcode nucleic acid molecule reference associated with each antigen, wherein counts for each uniquely mapped barcode nucleic acid molecule are summed for each cell in the barcoded antigen single cell matrix.

187. The method of claim 186, further comprising quantifying the barcode nucleic acid molecules in all cells and normalizing the quantification by taking the center log ratio of each barcode nucleic acid molecule in the plurality of barcode nucleic acid molecules in each sample capture.

188. The method of claim 187, further comprising removing background antigen signals by performing background denoising and scaling (DSB) on each of the plurality of barcoded nucleic acid molecules.

189. A method as described in claim 188, further comprising determining an antigen signal distribution, wherein when a well-separated bimodal distribution exists in the antigen signal distribution, a z-transform value is used to calculate the antigen specificity, and wherein when a well-separated bimodal distribution does not exist in the antigen signal distribution, a quantile value is used.

190. The method of claim 189, further comprising performing statistical modeling on the target antigen and negative control antigen distributions.

191. The method of any one of claims 131 to 190, wherein the first amplicon library, the second amplicon library, and the third amplicon library are analyzed simultaneously to obtain at least one candidate sequence of an antigen-positive target antibody captured on the modified cell surface.

192. The method of claim 191, further comprising grouping antibody secreting cells using an analysis of the first amplicon library and an analysis of the second amplicon library using an effective antibody constant region.

193. The method of claim 192, wherein the antibody constant region comprises an IgE constant region.

194. The method of claim 192 or 193, wherein the grouped antibody secreting cells comprise detectable levels of IgE heavy chain and CD79a, but lack detectable levels of Ms4a1 and CD19, wherein the grouped antibody secreting cells are IgE + Antibody-secreting cells.

195. The method of claim 194, further comprising evaluating the IgE + Differential expression of antibody secreting cells relative to IgA secreting cell isotypes, IgG secreting cell isotypes, and / or IgM secreting cell isotypes.

196. The method of claim 195, wherein the assessment of differential expression further characterizes the IgE + Distinct transcriptional signatures of antibody-secreting cells.

197. The method of claim 196, further comprising evaluating the IgE + Antigen specificity of antibody-secreting cells.

198. The method of claim 197, wherein the assessment comprises treating the IgE + The antigen specificity of the antibody secreting cells is compared relative to a plurality of antigens and a control antigen, and wherein the plurality of antigens comprises the target antigen.

199. The method of claim 198, wherein the comparing comprises calculating an empirical score for each of the plurality of antigens and the control antigen by subtracting the quantile value of the signal associated with the antigen (qT) from the quantile value of the signal associated with the control antigen (qC) by a penalty factor (x) to determine an antigen-specific score, wherein the antigen-specific score = qT - qC x .

200. The method of claim 199, wherein the penalty factor is the same for all antigens.

201. The method of claim 199, wherein the penalty factor is different for different antibody isotypes.

202. The method of claim 199, wherein the penalty factor is different when other antigens are used.

203. The method of claim 199, further comprising determining the antigen-specific score for each of the plurality of antigens and the control antigen.

204. The method of claim 203, further comprising calculating a control antigen-specific score, wherein the cell is selected when the antigen-specific score is high and the control antigen-specific score is low.

205. The method of claim 204, wherein the VDJ region identifies an antigen-specific antibody.

206. The method of claim 204, further comprising using a paired V H :V L Antibody sequences select antibody candidates from a hierarchical list of antigenic signals.

207. The method of any one of claims 191 to 206, further comprising determining differential expression between isotypes, conditions, and / or antigen specificities, wherein determining differential expression between isotypes comprises performing a paired Wilcox test on all cells in one isotype and performing a paired Wilcox test on all cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster; and wherein determining differential expression between antigen specificities comprises performing a paired Wilcox test on all cells in one isotype and performing a paired Wilcox test on all cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster.

208. The method of any one of claims 191 to 207, further comprising clustering VDJ sequence similarities in antigen-positive target cells, wherein clustering VDJ sequence similarities comprises grouping similar variable regions by amino acid sequence based on sequence alignment.

209. The method of any one of claims 191 to 208, wherein the antigen-positive target cells are IgE + .

210. The method of any one of claims 191 to 209, further comprising trimming redundant sequences.

211. A method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with a second component of a binding pair, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody, whereby the second component of the binding pair binds to the cell surface of the population of antibody-secreting cells; b) contacting the antibody-secreting cell population with an antibody capture complex, wherein the antibody capture complex comprises a first component of the binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to the second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; c) contacting the antibody-secreting cell population with a second anti-Ig antibody so that the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the second anti-Ig antibody; d) contacting the antibody-secreting cell population with an antigen comprising a barcode nucleic acid molecule so that the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the antigen; e) collecting a pool of antibody-secreting cells, wherein each cell in the pool secretes an antibody captured by the antibody-capture complex and bound by the second anti-Ig antibody and the antigen comprising the barcode nucleic acid molecule, and wherein the pool of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody; f) separating the antibody-secreting cell pool into single antibody-secreting cells, and for each single antibody-secreting cell: 1) hybridizing a portion of the barcode nucleic acid molecule to a portion of a first nucleic acid molecule attached to a solid surface, wherein the solid surface and the first nucleic acid molecule attached thereto are unique to each single antibody-secreting cell; 2) hybridizing a portion of the nucleic acid molecule comprising the region of the gene encoding the antigen-binding fragment of the target antibody with a portion of a second nucleic acid molecule attached to the solid surface; 3) preparing a first amplicon library of gene expression, a second amplicon library of variable region, diversity region and joining region (VDJ), and a third amplicon library of antigen barcode nucleic acid molecules; and 4) sequencing each of the three amplicon libraries; and g) identifying said region of said gene encoding said antigen-binding fragment of said target antibody.

212. The method of claim 211, wherein: The first component of the binding pair comprises avidin, streptavidin or avidin, and the second component of the binding pair comprises biotin; The first component of the binding pair comprises one of jun and fos, and the second component of the binding pair comprises the other of jun and fos; The first component of the binding pair comprises one of mad and max, and the second component of the binding pair comprises the other of mad and max; The first component of the binding pair comprises one of myc and max, and the second component of the binding pair comprises the other of myc and max; The first member of the binding pair comprises one of an azide and an alkyne, and the second member of the binding pair comprises the other of an azide and an alkyne; or The first component of the binding pair comprises one of an azide and a phosphine, and the second component of the binding pair comprises the other of an azide and a phosphine.

213. The method of claim 211, wherein the first component of the binding pair comprises avidin or streptavidin and the second component of the binding pair comprises biotin.

214. The method of claim 211, wherein: The second component of the binding pair comprises fluorescein isothiocyanate (FITC), and the first component of the binding pair comprises an anti-FITC antibody; The second component of the binding pair comprises phycoerythrin (PE), and the first component of the binding pair comprises an anti-PE antibody; The second component of the binding pair comprises an APC, and the first component of the binding pair comprises an anti-APC antibody; The second component of the binding pair comprises BV421, and the first component of the binding pair comprises an anti-BV421 antibody; The second component of the binding pair comprises BV510, and the first component of the binding pair comprises an anti-BV510 antibody; The second component of the binding pair comprises BV605, and the first component of the binding pair comprises an anti-BV605 antibody; The second component of the binding pair comprises BV650, and the first component of the binding pair comprises an anti-BV650 antibody; The second component of the binding pair comprises BV711, and the first component of the binding pair comprises an anti-BV711 antibody; or The second component of the binding pair comprises BV786, and the first component of the binding pair comprises an anti-BV786 antibody.

215. A method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, wherein the target antibody is secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells, wherein the population of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody, with an antibody capture complex, wherein the antibody capture complex comprises a first component of a binding pair linked to an antibody capture molecule, whereby the first component of the binding pair binds to a second component of the binding pair on the cell surface of the antibody-secreting cell population, and whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; b) contacting the antibody-secreting cell population with a second anti-Ig antibody so that the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the second anti-Ig antibody; c) contacting the antibody-secreting cell population with an antigen comprising a barcode nucleic acid molecule, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody-capture complex binds to the antigen; d) collecting a pool of antibody-secreting cells, wherein each cell in the pool secretes an antibody captured by the antibody-capture complex and bound by the second anti-Ig antibody and the antigen comprising the barcode nucleic acid molecule, and wherein the pool of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody; e) separating the antibody-secreting cell pool into single antibody-secreting cells, and for each single antibody-secreting cell: 1) hybridizing a portion of the barcode nucleic acid molecule to a portion of a first nucleic acid molecule attached to a solid surface, wherein the solid surface and the first nucleic acid molecule attached thereto are unique to each single antibody-secreting cell; 2) hybridizing a portion of the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody with a portion of a second nucleic acid molecule attached to the solid surface; 3) preparing a first amplicon library of gene expression in the antibody-secreting cells, a second amplicon library of variable regions, diversity regions, and joining regions (VDJ) in the antibody-secreting cells, and a third amplicon library of antigen barcode nucleic acid molecules; and 4) sequencing each of the three amplicon libraries; and f) identifying said region of said gene encoding said antigen-binding fragment of said target antibody.

216. The method of claim 215, wherein the second component of the binding pair comprises a surface marker of the antibody-secreting cell and the first component of the binding pair is an antibody that binds to the surface marker.

217. The method of claim 216, wherein the surface markers comprise CD27, CD38, CD45, CD138, CD98, CD78, CD319, CXCR4, BCMA, GPRC5D, FCRL5, CD19, Ly6D, CD52, or transmembrane activator calcium regulator and cyclophilin ligand interactor (TACI).

218. The method of claim 215, wherein the second component of the binding pair comprises an interleukin-6 receptor (IL-6R) and the first component of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6).

219. The method of claim 215, wherein the second component of the binding pair comprises CD27 and the first component of the binding pair comprises an anti-CD27 antibody or CD70.

220. The method of any one of claims 211 to 219, wherein the antibody capture molecule comprises a capture antibody.

221. The method of claim 220, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain κ antibody, or an anti-light chain λ antibody.

222. The method of claim 221, wherein the anti-Fc antibody comprises an anti-Fcγ antibody, an anti-Fcα antibody, or an anti-Fcε antibody.

223. The method of claim 222, wherein the anti-Fcγ antibody comprises an anti-FcγRI antibody, an anti-FcγRIIA antibody, an anti-FcγRIIB antibody, an anti-FcγRIIB1 antibody, an anti-FcγRIIB2 antibody, an anti-FcγRIIIA antibody, or an anti-FcγRIIIB antibody.

224. The method of claim 222, wherein the anti-Fca antibody comprises an anti-FcaRI antibody.

225. The method of claim 222, wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody.

226. The method of claim 220, wherein the capture antibody comprises an anti-IgM antibody.

227. The method of claim 220, wherein the capture antibody comprises an anti-IgG antibody.

228. The method of claim 227, wherein the anti-IgG antibody comprises an anti-IgG1 antibody, an anti-IgG2 antibody, an anti-IgG2a antibody, an anti-IgG2b antibody, an anti-IgG3 antibody, or an anti-IgG4 antibody.

229. The method of claim 220, wherein the capture antibody comprises an anti-IgA antibody.

230. The method of claim 229, wherein the anti-IgA antibody comprises an anti-IgA1 antibody, an anti-IgA2 antibody, an anti-secretory IgA antibody, or a polymeric anti-IgA antibody.

231. The method of claim 220, wherein the capture antibody comprises an anti-IgE antibody.

232. The method of any one of claims 211 to 219, wherein the antibody capture molecule comprises an Fc receptor or an extracellular domain of the Fc receptor.

233. The method of claim 232, wherein the Fc receptor comprises an Fcγ receptor, an Fcα receptor, or an Fcε receptor.

234. The method of claim 233, wherein the Fcγ receptor comprises an FcγRI receptor, an FcγRIIA receptor, an FcγRIIB receptor, an FcγRIIB1 receptor, an FcγRIIB2 receptor, an FcγRIIIA receptor, an FcγRIIIB receptor, or an FcRn receptor.

235. The method of claim 233, wherein the Fcα receptor comprises an FcαRI receptor or an Fcα / μR receptor.

236. The method of claim 233, wherein the Fcε receptor comprises an FcεRI receptor or an FcεRII receptor.

237. The method of any one of claims 211 to 219, wherein the antibody capture molecule comprises protein A or protein G.

238. The method of any one of claims 211 to 219, wherein the antibody capture molecule comprises protein L.

239. The method of any one of claims 211 to 238, wherein the second anti-Ig antibody is anti-IgE or anti-IgG.

240. The method of any one of claims 211 to 239, wherein the second anti-Ig antibody is detectably labeled.

241. The method of claim 240, wherein the second anti-Ig antibody is labeled with a radioactive compound, a fluorescent compound, or an enzyme.

242. The method of claim 241, wherein the radioactive compound comprises 3 H. 14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 Tc.

243. The method of claim 241, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or fluorescent protein.

244. The method of claim 241, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase.

245. The method of any one of claims 211 to 244, wherein the antibody-secreting cells are contacted with a plurality of barcoded antigens, wherein each barcoded antigen in the plurality of barcoded antigens comprises a unique antigen linked to a unique nucleic acid molecule.

246. The method of claim 245, wherein the unique nucleic acid molecule comprises DNA.

247. The method of claim 245, wherein the unique nucleic acid molecule comprises RNA.

248. The method of any one of claims 245 to 247, wherein each unique nucleic acid molecule comprises about 10 nucleotides to about 500 nucleotides.

249. The method of any one of claims 245 to 248, wherein each barcoded antigen is detectably labeled.

250. The method of claim 249, wherein each barcoded antigen is labeled with: Radioactive compounds, fluorescent compounds, or enzymes.

251. The method of claim 250, wherein the radioactive compound comprises 3 H. 14 C. 19 F. 35 S. 125 I. 131 I. 111 In or 99 Tc.

252. The method of claim 250, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or fluorescent protein.

253. The method of claim 250, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase.

254. The method of any one of claims 211 to 253, wherein the barcoded antigen comprises an allergen.

255. The method of any one of claims 211 to 254, wherein the antibody-secreting cell is a plasma cell or a plasmablast.

256. The method of any one of claims 211 to 255, further comprising enriching a cell population obtained from humans for immune cells prior to contacting the surface of the antibody-secreting cell population with the second component of a binding pair, or prior to contacting the surface of the antibody-secreting cell population with the antibody capture complex.

257. The method of claim 256, wherein the cell population obtained from the human is enriched for plasma cells or plasmablasts.

258. The method of claim 257, wherein pan-B cell enrichment is used to enrich the enriched immune cell population for plasma cells or plasmablasts.

259. The method of any one of claims 211 to 258, wherein the antibody-secreting cell population is obtained from human lymph nodes, lungs, bone marrow or blood.

260. The method of any one of claims 211 to 259, further comprising detecting one or more of the second anti-Ig antibody and / or the barcoded antigen.

261. The method of any one of claims 211 to 260, further comprising sorting the antibody-secreting cell population to obtain a pool of antibody-secreting cells bound by a second anti-Ig antibody and / or one or more of the barcoded antigens.

262. The method of any one of claims 211 to 261, further comprising contacting the antibody-secreting cell population with an Fc blocker prior to contacting the antibody-secreting cell population with the second component of the binding pair.

263. The method of any one of claims 211 to 262, wherein the portion of the barcode nucleic acid molecule is complementary to a first template switch oligonucleotide (TSO).

264. The method of any one of claims 211 to 263, wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first template switch oligonucleotide (TSO).

265. The method of any one of claims 211 to 264, wherein the 5' end of the first nucleic acid molecule attached to the solid surface comprises a first sequence of three consecutive riboguanosine residues.

266. The method of any one of claims 211 to 265, wherein the first nucleic acid molecule attached to the solid surface further comprises a first unique molecular identifier (UMI).

267. The method of any one of claims 211 to 266, wherein the first nucleic acid molecule attached to the solid surface further comprises a first surface barcode.

268. The method of any one of claims 211 to 267, wherein the first nucleic acid molecule attached to the solid surface further comprises a first sequencing primer.

269. The method of any one of claims 211 to 268, wherein the first nucleic acid molecule attached to the solid surface comprises, from 5' to 3', the first sequence of three riboguanosine residues, the first TSO, the first UMI, the first surface barcode, and the first sequencing primer.

270. The method of any one of claims 211 to 269, wherein the first nucleic acid molecule attached to the solid surface is attached via the 3' end of the first nucleic acid molecule attached to the solid surface.

271. The method of any one of claims 211 to 270, wherein the first nucleic acid molecule attached to the solid surface comprises a first DNA molecule attached to the solid surface.

272. The method of claim 271, wherein the first DNA molecule attached to the solid surface comprises a first single-stranded DNA molecule attached to the solid surface.

273. A method as claimed in claim 272, wherein the portion of the first single-stranded DNA molecule attached to the solid surface comprises a first TSO, and the method further comprises reverse transcribing the first single-stranded DNA molecule attached to the solid surface starting from the 3' end of the portion of the barcode nucleic acid molecule that is complementary to the first TSO.

274. The method of any one of claims 211 to 273, wherein the barcode nucleic acid molecule is a single-stranded DNA barcode nucleic acid molecule, and wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first TSO, and the method further comprises reverse transcribing the single-stranded DNA barcode nucleic acid molecule from the 3' end of the first TSO.

275. The method of any one of claims 211 to 274, wherein the 3' end of the portion of the barcode nucleic acid molecule comprises three consecutive cytidine residues.

276. A method as described in claim 275, which also includes reverse transcribing an mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody, thereby producing a single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody, and wherein the nucleotide sequence of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody differs from the complementary sequence of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody in that the 3' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody contains three additional consecutive cytidine residues.

277. The method of claim 276, wherein the 3' end of the mRNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody comprises a plurality of consecutive adenine residues.

278. The method of claim 276 or claim 277, wherein the 5' end of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody comprises a plurality of consecutive thymine residues.

279. The method of any one of claims 211 to 278, wherein the 5' end of the second nucleic acid molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues.

280. The method of any one of claims 211 to 279, wherein the second nucleic acid molecule attached to the solid surface further comprises a second UMI.

281. The method of any one of claims 211 to 280, wherein the second nucleic acid molecule attached to the solid surface further comprises a second surface barcode.

282. The method of any one of claims 211 to 281, wherein the second nucleic acid molecule attached to the solid surface further comprises a second sequencing primer.

283. The method of any one of claims 211 to 282, wherein the second nucleic acid molecule attached to the solid surface comprises, from 5' to 3', the second sequence of three riboguanosine residues, the second TSO, the second UMI, the second surface barcode, and the second sequencing primer.

284. The method of any one of claims 211 to 283, wherein the second nucleic acid molecule attached to the solid surface is attached via the 3' end of the second nucleic acid molecule attached to the solid surface.

285. The method of any one of claims 211 to 284, wherein the second nucleic acid molecule attached to the solid surface comprises a second DNA molecule attached to the solid surface.

286. The method of claim 285, wherein the second DNA molecule attached to the solid surface comprises a second single-stranded DNA molecule attached to the solid surface.

287. A method as claimed in claim 286, wherein the 5' end of the portion of the second single-stranded DNA molecule attached to the solid surface contains a second sequence of three consecutive riboguanosine residues, and wherein the 3' end of the portion of the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody contains three consecutive cytidine residues, and wherein the method further comprises reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting from the 3' end of the three consecutive cytidine residues.

288. The method of any one of claims 211 to 287, wherein the nucleic acid molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody comprises a second single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody.

289. A method as described in claim 287, wherein the 3' end of the portion of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody contains three consecutive cytidine residues, and wherein the 5' end of the portion of the second single-stranded DNA molecule attached to the solid surface contains a second sequence of three consecutive riboguanosine residues, and wherein the method further comprises reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting from the 3' end of the portion of the single-stranded DNA molecule encoding the region of the gene encoding the antigen-binding fragment of the target antibody.

290. The method of any one of claims 211 to 289, wherein the individual antibody-secreting cells are each disposed within a separator having a solid surface.

291. The method of any one of claims 211 to 290, wherein the solid surface comprises beads.

292. The method of claim 290 or claim 291, further comprising lysing the single antibody-secreting cell within the compartment.

293. The method of any one of claims 211 to 292, wherein the barcoded nucleic acid molecule further comprises a third sequencing primer.

294. The method of any one of claims 211 to 293, wherein each amplicon of the first amplicon library comprises a gene, wherein each amplicon does not comprise a nucleic acid molecule encoding the antigen-binding fragment of the target antibody comprising the region of the gene.

295. The method of claim 294, further comprising performing sample demultiplexing, alignment, filtering, and UMI counting on the first amplicon library.

296. The method of any one of claims 211 to 295, wherein sequencing of the first amplicon library comprises next generation sequencing.

297. The method of any one of claims 211 to 296, further comprising mapping each amplicon of the third amplicon library to a standard reference genome and aligning it.

298. The method of claim 297, wherein the standard reference genome comprises a human standard reference genome.

299. The method of claim 298, wherein the human standard reference genome is GRCh38.

300. The method of claim 297, wherein the standard reference genome comprises a mouse standard reference genome.

301. The method of claim 300, wherein the mouse standard reference genome is mm10.

302. The method of any one of claims 297 to 301, wherein the mapping and aligning comprises mapping each amplicon of the first amplicon library to the standard reference genome with a splicing-aware aligner (STAR), wherein each amplicon of the first amplicon library comprises a unique molecular identifier, wherein all amplicons of the first amplicon library mapped to annotated genes of the standard reference genome are classified, and wherein the classified amplicons are counted for each unique molecular identifier (UMI), thereby generating a single cell count matrix comprising mapped counts for each annotated gene and counts for each UMI.

303. The method of claim 302, further comprising calculating the total number of annotated genes divided by the log of the total number of UMIs counted. 10 The single cell count matrices of high-quality single antibody-secreting cells and widely distributed genes were filtered using a ratio of , wherein single antibody-secreting cells with a ratio lower than about 0.1 were filtered out, wherein single antibody-secreting cells with an interquartile range of more than about four times the total number of UMIs counted were filtered out, and single antibody-secreting cells with more than about 80% of the reads mapped to mitochondrial genes were filtered out.

304. The method of claim 303, further comprising normalizing the single cell count matrix so that a total number of UMIs counted is approximately 10,000.

305. The method of claim 303 or claim 304, further comprising computing a PCA embedding of the single cell count matrix, iteratively clustering major cell types to identify similarities between batches, thereby generating a unified manifold approximation and projection (UMAP).

306. The method of any one of claims 303 to 305, further comprising determining a linear adjustment function for each unit using cluster-specific centroids, applying the linear adjustment function for each unit to correct for differences in a batch, thereby generating a batch-corrected embedding, and using the batch-corrected embedding to generate a two-dimensional unified manifold approximation and projection (UMAP).

307. The method of claim 305 or claim 306, further comprising determining a weighted k-nearest neighbor graph and applying a Leiden algorithm to the weighted k-nearest neighbor graph having resolution values ​​of 0.1, 0.2, 0.5, and 1.0 to determine cell type clusters in an unsupervised manner.

308. The method of claim 305, further comprising performing a paired Wilcox test on all single antibody-secreting cells in a cluster and comparing the results of the first paired Wilcox test, and performing another paired Wilcox test on all single antibody-secreting cells in every other cluster to quantify the p-value and fold change of each gene in each cluster, wherein when a gene has a low p-value and a high fold change, the single antibody-secreting cells are marked by a common marker of plasma cells, plasmablasts, or B cells, and the single antibody-secreting cells are marked by a subtype of the cluster named by the top gene from the differential expression results.

309. The method of any one of claims 211 to 308, further comprising performing sample demultiplexing, reassembling read pairs into contigs, and aligning and annotating the contigs against a germline segment V(D)J reference sequence of a second amplicon family.

310. The method of claim 309, wherein alignment of the contigs to a germline segment V(D)J reference sequence comprises alignment relative to a human germline reference database or a mouse germline reference database.

311. The method of claim 310, wherein the human germline reference database comprises the IMGT database of human germline immunoglobulin sequences.

312. The method of claim 310, wherein the mouse germline reference database comprises the IMGT germline reference database.

313. The method of any one of claims 310 to 312, further comprising comparing the VDJ sequence against sequences in the human germline reference database or the mouse germline reference database, thereby marking the VDJ sequence for quality alignment by checking in-frame alignment, length of CDR3, and absence of stop codons in the variable region sequence and filtering the VDJ sequence based on the markings.

314. The method of claim 313, further comprising mapping the VDJ sequence to a reference database of immunoglobulin chains.

315. The method of claim 310, wherein the accuracy of the immunoglobulin isotypes, variable region maps, joining region maps, diversity region maps, and full-length variable regions of the heavy and light chain sequences of each cell are confirmed by the alignment.

316. The method of any one of claims 211 to 315, further comprising performing sample demultiplexing, alignment, filtering, and UMI counting on the third amplicon library.

317. The method of any one of claims 211 to 316, further comprising mapping the third amplicon library to a custom short read reference comprising the barcode nucleic acid molecule reference associated with each antigen, wherein the counts of each uniquely mapped barcode nucleic acid molecule are summed for each cell in the barcoded antigen single cell matrix.

318. The method of claim 317, further comprising quantifying the barcode nucleic acid molecules in all individual antibody-secreting cells and normalizing the quantification by taking the central logarithmic ratio of each barcode nucleic acid molecule in the multiple barcode nucleic acid molecules in each sample capture.

319. The method of claim 318, further comprising removing background antigen signals by performing background denoising and scaling (DSB) on each of the plurality of barcoded nucleic acid molecules.

320. A method as described in claim 319, further comprising determining an antigen signal distribution, wherein when a well-separated bimodal distribution exists in the antigen signal distribution, a z-transform value is used to calculate the antigen specificity, and wherein when a well-separated bimodal distribution does not exist in the antigen signal distribution, a quantile value is used.

321. The method of claim 320, further comprising performing statistical modeling of target antigen and negative control antigen distributions.

322. The method of any one of claims 211 to 321, wherein the first amplicon library, the second amplicon library, and the third amplicon library are analyzed simultaneously to obtain at least one candidate sequence of an antigen-positive target antibody.

323. The method of claim 322, further comprising grouping plasma cells or plasmablasts using an analysis of the first amplicon library and an analysis of the second amplicon library using an effective antibody constant region.

324. The method of claim 323, wherein the antibody constant region comprises an IgE constant region.

325. The method of claim 323 or claim 324, wherein the grouped plasma cells or plasmablasts comprise detectable levels of IgE heavy chain and CD79a, but lack detectable levels of Ms4a1 and CD19, wherein the grouped plasma cells or plasmablasts are IgE + Plasma cells or plasmablasts.

326. The method of claim 325, further comprising evaluating the IgE + Differential expression of plasma cells or plasmablasts relative to IgA secreting plasma cells or plasmablast isotypes, IgG secreting plasma cells or plasmablast isotypes, and / or IgM secreting plasma cells or plasmablast isotypes.

327. The method of claim 326, wherein the assessment of differential expression further characterizes the IgE + Distinct transcriptional signature of plasma cells or plasmablasts.

328. The method of claim 327, further comprising evaluating the IgE + Antigen specificity of plasma cells or plasmablasts.

329. The method of claim 328, wherein the assessment comprises treating the IgE + The antigen specificity of plasma cells or plasmablasts relative to a plurality of antigens and a control antigen is compared, and wherein the plurality of antigens comprises the target antigen.

330. The method of claim 329, wherein the comparing comprises calculating an empirical score for each of the plurality of antigens and the control antigen by subtracting the quantile value of the signal associated with the antigen (qT) from the quantile value of the signal associated with the control antigen (qC) by a penalty factor (x) to determine an antigen-specific score, wherein the antigen-specific score = qT - qC x .

331. A method as in claim 330, wherein the penalty factor is the same for all antigens.

332. The method of claim 330, wherein the penalty factor is different for different antibody isotypes.

333. The method of claim 330, wherein the penalty factor is different when other antigens are used.

334. The method of claim 331, further comprising determining the antigen-specific score for each of the plurality of antigens and the control antigen.

335. The method of claim 334, further comprising calculating a control antigen-specific score, wherein the cell is selected when the antigen-specific score is high and the control antigen-specific score is low.

336. The method of claim 335, wherein the VDJ region identifies an antigen-specific antibody.

337. The method of claim 335, further comprising using a paired V H :V L Antibody sequences select antibody candidates from a hierarchical list of antigenic signals.

338. The method of any one of claims 322 to 337, further comprising determining differential expression between isotypes, conditions, and / or antigen specificities, wherein determining differential expression between isotypes comprises performing a paired Wilcox test on all single antibody-secreting cells in one isotype and performing a paired Wilcox test on all single antibody-secreting cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster; and wherein determining differential expression between antigen specificities comprises performing a paired Wilcox test on all single antibody-secreting cells in one isotype and performing a paired Wilcox test on all single antibody-secreting cells in another isotype, thereby quantifying the p-value and fold change for each gene in each cluster.

339. The method of any one of claims 320 to 338, further comprising clustering VDJ sequence similarities in antigen-positive target cells, wherein clustering VDJ sequence similarities comprises grouping similar variable regions by amino acid sequence based on sequence alignment.

340. The method of any one of claims 320 to 339, wherein the antigen-positive target cell is IgE + .

341. The method of any one of claims 320 to 340, further comprising trimming redundant sequences.

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