Methods for mapping antigen specificity onto antibody-secreting cells

Abstract

The present disclosure provides an antibody capture complex and a method for capturing a target antibody secreted by an antibody-secreting cell. The present disclosure provides an antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, the first member of the binding pair being capable of binding to a second member of the binding pair, the antibody capture molecule being capable of binding to a target antibody, and the target antibody being secreted by an antibody-secreting cell. In some embodiments, when the second member of the binding pair is biotin and the first member of the binding pair is streptavidin, the antibody capture molecule does not bind to the kappa light chain of the target antibody.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing, filed electronically as an XML file named 381203733SEQ, created on March 10, 2023, with a size of 824 kilobytes, which is incorporated herein by reference. The present disclosure relates, in part, to antibody capture complexes and methods for mapping antigen specificity to antibody-secreting cells. [Background technology]

[0002] B cells express B cell receptors (BCRs) on their surface, thereby allowing the determination of antigen-specific B cell repertoire profiling. Although multiple platforms are available for antibody discovery from B cells expressing cell surface BCRs, the isolated antibodies vary from low to high affinity. After activation by cognate antigen, B cells undergo fine-tuning of their BCRs and can eventually differentiate into antibody-secreting cells (ASCs). Antibody-secreting cells are specialized cell types that represent the final stage of the B cell differentiation program and include plasmablasts, short-lived plasma cells, and long-lived plasma cells (or more commonly, simply "plasma cells" (PCs); Tellier and Nutt, Eur. J. Immunol., 2019, 49, 30-37). These ASCs can produce high-affinity antibodies with therapeutic or preventive potential. However, ASCs can also be responsible for antibody-mediated pathologies. While antigen-specific antibodies from B cells expressing BCRs on their cell surface can be easily cloned and sequenced after flow cytometric isolation, PCs, which are ASCs that do not express cell surface BCRs, cannot currently be easily profiled in a high-throughput fashion. Also, although PCs can be a source of very high affinity antibodies, the specificity of any given secreted antibody cannot be rapidly determined. Due to limitations in the application of current platforms to PCs, a given antibody must be identified by the BCR that secretes it. -There is a long felt and unmet need to efficiently combine with PC to efficiently determine the antigen specificity of the antibody. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Tellier and Nutt,Eur.J.Immunol.,2019,49,30-37 Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides an antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, the first member of the binding pair being capable of binding to a second member of the binding pair, the antibody capture molecule being capable of binding to a target antibody, and the target antibody being secreted by an antibody-secreting cell. In some embodiments, when the second member of the binding pair is biotin and the first member of the binding pair is streptavidin, the antibody capture molecule does not bind to the kappa light chain of the target antibody.

[0005] The disclosure also provides a method of capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting a population of antibody-secreting cells with a second member of a binding pair such that the second member of the binding pair can bind to a cell surface of the population of antibody-secreting cells, the population of antibody-secreting cells comprising antibody-secreting cells that secrete the target antibody; contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising a first member of the binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to the second member of the binding pair on the cell surface of the antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; and contacting the population of antibody-secreting cells with an antigen, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the antigen.

[0006] The disclosure also provides a method of capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting a population of antibody-secreting cells with an antibody capture complex, where the population of antibody-secreting cells comprises antibody-secreting cells that secrete a target antibody, and where the antibody capture complex comprises a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to a second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; and contacting the population of antibody-secreting cells with an antigen, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the antigen.

[0007] The disclosure also provides a method of capturing IgE antibodies secreted by antibody-secreting cells, the IgE antibodies being directed against an allergen, the method comprising: contacting a population of antibody-secreting cells with NHS-biotin to allow the biotin to bind to the cell surface, the population of antibody-secreting cells comprising antibody-secreting cells that secrete IgE antibodies; contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising streptavidin linked to the ectodomain of the high affinity IgE receptor (FcεRIa), 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, the antigen being an antigenic portion of an allergen or a mixture of antigenic portions, whereby the IgE antibodies captured by the antibody capture complex bind to the antigen.

[0008] In some embodiments of the methods disclosed herein, the method further comprises contacting the population of antibody-secreting cells with an antigen, followed by sorting the antibody-secreting cells to collect a pool of antibody-secreting cells, where each antibody-secreting cell in the pool secretes an antibody that is captured by the antibody capture molecule and bound by the antigen, and the collected pool of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody. In some embodiments of the methods disclosed herein, the method further comprises separating the collected pool of antibody-secreting cells into single cells and isolating the antibody-secreting cells that secrete the target antibody. In some embodiments, nucleic acid encoding the antibody is isolated from the antibody-secreting cells that secrete the target antibody.

[0009] In some embodiments of the methods disclosed herein, the step of contacting the population of antibody-secreting cells with an antigen includes (a) contacting the population of antibody-secreting cells with a first labeled form of the antigen such that the antigen binds to the antibody captured on the cell surface of the population of antibody-secreting cells that includes the target antibody captured on the cell surface of the antibody-secreting cells, where the antigen in the first labeled form is conjugated to a first detectable label; (b) washing the population of antibody-secreting cells to remove unbound antigen; and (c) contacting the population of antibody-secreting cells with (i) a first labeled form of the antigen such that the antigen binds to the antibody captured on the cell surface of the population of antibody-secreting cells that includes the target antibody captured on the cell surface of the antibody-secreting cells, where 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; and (c) contacting the population of antibody-secreting cells with (i) a first labeled form of the antigen. (ii) an unlabeled 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 the pool of antibody-secreting cells that remain bound to the first labeled form of the antigen, wherein the collected pool of secreting cells comprises antibody-secreting cells that secrete a 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.

[0010] In some embodiments of the methods disclosed herein, after collecting the pool of antibody-secreting cells that remain bound to the first labeled form of the antigen, the method further comprises separating the collected pool of antibody-secreting cells into single cells and isolating the antibody-secreting cells that secrete the target antibody. In some embodiments, nucleic acid encoding the antibody is isolated from the antibody-secreting cells that secrete the target antibody.

[0011] The 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 the target antibody is then 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, and the target antibody is then captured on the modified cell surface, the target antibody captured on the modified cell surface being bound to an antigen, the antigen being linked to a barcode nucleic acid molecule, and the antibody-secreting cell further comprises a nucleic acid molecule comprising a region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface; and b) attaching a portion of the barcode nucleic acid molecule to a solid surface of a first nucleic acid molecule. c) hybridizing a portion of a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface to a portion of a second nucleic acid molecule attached to a solid surface; d) preparing a first library of amplicons of gene expression in antibody-secreting cells, a second library of amplicons of variable regions, diversity regions, and joining regions (VDJ) in the antibody-secreting cells, and a third library of amplicons of antigen barcode nucleic acid molecules; e) sequencing each of the three amplicon libraries; and f) identifying the region of the gene encoding an antigen-binding fragment of the target antibody captured on the modified cell surface.

[0012] The disclosure also provides a method of identifying a region of a gene encoding an antigen-binding fragment of a target antibody, the target antibody being secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with a second member of a binding pair to allow the second member of the binding pair to bind to a cell surface, the population of antibody-secreting cells comprising antibody-secreting cells that secrete the target antibody; b) contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to the second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; c) contacting the population of antibody-secreting cells with a secondary anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the secondary anti-Ig antibody; and d) contacting the population of antibody-secreting cells with an antigen comprising a barcode nucleic acid molecule, whereby the antibody-secreting cells are bound to the second member of the binding pair. a) contacting said antibody-secreting cells with said antibody-secreting cells, said antibody being secreted and captured by said antibody capture complex, said antibody binding to said antigen; e) collecting said pool of antibody-secreting cells, said pool comprising antibody-secreting cells that secrete said target antibody; and f) separating said pool of antibody-secreting cells into single antibody-secreting cells, said single antibody-secreting cells comprising said ... 2) hybridizing a portion of the nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of a target antibody to a portion of a second nucleic acid molecule attached to the solid surface; 3) preparing a library of first amplicons of gene expression, a library of second amplicons of variable regions, diversity regions, and joining regions (VDJ), and a library of third amplicons of antigen barcode nucleic acid molecules.4) sequencing each of the three amplicon libraries; and g) identifying the region of the gene encoding the antigen-binding fragment of the target antibody.

[0013] The disclosure also provides 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 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, the antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to a second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; b) contacting the population of antibody-secreting cells with a secondary anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the secondary 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 cells and captured by the antibody capture complex binds to the antigen; and d) collecting a pool of antibody-secreting cells, wherein each cell in the pool a) collecting the antibody-secreting cells from the pool of antibody-secreting cells, the antibody-secreting cells secreting antibodies that are captured by the antibody capture complex and bound by the secondary anti-Ig antibody and by the antigen comprising the barcode nucleic acid molecule, the pool of antibody-secreting cells including the antibody-secreting cells secreting the target antibody; and b) 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, the solid surface and the first nucleic acid molecule attached thereto being unique to each single antibody-secreting cell; and f) identifying the region of the gene that encodes the antigen-binding fragment of the target antibody, the region comprising the antigen-barcode nucleic acid molecule, the antigen-barcode nucleic acid molecule comprising: a) a nucleic acid molecule encoding a region of the gene that encodes the antigen-binding fragment of the target antibody, hybridizing a portion of the nucleic acid molecule encoding a region of the gene that encodes the antigen-binding fragment of the target antibody to a portion of a second nucleic acid molecule attached to a solid surface; 3) preparing a first library of amplicons of gene expression, a second library of amplicons of variable regions, diversity regions, and joining regions (VDJ), and a third library of amplicons of antigen barcode nucleic acid molecules; 4) sequencing each of the three libraries of amplicons; and f) identifying the region of the gene that encodes the antigen-binding fragment of the target antibody.

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0015] [Figure 1] A representative workflow of the Ig secretion capture method is shown. Step 1 shows biotinylation of cells with N-hydroxysuccinimide biotin (NHS-biotin). Step 2 shows the Ig secretion capture reagent, including streptavidin coupled to anti-Fc. αIgK was used for antibodies expressing kappa light chain or FcεRIα ectodomain for specific IgE capture. Step 3 shows secreted αIgE or αIgG antibodies bound to oligo-barcoded and fluorescently tagged antigens bound by anti-IgG or anti-IgE antibodies. [Diagram 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 that includes the ectodomain (secreted IgE capture reagent) of the high affinity IgE receptor (external FcεRIα) conjugated with streptavidin, and streptavidin bound to biotin on the PC. Step 2 also shows the external FcεRIα of the secreted IgE capture reagent capturing the IgE secreted by the PC. Step 3 shows the captured IgE bound to the barcoded antigen and bound by the anti-IgE antibody. [Diagram 3] A representative workflow of the IgG secretion capture method is shown. Step 1 shows the biotinylation of cells with NHS-biotin. Step 2 shows the IgG secretion capture reagents including streptavidin-conjugated anti-mouse IgK assembled on biotinylated cells. Step 3 shows the secreted IgG antibody bound to the barcoded antigen and bound by the anti-IgG antibody. [Figure 4A]In Figure 4A, detection of secreted IgG captured on the surface of ARH77 cells is shown. In Figure 4B, detection of secreted IgE captured on the surface of U266 cells is shown. In Figure 4C, secreted IgE captured on the surface of U266 cells is detected, but surface IgE as a B cell receptor is not detected. In Figure 4D, titration of streptavidin-αIgk (StAv-αIgk) on ARH77 cells is shown. In Figure 4E, titration of streptavidin-FceRIα (StAv-FcεRIα) on U266 cells is shown. In Figure 4F, high specificity of detection of secreted IgE captured on the surface of U266 cells, but not IgM secreted by Ramos cells is shown. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 5A] Figure 5A shows a representative workflow of the procedure to determine the sequence of the V(D)J region of the barcoded antigen and specific antibody, and the sequence of the barcode, and then confirm the antigen specificity of the antibody by enzyme-linked immunosorbent assay (ELISA). Figure 5B shows flow cytometry of antigen-specific IgG captured on the surface of PCs in hIL-4Rα-immunized mice. Figure 5C shows visualization of antigen specificity based on barcode signals of hIL-6Rα (non-specific) and hIL-4Rα (specific) in bone marrow (BM) PCs, and splenic draining lymph node (dLN) B cells and PCs. Figure 5D shows UMAP of B cells and PCs isolated from spleen / dLN and bone marrow of challenged mice. Figure 5E is a graph showing total IgG, ELISA antibody levels of anti-hIL-4Rα, and the ratio of anti-hIL-4Rα to total IgG in BM-PCs, SP-PCs, and SP-B cells. Figures 5F and 5G show graphs from two separate experiments showing the binding affinity of antibodies cloned from BM-PCs, SP-PCs, and SP-B cells. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 5G] Same as above. [Figure 6A] FIG. 6A shows a graph depicting the percentage of similarity to the germline V region between hIL-4Rα-specific and non-specific cells isolated from BM-PCs, spleen / dLN-PCs, and spleen / dLN-B cells from challenged mice. FIG. 6B shows a Venn diagram depicting clonal overlap based on CDR3 nucleotide sequences from heavy and light chains of clones isolated from BM-PCs, spleen / dLN-PCs, and spleen / dLN-B cells. FIG. 6C shows a volcano plot comparison of differential gene expression between hIL-4Rα-specific and non-antigen-specific BM-PCs. FIG. 6D shows a heat map depicting the transcriptional profile of differentially expressed genes expressed in hIL-4Rα-specific and non-antigen-specific BM-PCs. FIG. 6E shows a UMAP depicting the location and binding affinity (Kd) of hIL-4Rα-specific antibodies derived from BM-PCs to total BM-PCs. FIG. 6F shows a UMAP depicting scaled gene expression of Cd24a, Ppob, Fkbp11, Ssr2, Tmem176a, Tmem176b, Ly6d, and CD74 in BM-PCs. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 7A]In Figure 7A, a representative workflow of the procedure to sequence the V(D)J region of the barcoded antigen and specific antibody and the barcode, followed by confirming the antigen specificity of the antibody by enzyme-linked immunosorbent assay (ELISA) is shown. Figure 7B shows the gating of PCs as cells, single cells, live cells, DUMP- cells, and CD138+Blimp-1+ cells from left to right. Figure 7C shows IgEVenus+ PCs secreting IgE from the BM and LDLN of house dust mite (HDM) challenged mice. Figure 7D shows the UMAP of IgE+ PCs based on VDJ-BCR expression on non-IgE PCs. Figure 7E shows the percentage of IgE, IgG, IgA, and IgM PCs and the average expression of the top four genes expressed with different isotypes from both dLN and BM. Figure 7F shows the visualization of antigen specificity based on barcode expression of Der p1, Der p2, Der f1 against the negative control Ole e1. Figure 7G shows representative binding curves of IgE antibodies to Der p 1, Der p 2, Der f 1, and the negative control antigen Ole e 1. Figure 7H shows antigen specificity verified by ELISA for a subset of IgE antibodies generated from IgE-secreting cells. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 7F] Same as above. [Figure 7G] Same as above. [Figure 7H] Same as above. [Figure 8A]In Figure 8A, flow plots of BM from an allergic bone marrow donor, BM from a non-allergic bone marrow control donor, and a non-streptavidin FcεR1α capture control are shown. Figure 8B shows the results of tests performed on serum to detect levels of IgE specific to cat dander e1, dog dander e5, common silver birch t3, Dermatophthora pteronyssinus d1, timothy g6, and alternaria alternaria m6. Figure 8C shows expression dot plots showing scaled expression of immunoglobulin genes in total PCs, as well as PC and B cell markers. Figure 8D shows a graph showing scaled expression of IGHE transcripts in IgE, IgG, IgA, and IgM PCs. Figure 8E shows a table of clustered BCR clonotypes showing the five IgE clonotypes, their clonal sizes, and the number of IgE cells relative to non-IgE cells within each clonotype. Figure 8F shows an alignment plot comparing the IgE sequence from the IgE PC with the IgG sequence in clonotype 21. Figure 8G shows the antigen specificity tested by ELISA for one human IgE antibody isolated by the IgE capture complex of the present disclosure from allergic BM. Representative binding curves of human IgE antibodies to Fel d1, Can e1, Der p1, and DNP-IgE are shown. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 8F] Same as above. [Figure 8G] Same as above. [Figure 9-1]Single cell capture and library preparation for mRNA, V(D)J regions, and antigen barcodes are shown. In the bottom row of Figure 9, (i) the light blue rectangle represents the template switch oligo, (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 box immediately to the right of the purple rectangle represents a sequencing primer. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 10] A representative flow chart of the analysis of single-cell mRNA, V(D)J region, and antigen barcode libraries is shown. [Figure 11] Visualization of antigen specificity is shown. Antibody candidates were selected in favor of cells with higher antigen specificity scores, i.e. strong signals for the target antigens and low signals for the control antigens. The target antigens were HDM allergens Der p 1, Der p 2, and Der f 1, and the negative control was olive antigen Ole e 1. The antigen specificity score for each target antigen was determined by subtracting the quantile rank of the control antigen (qC) raised to the power of x from the quantile rank of the target antigen (qT), i.e. qT-qCx. The cells with the highest antigen specificity scores were selected as antigen-specific candidates. [Figure 12-1] 1 shows a table of IgE candidates based on antigen specificity score. For each candidate, the table records the sample from which the cells were obtained, the raw antigen barcode counts for each of the target antigens, the normalized antigen barcode counts, and the antigen specificity score. Data was obtained for 1,069 IgE+ cells. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 13A]Figure 13 shows the binding of IgE antibodies from PC secretory IgE captured with the secretory IgE capture reagent, PC from mice challenged with HDM extract. Figure 13A shows representative binding curves of IgE antibodies to Der p 1 (left chart), Der p 2 (middle chart), and Der f 1 (right chart). Figure 13B shows representative binding curves of IgE antibodies to the negative control antigen Ole e 1. [Figure 13B] Same as above. [Figure 14-1] Figure 14 shows that antigen-specific secreted IgG can be captured on the surface of PCs derived from loaded VelocImmune® mice. Figure 14A shows the sequential flow cytometry gating strategy to detect antigen-specific IgG+ splenic B cells. Figure 14B shows the sequential flow cytometry gating strategy to identify BM-derived PCs with captured antigen-specific secreted IgG. Figure 14C shows, from left to right, the four controls used to precisely define the BM-derived antigen-specific PC populations, as well as a splenic positive control. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above. [Figure 14-5] Same as above. [Figure 14-6] Same as above. [Figure 15A] Normalized values ​​of target antigen barcode counts on the y-axis are shown relative to normalized barcode counts of control antigen barcode counts in all PCs derived from immunized mice and colored by isotype. Figures 15A and 15B show data for bone marrow-derived cells, and Figures 15C and 15D show data for spleen cells. [Figure 15B] Same as above. [Figure 15C-D] Same as above. [Figure 16A]Figure 16A shows a flow cytometry plot of ARH77 cells stained with αIgG, Figure 16B shows a flow cytometry plot of U266 cells stained with αIgE, and Figure 16C shows flow cytometry analysis of Ramos, U266, or a 100:1 mixture of these cells in the presence of an embodiment of the StAv-FceRIα capture complex. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 17A] Figure 17A shows the immunization regimen that mice received prior to tissue harvest for downstream antibody specificity mapping via antibody secretion trap and sequencing work. Figure 17B shows IgG-secreting PCs gated as live cells, lymphocytes, single cells, DUMP- cells (TCRβ, CD200R3, Ly6G, CD49b, CD11b, and IgM), CD98+TACI+, and IgG+Ag+ cells. Figure 17C shows Ag+IgG+ cells from spleen and dLN gated as live cells, lymphocytes, single cells, CD19+CD3-, IgD-IgM-, and IgG+Ag+ cells. Figure 17D shows the gating controls to identify Ag+IgG+BMPCs. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 18A] Figure 18A shows combined transcriptional and unbiased clustering analysis of splenic plasma cells, BM plasma cells, and splenic B cells. Figures 18B and 18C show three distinct cell populations obtained by scRNA-seq: bone marrow plasma cells (BMPC), splenic / dLN plasmablasts / plasmacytoid (Sp / dLN PB / PC), and splenic / dLN B cells (Sp / dLN B cells). Figure 18D shows Biacore analysis of supernatant and purified antibodies of the present disclosure showing affinity of BMPC, Sp / dLN PB / PC, and Sp / dLN B cells as measured by the negative log10 of binding Kd. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 18D]Same as above. [Figure 19A] Figure 19A shows representative binding curves of IgE antibodies to Der p1, Der p2, Der f1, and the negative control Ole e1. Figure 19B shows a Fel d1-specific ELISA to test the binding of human IgE mAb 12_2 and its cross-reactivity to Fel d1. [Figure 19B] Same as above. [Figure 20A] Figure 20A shows the gating strategy for sorting plasma cells from human bone marrow using CD38 staining, and Figure 20B shows the results of an exemplary serum ImmunoCAP® test on an allergic donor. [Figure 20B] Same as above. [Figure 21A] The antibody secretion trap and sequencing method disclosed herein allows for the detection of vaccine-specific IgG-secreting plasma cells from bone marrow donors. Figure 21A is a representative workflow of the procedure for gating and determining PCs as cells, single cells, live cells, DUMP- cells, and CD20-CD38++ cells. Figure 21B shows IgG-secreting cells and antigen-positive cells found within IgG-secreting cells. Figure 21C shows the lack of detection of IgG-secreting PCs without NHS-biotin components, or StAv-Ig kappa, or anti-IgG antibodies. Figure 21D shows the lack of detection of antigen-specific IgG-secreting cells without antigen staining. [Figure 21B] Same as above. [Figure 21C] Same as above. [Figure 21D] Same as above. [Figure 22]The workflow of secretion trap combined with antigen tracking (STAC) is shown. Step 1: Antibody-secreting cells were biotinylated with amine-reactive NHS-biotin. Step 2: Secreted antibodies were captured on the surface of antibody-secreting cells using streptavidin coupled to anti-hIgG. Anti-hIgG was used here because all antibodies have human constant regions. Step 3: Cells were incubated with monomeric hIL-4Rα conjugated with AlexaFluor647 for 30 min at room temperature. Cells were washed and then incubated with hIL-4Rα antigen pre-incubated with StAv-PE for 2 × 45 min at room temperature. [Figure 23A] Flow cytometric analysis of cells undergoing the STAC protocol. Figure 23A shows a table of antibodies used in the experiment. Antibodies 1-3 are specific for hIL-4Rα with different affinities (KD), and antibody 4 is specific for a different target and serves as a negative control for the experiment. Figure 23B shows that a model B cell line (A25 cell line) that does not contain a surface BCR was incubated with NHS-biotin and anti-hIgG trap reagent (steps 1 and 2 in Figure 22). These cells were then incubated with antibodies from the table in Figure 23A, followed by antigen tracking incubation (step 3 in Figure 22). Figure 23C shows flow cytometric analysis of cells undergoing the STAC protocol, showing that antibody-secreting cells can be separated based on affinity. Antibodies 1-4 are color-coded on the flow cytometric plot according to the colors in Figure 23A. The high affinity antibodies (ab_1, ab_2) are biased towards monomeric hIL-4Rα-AlexaFluor647 (y-axis), the low affinity antibody (ab_3) is biased towards tetrameric hIL-4Rα-biotin-StAv-PE (x-axis), and the negative control antibody (ab_4) is negative for both antigens. Figure 23D shows flow cytometry analysis of cells undergoing the STAC protocol. All cells are hIgK+, showing that all antibodies, including the negative control (ab_4), are bound to the surface by the secretion trap. The anti-hIgK FMO is shown in grey, all other colors match those in Figure 23A. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0017] Various terms are used throughout the specification and claims with respect to the present disclosed embodiments. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0018] Unless otherwise expressly stated, it is not intended that any method or aspect described herein should be construed as requiring that its steps be performed in a particular order. Thus, a claim to a method is not intended to infer a certain order in any respect unless the claim or specification otherwise expressly states that the steps are limited to a particular order. This holds true against all possible implicit bases for interpretation, including questions of logic regarding the placement of steps or operational flow, obvious meaning derived from grammatical organization or punctuation, and the number or type of aspects described herein.

[0019] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0020] As used herein, the term "about" means that the recited numerical value is approximate and that small variations do not significantly affect the practice of the disclosed embodiments. When numerical values ​​are used, unless otherwise indicated by the context, the term "about" means that the numerical value can vary by ±10% and remain within the range of the disclosed embodiments.

[0021] As used herein, the term "comprising" may be replaced, where appropriate, with "consisting of" or "consisting essentially of" in certain embodiments.

[0022] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complementary strand.

[0023] As used herein, the term "antibody" can include intact immunoglobulin molecules, fragmented immunoglobulin molecules that contain an antigen-binding fragment, and antigen-binding fragments that do not include any other fragments of the immunoglobulin molecule.

[0024] The present disclosure provides an antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, the first member of the binding pair being capable of binding to a second member of the binding pair, the antibody capture molecule being capable of binding to a target antibody, and the target antibody being secreted by an antibody-secreting cell. In some embodiments, when the second member of the binding pair is biotin and the first member of the binding pair is streptavidin, the antibody capture molecule does not bind to the kappa light chain of the target antibody.

[0025] In some embodiments, a first member of the binding pair comprises a first reagent and a second member comprises a second reagent, and the first reagent and the second reagent form a bond.

[0026] In some embodiments, the first member of the binding pair comprises avidin, streptavidin, or anti-biotin, and the second member of the binding pair comprises biotin. In some embodiments, the first member of the binding pair comprises biotin, and the second member of the binding pair comprises avidin and / or streptavidin. In some embodiments, the first member of the binding pair comprises biotin, and the second member of the binding pair comprises anti-biotin. In some embodiments, the first member of the binding pair comprises one of jun and fos, and the second member of the binding pair comprises the other of jun and fos. In some embodiments, the first member of the binding pair comprises one of mad and max, and the second member of the binding pair comprises the other of mad and max. In some embodiments, the first member of the binding pair comprises one of myc and max, and the second member of the binding pair comprises the other of myc and max. In some embodiments, 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, 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.

[0027] In some embodiments, the second member of the binding pair comprises a surface marker of an antibody-secreting cell and the first member of the binding pair comprises an antibody that binds to the 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 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.

[0028] In some embodiments, the second member of the binding pair comprises interleukin-6 receptor (IL-6R) and the first member of the binding pair comprises an anti-IL-6R antibody, interleukin-6 (IL-6), and / or a fragment of IL-6 capable of binding to IL-6R. In some embodiments, the second member of the binding pair comprises CD27 and the first member of the binding pair comprises an anti-CD27 antibody, CD70, and / or a fragment of CD70 capable of binding to CD27.

[0029] In some embodiments, the second member of the binding pair comprises fluorescein isothiocyanate (FITC) and the first member of the binding pair comprises an anti-FITC antibody. In some embodiments, the second member of the binding pair comprises fluorescein and the first member of the binding pair comprises an anti-fluorescein antibody. In some embodiments, the second member of the binding pair comprises phycoerythrin (PE) and the first member of the binding pair comprises an anti-PE antibody. In some embodiments, the second member of the binding pair comprises APC and the first member of the binding pair comprises an anti-APC antibody. In some embodiments, the second member of the binding pair comprises BV421 and the first member of the binding pair comprises an anti-BV421 antibody. In some embodiments, the second member of the binding pair comprises BV510 and the first member of the binding pair comprises an anti-BV510 antibody. In some embodiments, the second member of the binding pair comprises BV605 and the first member of the binding pair comprises an anti-BV605 antibody. In some embodiments, the second member of the binding pair comprises BV650 and the first member of the binding pair comprises an anti-BV650 antibody. In some embodiments, the second member of the binding pair comprises BV711 and the first member of the binding pair comprises an anti-BV711 antibody. In some embodiments, the second member of the binding pair comprises BV786 and the first member of the binding pair comprises an anti-BV786 antibody.

[0030] In some embodiments, the antibody capture molecule comprises a capture antibody.

[0031] 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.

[0032] In some embodiments, the capture antibody comprises an anti-light chain kappa antibody and / or an anti-light chain lambda antibody. In some embodiments, the capture antibody comprises an anti-light chain kappa antibody. In some embodiments, the capture antibody comprises an anti-light chain lambda antibody.

[0033] In some embodiments, the capture antibody comprises an anti-Ig antibody.

[0034] In some embodiments, the capture antibody comprises an anti-IgM antibody.

[0035] 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.

[0036] 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 multimeric 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 multimeric anti-IgA antibody.

[0037] In some embodiments, the capture antibody comprises an anti-IgE antibody.

[0038] In some embodiments, the antibody capture molecule comprises an Fc receptor or an ectodomain of an Fc receptor. In some embodiments, the Fc receptor comprises an Fcγ receptor, an Fcα receptor, and / or an Fcε receptor. In some embodiments, the Fc receptor comprises an Fcγ receptor. In some embodiments, the Fc receptor comprises an Fcα receptor. In some embodiments, the Fc receptor comprises an Fcε receptor. In some embodiments, 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, and / or an FcRn receptor. In some embodiments, the Fcγ receptor comprises an FcγRI receptor. In some embodiments, the Fcγ receptor comprises an FcγRIIA receptor. In some embodiments, the Fcγ receptor comprises an FcγRIIB receptor. In some embodiments, the Fcγ receptor comprises an FcγRIIB1 receptor. In some embodiments, the Fcγ receptor comprises an FcγRIIB2 receptor. In some embodiments, the Fcγ receptor comprises an FcγRIIIA receptor. In some embodiments, the Fcγ receptor comprises an FcγRIIIB receptor. In some embodiments, the Fcγ receptor comprises an FcRn receptor. In some embodiments, the Fcα receptor comprises an FcαRI receptor and / or an Fcα / μR receptor. In some embodiments, the Fcα receptor comprises an FcαRI receptor. In some embodiments, the Fcα receptor comprises an Fcα / μR receptor. In some embodiments, the Fcε receptor comprises an FcεRI receptor and / or an FcεRII receptor. In some embodiments, the Fcε receptor comprises an FcεRI receptor. In some embodiments, the Fcε receptor comprises an FcεRII receptor. In some embodiments, the Fc receptor comprises a fragment of an Fc receptor capable of binding to Fc.

[0039] In some embodiments, the antibody capture molecule comprises Protein A, a fragment of Protein A capable of binding to a portion of an Fc region, Protein G, and / or a fragment of Protein G capable of binding to a portion of an 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 an 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 an 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 a 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 a light chain.

[0040] In some embodiments, the antibody capture complex comprises avidin or streptavidin linked to the ectodomain of the high affinity IgE receptor (FcεRIa). In some embodiments, the antibody capture complex comprises avidin linked to FcεRIa. In some embodiments, the antibody capture complex comprises streptavidin linked to FcεRIa.

[0041] target antibody In some embodiments, the targeting antibody comprises an IgM antibody.

[0042] In some embodiments, the targeting 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.

[0043] 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.

[0044] In some embodiments, the targeting antibody comprises an IgE antibody.

[0045] target antigen In some embodiments, the target of the targeting antibody, i.e., the antigen to which the targeting 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 a venom, a substance present in the saliva of a biting insect, a mold spore, a cosmetic, a metal, latex, wood, and / or a 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 a venom. In some embodiments, the allergen comprises a substance present in the saliva of a biting insect. In some embodiments, the allergen comprises a mold spore. In some embodiments, the allergen comprises a cosmetic. 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 a plant pollen.

[0046] 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, Fel d 1, Fel d 4, Mus m 1, Rat n 1, Chilida nitropomyosin, 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 Equ c 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 Chillida nitropomyosin. In some embodiments, the animal product comprises shellfish tropomyosin. In some embodiments, the animal product comprises cockroach tropomyosin.

[0047] In some embodiments, the animal products include animal fur, animal dander, cockroach cups, wool, and / or dust mite secretions. In some embodiments, the animal products include animal fur. In some embodiments, the animal products include animal dunder. In some embodiments, the animal products include cockroach cups. In some embodiments, the animal products include wool. In some embodiments, the animal products include dust mite secretions.

[0048] In some embodiments, the drug comprises a penicillin, a sulfonamide, a salicylate, and / or a neomycin. In some embodiments, the drug comprises a penicillin. In some embodiments, the drug comprises a sulfonamide. In some embodiments, the drug comprises a salicylate. In some embodiments, the drug comprises a neomycin.

[0049] In some embodiments, the food product comprises celery, celeriac, corn, maize, eggs, fruit, pumpkin, eggplant, legumes, milk, seafood, sesame, soy, nuts, wheat, and / or balsam of Peru. In some embodiments, the food product comprises celery. In some embodiments, the food product comprises celeriac. In some embodiments, the food product comprises corn. In some embodiments, the food product comprises maize. In some embodiments, the food product comprises eggs. In some embodiments, the food product comprises fruit. In some embodiments, the food product comprises pumpkin. In some embodiments, the food product comprises eggplant. In some embodiments, the food product comprises legumes. In some embodiments, the food product comprises milk. In some embodiments, the food product comprises seafood. In some embodiments, the food product comprises sesame. In some embodiments, the food product comprises soy. In some embodiments, the food product comprises nuts. In some embodiments, the food product comprises wheat. In some embodiments, the food product comprises balsam of Peru. In some embodiments, the legumes include beans, peas, peanuts, or soybeans. In some embodiments, the legume comprises a bean. In some embodiments, the legume comprises a pea. In some embodiments, the legume comprises a peanut. In some embodiments, the legume comprises a soybean. In some embodiments, the nut comprises a pecan and / or almond. In some embodiments, the nut comprises a pecan. In some embodiments, the nut comprises an almond.

[0050] In some embodiments, the substance present in the venom comprises a substance present in a honeybee sting venom and / or a substance present in a wasp sting venom. In some embodiments, the substance present in the venom comprises a substance present in a honeybee sting venom. In some embodiments, the substance present in the venom comprises a substance present in a wasp sting venom.

[0051] 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 of 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-alkyl-1,3-galactose-beta-1,4-N-acetylglucosamine.

[0052] In some embodiments, the cosmetic comprises a fragrance and / or Quaternium-15. In some embodiments, the cosmetic comprises a fragrance. In some embodiments, the cosmetic comprises Quaternium-15.

[0053] In some embodiments, the metal comprises nickel and / or chromium. In some embodiments, the metal comprises nickel. In some embodiments, the metal comprises chromium.

[0054] In some embodiments, the plant pollen comprises grass pollen, weed pollen, and / or tree pollen. In some embodiments, the plant pollen comprises grass pollen. In some embodiments, the plant pollen comprises weed pollen. In some embodiments, the plant pollen comprises tree pollen. In some embodiments, the grass pollen comprises ryegrass pollen and / or timothy grass pollen. In some embodiments, the grass pollen comprises ryegrass pollen. In some embodiments, the grass pollen comprises timothy grass pollen. In some embodiments, the weed pollen comprises ragweed pollen, plantain pollen, nettle pollen, Artemisia vulgaris species pollen, Chenopodium album species pollen, or sorrel pollen. In some embodiments, the weed pollen comprises ragweed pollen. In some embodiments, the weed pollen comprises plantain pollen. In some embodiments, the weed pollen comprises nettle pollen. In some embodiments, the weed pollen comprises Artemisia vulgaris species pollen. In some embodiments, the weed pollen comprises Chenopodium album species pollen. In some embodiments, the weed pollen comprises sorrel pollen. In some embodiments, the tree pollen comprises birch pollen, alder pollen, hazel pollen, hornbeam pollen, Aesculus spp. pollen, willow pollen, poplar pollen, Platanus spp. pollen, Tilia spp. pollen, Olea spp. pollen, Ashei Juniperus pollen, or Alstonia scholaris species pollen. In some embodiments, the tree pollen comprises birch pollen. In some embodiments, the tree pollen comprises alder pollen. In some embodiments, the tree pollen comprises hazel pollen. In some embodiments, the tree pollen comprises hornbeam pollen. In some embodiments, the tree pollen comprises Aesculus spp. pollen. In some embodiments, the tree pollen comprises willow pollen. In some embodiments, the tree pollen comprises poplar pollen. In some embodiments, the tree pollen comprises Platanus pollen. In some embodiments, the tree pollen comprises Tilia pollen. In some embodiments, the tree pollen comprises Olea pollen. In some embodiments, the tree pollen comprises Aschae juniperus pollen.In some embodiments, the tree pollen comprises pollen of the species Alstonia scholaris. In some embodiments, the plant pollen comprises olive tree pollen. In some embodiments, the allergen comprises Ole e 1.

[0055] In some embodiments, allergens include those derived from oak trees (Quercus albus), cedar trees (Cryptomeria japonica), or elm trees or hickory trees.

[0056] In some embodiments, the allergen comprises a dog allergen, e.g., Can f protein. In some embodiments, the allergen comprises a cow's milk allergen or a beef protein, e.g., 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 a serum albumin. In some embodiments, the allergen comprises a fish allergen (e.g., Gad c / Gad m / Pan h / Sals s protein), and in some embodiments, the allergen comprises parvalbumin.

[0057] In any of the embodiments described herein, the allergen may be replaced with a non-allergenic substance, such as, for example, a foreign antigen. In some embodiments, the foreign antigen comprises a coronavirus spike protein. In some embodiments, the foreign antigen comprises a mutant peptide derived from the host. In some embodiments, the foreign antigen comprises a protein from another species. In some embodiments, the foreign antigen comprises a substance from an infectious disease, such as, for example, SARS-CoV-2 or influenza virus.

[0058] In some embodiments, the antibody secreting cells are plasma cells. In some embodiments, the antibody secreting cells are plasmablasts. In some embodiments, the plasma cells are short-lived plasma cells. In some embodiments, the plasma cells are long-lived plasma cells.

[0059] Method for capturing target antibodies secreted by antibody-secreting cells The disclosure also provides a method of capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting a population of antibody-secreting cells with a second member of a binding pair to allow the second member of the binding pair to bind to the cell surface, the population of antibody-secreting cells comprising antibody-secreting cells that secrete the target antibody; contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising a first member of the binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to the second member of the binding pair on the cell surface of the antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; and contacting the population of antibody-secreting cells with an antigen to allow the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex to bind to the antigen. In some embodiments, the method further comprises contacting the population of antibody-secreting cells with a secondary anti-Ig antibody after contacting the population of antibody-secreting cells with the antibody capture complex. The step of contacting with the second anti-Ig antibody can be carried out before, simultaneously with, or after the step of contacting the population of antibody-secreting cells with the antigen.

[0060] The present disclosure also provides a method of capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting a population of antibody-secreting cells with an antibody capture complex, the population of antibody-secreting cells comprising antibody-secreting cells secreting a target antibody, the antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to a second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells, and contacting the population of antibody-secreting cells with an antigen, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the antigen. In some embodiments, the method further comprises contacting the population of antibody-secreting cells with a secondary anti-Ig antibody after contacting the population of antibody-secreting cells with the antibody capture complex. The step of contacting with the secondary anti-Ig antibody can be performed before, simultaneously with, or after the step of contacting the population of antibody-secreting cells with the antigen.

[0061] In some embodiments, after the population of antibody-secreting cells has contacted the antigen, the method may further comprise sorting the population of antibody-secreting cells to collect a pool of antibody-secreting cells, wherein each antibody-secreting cell in the pool secretes an antibody that is captured by the antibody capture molecule and bound by the antigen, and the collected pool of antibody-secreting cells comprises antigen-secreting cells that secrete the target antibody, and in some such embodiments, the method may further comprise separating the collected pool of antibody-secreting cells into single cells and isolating the antigen-secreting cells that secrete the target antibody.

[0062] 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, the portion of the barcode nucleic acid molecule comprises a sequencing primer. In some embodiments, the sequencing primer is upstream of a portion of the nucleotide sequence of the barcode nucleic acid molecule that is unique to the antigen. In some embodiments, the 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, upstream of a portion of the barcode nucleic acid molecule, and complementary to a 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.

[0063] In some embodiments, the population of antibody-secreting cells is contacted with a plurality of barcoded antigens, each barcoded antigen of the plurality of barcoded antigens comprising 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.

[0064] In some embodiments, each barcoded antigen of the plurality of barcoded antigens is detectably labeled. In some embodiments, each barcoded antigen of the plurality of barcoded antigens is labeled with a radioactive compound, a fluorescent compound, or an enzyme. In some embodiments, each barcoded antigen of the plurality of barcoded antigens is labeled with a radioactive compound. In some embodiments, each barcoded antigen of the plurality of barcoded antigens is labeled with a fluorescent compound. In some embodiments, each barcoded antigen of the plurality of barcoded antigens is labeled with an enzyme. In some embodiments, the radioactive compound is 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 In some embodiments, the radioactive compound comprises: 19 In some embodiments, the radioactive compound comprises: 35 In some embodiments, the radioactive compound comprises: 125 In some embodiments, the radioactive compound comprises: 131 In some embodiments, the radioactive compound comprises: 111 In some embodiments, the radioactive compound comprises 99 In some embodiments, the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or a 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 a 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.

[0065] In embodiments where a secondary anti-Ig antibody is used, in some such embodiments the secondary 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 secondary anti-Ig antibody comprises an anti-IgM antibody. In some embodiments the secondary anti-Ig antibody comprises an anti-IgG antibody. In some embodiments the secondary anti-Ig antibody comprises an anti-IgA antibody. In some embodiments the secondary anti-Ig antibody comprises an anti-IgE antibody.

[0066] In some embodiments, the secondary anti-Ig antibody is detectably labeled. In some embodiments, the secondary anti-Ig antibody is labeled with a radioactive compound, a fluorescent compound, or an enzyme. In some embodiments, the secondary anti-Ig antibody is labeled with a radioactive compound. In some embodiments, the secondary anti-Ig antibody is labeled with a fluorescent compound. In some embodiments, the secondary anti-Ig antibody is labeled with an enzyme. In some embodiments, the radioactive compound is 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 In some embodiments, the radioactive compound comprises: 19 In some embodiments, the radioactive compound comprises: 35 In some embodiments, the radioactive compound comprises: 125 In some embodiments, the radioactive compound comprises: 131 In some embodiments, the radioactive compound comprises: 111 In some embodiments, the radioactive compound comprises 99In some embodiments, the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or a 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 a 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.

[0067] In some embodiments, the population of antibody-secreting cells is obtained by enriching a population of cells obtained from a human for immune cells, hi some embodiments, the population of cells obtained from a human is enriched for plasma cells or plasmablasts.

[0068] In some embodiments, the population of antibody-secreting cells is obtained from lymph nodes, lungs, bone marrow, and / or blood of a human. In some embodiments, the population of antibody-secreting cells is obtained from lymph nodes. In some embodiments, the population of antibody-secreting cells is obtained from lungs. In some embodiments, the population of antibody-secreting cells is obtained from bone marrow. In some embodiments, the population of antibody-secreting cells is obtained from blood.

[0069] In some embodiments, the antigen and / or the secondary anti-Ig antibody are detected. In some embodiments, the antibody-secreting cells are sorted to collect a pool of antibody-secreting cells bound by the antigen and / or the secondary anti-Ig antibody, and the collected pool of antibody-secreting cells includes antibody-secreting cells that secrete the target antibody.

[0070] In some embodiments, after contacting the antibody-secreting cells with the Fc block, the population of antibody-secreting cells is contacted with a second member of the binding pair.

[0071] The disclosure also provides a method of capturing IgE antibodies secreted by antibody-secreting cells, the IgE antibodies being directed against an allergen, the method comprising: contacting a population of antibody-secreting cells with NHS-biotin to allow the biotin to bind to the cell surface, the population of antibody-secreting cells comprising antibody-secreting cells that secrete IgE antibodies; contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising streptavidin linked to an ectodomain of FcεRIa, 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, the antigen being 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. In some embodiments, the antigen comprises multiple barcoded antigens, each of the multiple barcoded antigens being a different antigenic portion of an allergen. In some embodiments, the method further comprises sorting the population of antibody-secreting cells to collect a pool of antibody-secreting cells, wherein each antibody-secreting cell in the pool secretes an IgE antibody that is captured by the antibody capture complex and bound by the antigen, and wherein the collected pool of antibody-secreting cells comprises antigen-secreting cells that secrete IgE antibodies.

[0072] In some embodiments, the method further comprises contacting the population of antibody-secreting cells with an anti-IgE antibody to allow the IgE antibody secreted by the antibody-secreting cells and captured by the antibody capture complex to bind to the anti-IgE antibody. In some embodiments, the method may further comprise sorting the population of antibody-secreting cells to collect a pool of antibody-secreting cells, each of the antibody-secreting cells in the pool secreting an IgE antibody that is captured by the antibody capture complex and bound by the antigen and the anti-IgE antibody, and the collected pool of antibody-secreting cells comprises antigen-secreting cells that secrete IgE antibodies.

[0073] 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.

[0074] Antigen Tracking The population of antibody-secreting cells may include a plurality of antibody-secreting cells each secreting an antibody captured by the antibody capture complex, the plurality of antibody-secreting cells including antibody-secreting cells secreting a target antibody. Some of the antibodies secreted by the plurality of antibody-secreting cells bind to the antigen to which the target antibody binds, but the antibodies may have different affinities for the antigen. Antigen tracking can be used to obtain a cell population enriched for antibody-secreting cells secreting antibodies with high affinity for the antigen, which allows for the separation of antibody-secreting cells secreting target antibodies with high affinity. Thus, in some embodiments of the methods disclosed herein, the step of contacting the population of antibody-secreting cells with an antigen includes antigen tracking. In these embodiments, as an initial binding step, a population of antibody-secreting cells is contacted with a first labeled form of the antigen so that the antigen can bind to the antibody, including the target antibody captured on the surface of the antibody-secreting cells, and form an antigen-antibody complex, followed by a "tracking" step in which the cells are contacted with one 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 a combination of an unlabeled form of the antigen and a second labeled form of the antigen ("combination tracking"). Cells that remain bound to the first labeled form of the antigen after tracking represent cells that secrete antibodies with high binding affinity. Antigen tracking thus allows selection between cells secreting antibodies with different affinities to enrich for cells secreting antibodies with high affinity, allowing the isolation of antibody-secreting cells that secrete high affinity target antibodies.

[0075] The term "enrichment" refers to increasing the frequency or proportion of desired cells in a cell population, e.g., increasing the proportion of antibody-secreting cells that secrete high affinity antibodies within an antibody-secreting cell population that includes cells that secrete antibodies with various affinities (e.g., high affinity, medium affinity, and low affinity). Thus, an antibody-secreting cell population enriched for cells that secrete high affinity antibodies encompasses a cell population that has a higher frequency and / or a higher proportion of antibody-secreting cells that secrete high affinity antibodies as a result of the enrichment process. In the present context, the enrichment process is a process that involves antigen tracking, whereby, from a population of cells secreting antibodies of various affinities to the antigen, cells that secrete antibodies with high affinity are selected, and the cells secreting the high affinity antibodies are separated from cells secreting antibodies that are not high affinity.

[0076] The cell population resulting from the tracking is enriched for antibody-secreting cells that secrete antibodies with high binding affinity to the antigen of interest. In other words, the enriched cell population has a higher percentage of cells secreting antibodies that bind to the antigen with high binding affinity compared to the cell population before tracking or without tracking. In some embodiments, at least 40% of the collected cells secrete high affinity antibodies, e.g., antibodies with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population can be a population with at least 50% of the cells in the population that secrete antibodies that bind to the antigen of interest with high binding affinity, e.g., antibodies with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population can be a population with at least 60% of the cells in the population that secrete antibodies that bind to the antigen of interest with high binding affinity, e.g., antibodies with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population can be a population with at least 70% of the cells in the population that secrete antibodies that bind to the antigen of interest with high binding affinity, e.g., antibodies with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population can be a population having at least 80% of the cells in the population secreting an antibody that binds to an antigen of interest with high binding affinity, e.g., an antibody having a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population can be a population having at least 90% of the cells in the population secreting an antibody that binds to an antigen of interest with high binding affinity, e.g., an antibody having a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population can be a population having at least 95% of the cells in the population secreting an antibody that binds to an antigen of interest with high binding affinity, e.g., an antibody having a KD of 0.1 pM to 25 nM. In some embodiments, the frequency of cells secreting high affinity antibodies (e.g., antibodies having a KD of 0.1 pM to 25 nM) in the cell population after tracking is increased by at least 30% compared to the frequency of the cell population before 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 25 nM) in the cell population after tracking is increased 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 25 nM) in the cell population after tracking is increased by at least 50% compared to the frequency of the cell population before 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 25 nM) in the cell population after tracking is increased by at least 75% compared to the frequency of the cell population before 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 25 nM) in the cell population after tracking is increased by at least 100% compared to the frequency of the cell population before 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 25 nM) in the cell population after tracking is increased by at least 200% compared to the frequency of the cell population before 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 is increased by at least 40% compared to the frequency of the cell population before 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 is increased by at least 50% compared to the frequency of the cell population before 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 is increased by at least 75% compared to the frequency of the cell population before 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 is increased by at least 100% compared to the frequency of the cell population before 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 is increased 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 is increased by at least 40% compared to the frequency of the cell population before 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 is increased by at least 50% compared to the frequency of the cell population before 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 is increased by at least 75% compared to the frequency of the cell population before 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 is increased by at least 100% compared to the frequency in the cell population before 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 is increased by at least 200% compared to the frequency in the cell population before or without tracking.

[0077] The term "binding affinity," as is readily understood in the art, generally refers to the strength of 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, as used herein, "binding affinity" refers to the intrinsic binding affinity that 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 is generally measured by the dissociation equilibrium constant (KD or K D ) can be expressed as: K D There is an inverse relationship between the (molar) value and binding affinity, K DThe smaller the value (M), the higher the binding affinity. Thus, "higher affinity" generally refers to an antibody that binds to an antigen more strongly and / or faster and / or remains bound longer. Generally, due to its strong binding interaction, a lower concentration (M) of antigen is required to achieve the desired effect.

[0078] The term "kd" (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody, Ig, antibody binding fragment, or molecular interaction. This value is known as the k off Also called value.

[0079] The term "ka" (M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody, Ig, antibody binding fragment, or molecule interaction.

[0080] "KD" or "K D The term "(M)" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, or the equilibrium dissociation constant of an antibody, Ig, antibody binding fragment, or molecule interaction. The equilibrium dissociation constant is obtained by dividing ka by kd.

[0081] Various methods for measuring binding affinity are known in the art, any of which may be used for the purposes of the present disclosure. The binding affinity obtained using the method is typically within the range of about 0.1 pM to about 25 nM as determined by surface plasmon resonance. In some embodiments, the binding affinity is less than about 10 nM as determined by surface plasmon resonance.

[0082] The term "high affinity" antibodies refers to those antibodies that have a binding affinity, expressed as K, of 25 nM or less, e.g., values ​​between about 0.1 pM and about 25 nM. For this purpose, a high affinity antibody has a binding affinity of about 25×10 as measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA. -9 M (25nM) or less, approximately 10×10 -9M (10nM) or less, approximately 1×10 -9 M (1nM) or less, approximately 1×10 -10 M (0.1nM) or less, approximately 0.5×10 -10 M (0.05nM) or less, approximately 0.05×10 -10 The antibody may have a measured KD of nE M (5 pM) or less, about 1 pM or less, or about 0.5 pM or less. One of skill in the art will appreciate that the value of the KD of an antibody can be calculated using a -z or n x 10 -z For example, 3.2E -12 is 3.2 x 10 -12It will be appreciated that the high affinity antibody has a measured KD in the range of about 0.1 pM to about 25 nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1 pM to about 20 nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1 pM to about 15 nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1 pM to about 10 nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1 pM to about 5 nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1 pM to about 1 nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1 pM to about 0.5 nM. In some embodiments, the high affinity antibody has a measured KD in the range of about 0.1 pM to about 0.1 nM. In some embodiments, a high affinity antibody has a measured KD of less than about 20 nM. In some embodiments, a high affinity antibody has a measured KD of less than about 15 nM. In some embodiments, a high affinity antibody has a measured KD of less than about 10 nM. In some embodiments, a high affinity antibody has a measured KD of less than about 5 nM. In some embodiments, a high affinity antibody has a measured KD of less than about 1 nM. In some embodiments, a high affinity antibody has a measured KD of less than about 0.1 nM. In some embodiments, a high affinity antibody has a measured KD of less than about 0.5 nM. In some embodiments, a high affinity antibody has a measured KD of less than about 0.01 nM. In some embodiments, a high affinity antibody has a measured KD of less than about 0.001 nM (or 1 pM). In some embodiments, a high affinity antibody has a measured KD of less than about 0.5 pM.

[0083] In some embodiments, the antigen is a protein that exists in a monomeric form. An example of a protein that exists in a monomeric form includes an interleukin molecule, such as IL-13. In some embodiments, the antigen is a protein that exists in a multimeric form, including homomers and heteromers. 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.

[0084] Regardless of whether the antigen is a protein that exists in a monomeric form, a multimeric form, or a mixture thereof, the antigen may be used in the methods described herein in monovalent or multivalent form. The terms "monovalent" and "multivalent" refer to the number of units of antigen that are presented, and are used to distinguish the antigen itself from being a protein in monomeric form, multimeric form, or a mixture thereof. Thus, a monovalent form of an antigen refers to a single unit form of the antigen, which may itself be a protein in monomeric form, multimeric form, or a mixture thereof. A multivalent form of an antigen typically refers to multiple units of the antigen presented by a multivalent molecule to which the antigen is bound or linked. The multivalent molecule may be a dimer, trimer, tetramer, pentamer, hexamer, etc., or a mixture thereof. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which may be complexed with biotin and then linked to an antigen, thereby providing a multivalent (e.g., tetravalent) form of the antigen. In some embodiments, the streptavidin multimer comprises a tetramer, and may additionally comprise a trimer and / or a dimer. In some embodiments, the streptavidin multimer is conjugated to a fluorophore, such as phycoerythrin.

[0085] In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment to which an antigen can be linked to provide a bivalent form of the antigen. In some embodiments, the multivalent molecule is a trimer of a trimerizing molecule, such as a Foldon, to which an antigen can be linked to provide a trivalent form of the antigen.

[0086] In some embodiments, the step of contacting the 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 to allow the antigen to bind to the antibody, including the target antibody captured on the cell surface, where 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 either (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 the pool of antibody-secreting cells that remain bound to the first labeled form of the antigen, where the collected pool of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody, and where 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.

[0087] In some embodiments, the first labeled form of the antigen is at a concentration of 0.001 nM to 1 uM. In some embodiments, the first labeled form of the antigen is at a concentration of 0.01 nM to 100 nM. In some embodiments, the first labeled form of the antigen is at a concentration of 0.05 nM to 10 nM. In some embodiments, the first labeled form of the antigen is at a concentration of 0.05 nM to 9 nM, 0.05 nM to 8 nM, 0.05 nM to 7 nM, 0.05 nM to 6 nM, 0.05 nM to 5 nM, 0.05 nM to 4 nM, 0.05 nM to 3 nM, 0.05 nM to 2 nM, or 0.05 nM to 1 nM. In some embodiments, the first labeled form of the antigen is at a concentration of 0.1 nM to 7.5 nM. In some embodiments, the first labeled form of the antigen is at a concentration of 0.1 nM to 7 nM, 0.1 nM to 6 nM, 0.1 nM to 5 nM, 0.1 nM to 4 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, or 0.1 nM to 1 nM. In some embodiments, the first labeled form of the antigen is at a concentration of 0.2 nM to 7.5 nM. In some embodiments, the first labeled form of the antigen is at a concentration of between 0.2 nM and 7 nM, between 0.2 nM and 6 nM, between 0.2 nM and 5 nM, between 0.2 nM and 4 nM, between 0.2 nM and 3 nM, between 0.2 nM and 2 nM, between 0.2 nM and 1 nM, between 0.3 nM and 7 nM, between 0.3 nM and 6 nM, between 0.3 nM and 5 nM, between 0.3 nM and 4 nM, between 0.3 nM and 3 nM, between 0.3 nM and 2 nM, between 0.3 nM and 1 nM, between 0.5 nM and 7 nM, between 0.5 nM and 6 nM, between 0.5 nM and 5 nM, between 0.5 nM and 4 nM, between 0.5 nM and 3 nM, between 0.5 nM and 2 nM, between 0.5 nM and 1 nM. In some embodiments, the first labeled form of the antigen is at a concentration of 1.0 nM to 10 nM, 1.0 nM to 9 nM, 1.0 nM to 8.0 nM, 1.0 nM to 7 nM, 1.0 nM to 6 nM, 1.0 nM to 5 nM, 1.0 nM to 4 nM, 1.0 nM to 3 nM, 1.0 nM to 2 nM, 2.0 nM to 10.0 nM, or 5.0 nM to 10.0 nM. In specific embodiments, antibody secreting cells can be contacted with the first labeled form of the antigen, and the first labeled form of the antigen is at a concentration of 0.2 nM. In specific embodiments, antibody secreting cells can be contacted with the first labeled form of the antigen, and the first labeled form of the antigen is at a concentration of 5.0 nM.In a specific embodiment, antibody secreting cells can be contacted with a first labeled form of the antigen, where the first labeled form of the antigen is at a concentration of 7.5 nM. In a specific embodiment, antibody secreting cells can be contacted with a first labeled form of the antigen, where the first labeled form of the antigen is at a concentration of 10 nM. In specific embodiments, antibody secreting cells can be contacted with a first labeled form of the antigen, where the first labeled form of the antigen is at a concentration of 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.

[0088] In some embodiments, contacting of the antibody secreting cells with the first labeled form of the antigen occurs for about 5 to about 60 minutes, for example, about 20 minutes, about 30 minutes, about 40 minutes, or about 50 minutes.

[0089] 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. The antigen itself, whether in monovalent form, multivalent form, or mixture thereof, can be a protein that is a monomer, a multimer, or a mixture of monomers and multimers.

[0090] In some embodiments, the first labeled form of antigen is an antigen conjugated to a first detectable label.Antigen can be labeled with small molecule, radioisotope, enzyme protein, and fluorescent dye.In some embodiments, the detectable label is a small molecule.Detectable small molecule label allows easy labeling of protein and can be used in many regular introduction detection assays known in the art.

[0091] In some embodiments, the detectable label is an enzyme reporter. Although enzyme labels are larger than biotin, they rarely disrupt antibody function. Commonly used enzyme labels are horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase. To use an enzyme-labeled antibody, the sample is incubated with an enzyme-specific substrate that 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 be reacted with diaminobenzidine to produce a brown product or with luminol to produce light. In contrast, AP can be reacted with para-nitrophenyl phosphate (pNPP) to produce a yellow product that is detected by a spectrophotometer, or with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) to produce a purple precipitate.

[0092] In some embodiments, the detectable label is a fluorescent label. The fluorescent label is directly conjugated with the antibody and does not require enzyme / substrate or binding interaction for detection. Therefore, the amount of fluorescent signal detected is directly proportional to the amount of target protein in the sample. The fluorescent tag can be covalently bound to the antibody via primary amine or thiol.

[0093] After incubation of the population of antibody-secreting cells with the first labeled form of antigen (initial binding step), any unbound antigen can be removed from the primary antibody-producing cells. In some embodiments, the unbound antigen is removed by washing. As known in the art, washing is a technique that uses a washing buffer to remove undesired components, including unbound antigen. Washing buffers are known in the art. In some embodiments, the washing buffer is a phosphate-buffered saline (PBS)-based washing buffer. In some embodiments, the washing buffer is a Tris-buffered saline (TBS) washing buffer. In some embodiments, the washing buffer includes a detergent. In some embodiments, the detergent is Tween®-20. The cells are washed with the washing buffer for an allotted time to remove unbound antigen. This allotted time is sufficient to remove unbound antigen. In some embodiments, the allotted time may be about 10 to about 60 minutes to remove unbound antigen, and multiple washes of 10 to 60 minutes total may be used, for example, three 10-minute washes, or one 30-minute wash, two to four 5-15-minute washes each, etc. After washing and aspirating the wash buffer and the supernatant containing unbound antigen, the pellet containing the antigen-bound cells may be used in a subsequent step. In some embodiments, the pellet containing the bound antigen may be resuspended in a buffer and used in a subsequent step. In some embodiments, the buffer used to resuspend the pellet may be the same wash buffer. In some embodiments, the buffer used to resuspend the pellet may be a different buffer than the wash buffer.

[0094] After contacting a population of antibody-secreting cells with a first labeled form of the antigen (initial binding / contacting step) and removing the unbound first labeled form of the antigen, the cells are subjected to antigen tracking, i.e., the cells are contacted again with the antigen or "tracked" to selectively enrich for cells secreting antibodies with high affinity to the antigen. This tracking can be done using any of several forms of 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). This tracking allows the tracking antigen to bind to the antibody originally bound by the first labeled form of the antigen, thereby tracking the first labeled form of the antigen away from 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.

[0095] In some embodiments, tracking is performed using an unlabeled form of the antigen (cold tracking). In some embodiments, the unlabeled form of the antigen is a monovalent form of the antigen, i.e., the monovalent form of the antigen is the antigen itself without further secondary multimer formation, while the antigen itself may be a protein in monomeric, multimeric, or a mixture of monomeric and multimeric forms. In some embodiments, the unlabeled form of the antigen is a multivalent form of the antigen, where the antigen itself may be a protein in monomeric, multimeric, or a mixture of monomeric and multimeric forms. In some embodiments, the unlabeled form of the antigen is a mixture of monovalent and multivalent forms of the antigen. In embodiments where a multivalent form of the antigen is used, such a form may be provided by a multivalent molecule to which the antigen is bound or linked. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which may be complexed with biotin and then linked to the antigen, thereby providing a multivalent (e.g., tetravalent) form of the antigen. In some embodiments, streptavidin multimers may include trimers and / or dimers in addition to tetramers. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment to which an antigen can be linked to provide a bivalent form of the antigen. In some embodiments, the multivalent molecule is a trimer of a trimer-forming molecule, such as Foldon, to which an antigen can be linked to provide a trivalent form of the antigen.

[0096] In some embodiments, tracking is performed using a second labeled form of the antigen (hot tracking). In such embodiments, after the initial binding and washing steps, the antibody-secreting cells are tracked with the second labeled form of the antigen. The second labeled form of the antigen has a label that provides a detectable signal that is different from the label on the first labeled form of the antigen. Suitable choices of labels are described herein, as long as the label on the second labeled form of the antigen is different from the label on the first labeled form of the antigen. Such labels include small molecules, radioisotopes, enzyme proteins, and fluorescent dyes. In some embodiments, the first label is AlexaFluor647 and the second label is phycoerythrin.

[0097] In some embodiments, the second labeled form of the antigen is a monovalent form of the antigen. In some embodiments, the second labeled form of the antigen is a multivalent form of the antigen. In some embodiments, the second labeled form of the antigen comprises a mixture of monovalent and multivalent forms of the antigen. In embodiments in which a multivalent form of the antigen is used, such a form may be provided by a multivalent molecule to which the antigen is bound or linked. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which may be complexed with biotin and then linked to the antigen, thereby providing a multivalent (e.g., tetravalent) form of the antigen. In some embodiments, the streptavidin multimer may include trimers and / or dimers in addition to tetramers. 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 the antigen may be linked to provide a bivalent form of the antigen. In some embodiments, the multivalent molecule is a trimer of a trimerizing molecule, such as a Foldon, to which an antigen can be linked to provide a trivalent form of the antigen.

[0098] The first labeled form of antigen and the second labeled form of antigen can be the same or different valence forms of antigen, but must have labels that emit different detectable signals.In some embodiments, the first labeled form of antigen is a monovalent form of antigen, and the second labeled form of antigen is also a monovalent form.In some embodiments, the first labeled form of antigen is a monovalent form of antigen, while the second labeled form of antigen is a multivalent form.

[0099] 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 combination tracking. In such embodiments, after an initial binding step and a washing step, the antibody-secreting cells are tracked with an unlabeled form of the antigen and a second labeled form of the antigen. The two forms of the tracking antigen: the unlabeled form of the antigen ("cold tracking antigen") and the second labeled form of the antigen ("hot tracking antigen") can be contacted with the cells simultaneously or sequentially (e.g., the cold tracking antigen is added first, followed by the hot tracking 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 monovalent and multivalent forms. 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 comprises a mixture of monovalent and multivalent forms. In embodiments where a multivalent form of an antigen is used for combinatorial tracking, such a form may be provided by a multivalent molecule to which the antigen is bound or linked. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which may be complexed with biotin and then linked to the antigen, thereby providing a multivalent (e.g., tetravalent) form of the antigen. In some embodiments, the streptavidin multimer may include trimers and / or dimers in addition to tetramers. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment, to which the antigen may be linked to provide a bivalent form of the antigen. In some embodiments, the multivalent molecule is a trimer of a trimer-forming molecule, such as Foldon, to which the antigen may be linked to provide a trivalent form of the antigen.

[0100] For either type of tracking, the tracking antigen concentration used is in excess compared to the concentration of the first labeled form of the antigen, regardless of the form of antigen used for tracking (i.e., unlabeled form, second labeled form, or unlabeled form and second labeled form). In embodiments where an unlabeled form of the antigen is used (in a cold or combination tracking), the unlabeled form of the antigen is at least 2-fold, i.e., 2-fold up to, for example, 2500-fold molar ratio to the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is at 2-fold molar ratio to the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is at 3-fold molar ratio to the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is at 4-fold molar ratio to the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is at 5-fold molar ratio to the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is at 6-fold, 7-fold, 8-fold, or 9-fold molar ratio to the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is at a 10x, 15x, 20x, or 25x molar ratio relative to the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is at a 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x, or more molar ratio relative to the first labeled form of the antigen. In some embodiments, depending on the concentration of the first labeled form of the antigen, the unlabeled form of the antigen can be at a concentration of 0.4nM to 1uM or 10 to 600nM. In some embodiments, depending on the concentration of the first labeled form of the antigen, the unlabeled form of the antigen is at a concentration of 10 nM to 600 nM, 10 nM to 500 nM, 10 nM to 400 nM, 10 nM to 300 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 75 nM, 10 nM to 65 nM, 10 nM to 50 nM, 10 nM to 40 nM, 10 nM to 30 nM, 10 nM to 25 nM, or 10 nM to 20 nM. In embodiments where a second labeled form of the antigen is used (in hot or combinatorial tracking), the second labeled form of the antigen is at a molar ratio of at least 2-fold relative to the first labeled form of the antigen, i.e., 2-fold up to, for example, 2500-fold.In some embodiments, the second labeled form of the antigen is in a 2x molar ratio to the first labeled form of the antigen. In some embodiments, the second labeled form of the antigen is in a 3x molar ratio to the first labeled form of the antigen. In some embodiments, the second labeled form of the antigen is in a 4x molar ratio to the first labeled form of the antigen. In some embodiments, the second labeled form of the antigen is in a 5x molar ratio to the first labeled form of the antigen. In some embodiments, the second labeled form of the antigen is in a 6x, 7x, 8x, or 9x molar ratio to the first labeled form of the antigen. In some embodiments, the second labeled form of the antigen is in a 10x, 15x, 20x, or 25x molar ratio to the first labeled form of the antigen. In some embodiments, the second labeled form of the antigen is in a 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x, or more molar ratio to the first labeled form of the antigen. In some embodiments, depending on the concentration of the first labeled form of the antigen, the second labeled form of the antigen can be at a concentration of 0.4 nM to 1 uM or 10 nM to 600 nM. In some embodiments, depending on the concentration of the first labeled form of the antigen, the second labeled form of the antigen can be at a concentration of 10 nM to 600 nM, 10 nM to 500 nM, 10 nM to 400 nM, 10 nM to 300 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 75 nM, 10 nM to 65 nM, 10 nM to 50 nM, 10 nM to 40 nM, 10 nM to 30 nM, 10 nM to 25 nM, or 10 nM to 20 nM.

[0101] In some embodiments where combination tracking is performed, the cold and hot tracking antigens may be at the same or different concentrations.

[0102] In some embodiments of combination tracking, cells are contacted sequentially with cold and hot tracking antigens, and in some such embodiments, a wash step may be included between these two tracking antigens. In such embodiments, cells are washed with a wash buffer for an allotted time sufficient to remove unbound antigens. In some embodiments, cells are washed with one or more washes for a time period between about 10 minutes and about 60 minutes.

[0103] The tracking is performed for a period of time sufficient to allow the tracking antigen to bind to the antibody. In some embodiments, the tracking is performed with the unlabeled form of the antigen or the second labeled form for about 5 to about 60 minutes, e.g., about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 45 minutes, or about 50 minutes. In some embodiments of the combined tracking, the tracking is performed with the unlabeled form of the antigen for about 5 to about 60 minutes, followed by incubation with the second labeled form of the antigen for about 5 to about 60 minutes. The incubation times with the unlabeled form of the antigen and the second labeled form of the antigen may be the same length or different lengths. As a non-limiting example, the unlabeled form of the antigen may be contacted with the cells for about 30 minutes, while the second labeled form of the antigen may then be contacted with the cells for about 45 minutes, or vice versa. In some embodiments of the combined tracking, the tracking is performed with the second labeled form of the antigen for about 5 to about 60 minutes. In some embodiments of the combined tracing, tracing is performed simultaneously with an unlabeled form of the antigen and a second labeled form of the antigen for about 5 to about 60 minutes.

[0104] In some embodiments, the chases are performed more than once. In some embodiments, the cold chases are performed more than once. In some embodiments, the hot chases are performed more than once. In some embodiments, one cold chase is performed followed by two or more hot chases. In some embodiments, two or more cold chases are performed followed by one hot chase. In some embodiments, two cold chases are performed followed by two or more hot chases. In some embodiments, a combination chase is performed more than once. In some embodiments, a wash step is included after each chase step. In some embodiments, two or more wash steps, for example two washes or three washes, are included after each chase step.

[0105] After the chase is completed, unbound antigen is again removed by washing, and the cells that remain bound to the first labeled form of the antigen are collected.

[0106] In some embodiments where 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 first labeled form of the antigen. In some embodiments where the first detectable label is a first fluorescent label and the second detectable label is a second fluorescent label that is different from the first fluorescent label, two-dimensional FACS is used to collect cells that remain bound to the first labeled form of the antigen. In a specific embodiment, the first detectable label is A647 and the second detectable label is phycoerythrin.

[0107] Isolation of Antibody-Encoding Nucleic Acids The pool of antibody-secreting cells can be sorted or separated into single cells. Protocols for single cell isolation by flow cytometry are well known (Huang, J. et al, 2013, supra). Single cells may also 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), all of which are well known in the art. See, e.g., 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 (e.g., the 10X Chromium System from 10x Genomics (Pleasanton, Calif.)).

[0108] Once obtained, single antibody-secreting cells can be grown by common cell culture techniques for subsequent DNA preparation. Alternatively, antibody genes can be directly amplified from single antibody-producing cells and then cloned into DNA vectors.

[0109] Nucleic acids encoding antibodies or fragments thereof can be isolated from the antibody-secreting cells obtained herein. For example, genes or nucleic acids encoding immunoglobulin variable heavy and variable light chains (i.e., VH, VL, VL can be Vκ or Vλ) can be recovered using RT-PCR protocols with nucleic acids isolated from antibody-secreting cells. For example, nucleic acids encoding antibody fragments are first reverse transcribed (RT) into complementary DNA (cDNA). The cDNA is then amplified in a PCR reaction using primers specific to the antibody gene sequence, e.g., the constant region of the antibody chain. All chains can be amplified in multiplex or separately using chain-specific primers. The amplicons are then sequenced to obtain the nucleic acid sequence encoding the antibody fragment. These RT-PCR protocols are well known and conventional techniques, as described, for example, in Wang et al., J. Immunol. Methods (2000) 244:217-225 and described herein.

[0110] In some embodiments, the nucleic acid encodes a fragment of an antibody, such as a variable domain, a constant domain, or a combination thereof. In certain embodiments, the nucleic acid isolated from the antibody-producing cell encodes a variable domain of an antibody. In some embodiments, the nucleic acid encodes an antibody heavy chain or a fragment thereof. In other embodiments, the nucleic acid encodes an antibody light chain or a fragment thereof.

[0111] Once recovered, the gene or nucleic acid encoding the antibody can be cloned into IgG heavy and light chain expression vectors and expressed via transfection of a host cell. For example, the gene or nucleic acid encoding the antibody can be inserted into a replicable vector for further cloning (amplification of the DNA) or expression in cells (stable or transient). Many vectors, particularly expression vectors, are available or can be engineered to contain the appropriate regulatory elements necessary to regulate expression of the antibody encoding gene or nucleic acid.

[0112] An expression vector in the context of the present disclosure may be any suitable vector, including chromosomal nucleic acid vectors, non-chromosomal nucleic acid vectors, and synthetic nucleic acid vectors (nucleic acid sequences that include a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors.

[0113] In some embodiments, the nucleic acid molecule is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech (1997) 12:355-59), a compressed nucleic acid vector (e.g., as described in U.S. Pat. No. 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, e.g., U.S. Pat. Nos. 5,589,466 and 5,973,972.

[0114] In certain embodiments, the expression vector may be a vector suitable for expression in yeast systems. Any vector suitable for expression in yeast systems may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as yeast alpha factor, alcohol oxidase, and PGH. See F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516-544 (1987).

[0115] In some embodiments, an expression vector having a nucleic acid isolated from an antibody-secreting cell and encoding an antibody or a fragment thereof is introduced into a host cell for expression of the antibody or a fragment thereof. The host cell may include, for example, a mammalian cell, a yeast cell, a bacterial cell, or an insect cell. In some embodiments, the host cell is cultured under conditions to express the nucleic acid, and the antibody or a portion thereof may then be produced and isolated for further use. In some embodiments, the host cell containing one or more of the above nucleic acids is cultured under conditions to express a full-length antibody, and the antibody may then be produced and isolated for further use. In certain embodiments, the host cell contains a nucleic acid encoding a variable domain of an antibody, and the cell is cultured under conditions to express the variable domain. In other embodiments, the host cell contains a nucleic acid encoding a variable heavy (VH) domain of an antibody, and the cell is cultured under conditions to express the VH domain. In another embodiment, the host cell contains a nucleic acid encoding a variable light (VL) domain of an antibody, and the cell is cultured under conditions to express the VL domain. In a specific embodiment, the host cell comprises a nucleic acid encoding the VH domain of the antibody and a nucleic acid encoding the VL domain of the antibody, and the cells are cultured under conditions to express the VH domain and the VL domain.

[0116] In some embodiments, the host cell is a bacterial cell or a yeast cell. In some embodiments, the host cell is a mammalian cell. In other embodiments, the host cell may be, for example, a Chinese Hamster Ovary cell (CHO), such as CHO K1, DXB-11 CHO, Veggie-CHO cell; COS (e.g., COS-7); stem cell; retinal cell; Vero cell; CV1 cell; kidney cell, such as HEK293, 293 EBNA, MSR 293, MDCK, aHaK, BHK21 cell; HeLa cell; HepG2 cell; WI38, MRC 5; Colo25; HB 8065; HL-60; Jurkat or Daudi cell; A431 (epidermal) cell; CV-1, U937, 3T3, or L cell; C127 cell, SP2 / 0, NS-0, or MMT cell; tumor cell; and cell line derived from any of the aforementioned cells. In certain embodiments, the host cell is a CHO cell. In a specific embodiment, the host cell is a CHO K1 cell.

[0117] The 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 the target antibody is then 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, and the target antibody is then captured on the modified cell surface, the target antibody captured on the modified cell surface being bound to an antigen, the antigen being linked to a barcode nucleic acid molecule, and the antibody-secreting cell further comprises a nucleic acid molecule comprising a region of the gene encoding an antigen-binding fragment of the target antibody captured on the modified cell surface; and b) providing a first nucleic acid molecule having a portion of the barcode nucleic acid molecule attached to a solid surface. c) hybridizing a portion of a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface to a portion of a second nucleic acid molecule attached to a solid surface; d) preparing a first library of amplicons of gene expression in antibody-secreting cells, a second library of amplicons of variable regions, diversity regions, and joining regions (VDJ) in the antibody-secreting cells, and a third library of amplicons of antigen barcode nucleic acid molecules; e) sequencing each of the three libraries of amplicons; and f) identifying the region of the gene encoding an antigen-binding fragment of the target antibody captured on the modified cell surface.

[0118] In the above method, with respect to step b) of hybridizing a portion of the barcode nucleic acid molecule within a partition (e.g., a droplet) with a portion of a first nucleic acid molecule attached to a solid surface, the partition (e.g., a bead) dissolves and the cell barcode nucleic acid molecule / TSO oligo floats freely.

[0119] In the above method, with respect to step c) of hybridizing 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 with a portion of a second nucleic acid molecule attached to a solid surface, the nucleic acid molecule (e.g., BCR mRNA) containing a region of a gene encoding an antigen-binding fragment of a target antibody captured on a modified cell surface does not hybridize directly. Rather, it first becomes a first strand cDNA, and then a CCC is added to the end of the cDNA by reverse transcriptase. The CCC then hybridizes to the rGrGrG portion of TSO. In addition, with respect to the second nucleic acid molecule attached to a solid surface within a partition (e.g., a droplet), the partition (e.g., a bead) dissolves and the second nucleic acid molecule floats freely.

[0120] In some embodiments, the modified cell surface modification comprises avidin, streptavidin, antibiotin, 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 modified cell surface modification comprises avidin. In some embodiments, the modified cell surface modification comprises streptavidin. In some embodiments, the modified cell surface modification comprises biotin. In some embodiments, the modified cell surface modification comprises jun protein or a portion thereof. In some embodiments, the modified cell surface modification comprises fos protein or a portion thereof. In some embodiments, the modified cell surface modification comprises mad protein or a portion thereof. In some embodiments, the modified cell surface modification comprises max protein or a portion thereof. In some embodiments, the modified cell surface modification comprises myc protein or a portion thereof. In some embodiments, the modified cell surface modification comprises azide. In some embodiments, the modification of the modified cell surface comprises an alkyne. In some embodiments, the modification of the modified cell surface comprises a phosphine.

[0121] 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 ligated to the first TSO. In some embodiments, a 3' barcoding system can be used. In this case, the antigen barcode will have polyA. VDJ enrichment and sequencing can be performed differently.

[0122] In some embodiments, the portion of the first nucleic acid molecule attached to the solid surface comprises a first template switch oligo (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, in a 3' to 5' direction, a first sequence of three riboguanosine residues, a first TSO, a first UMI, a first surface barcode, and a first sequencing primer. In some embodiments, the first nucleic acid molecule attached to the solid surface is attached at the 5' end of the first nucleic acid molecule attached to the solid surface.

[0123] 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, a 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 a portion of the barcode nucleic acid molecule that is complementary to the first TSO. Reverse transcription also occurs on the other strand, with the antigen barcode nucleic acid extending from its 3' and the first nucleic acid molecule attached to the solid surface also extending from its 3'.

[0124] In some embodiments, the barcode nucleic acid molecule is a single-stranded DNA barcode nucleic acid molecule, 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 starting from the 3' end of the first TSO.

[0125] In some embodiments, the 3' end of a portion of the barcode nucleic acid molecule comprises three consecutive cytidine or ribocytidine residues.

[0126] In some embodiments, mRNA molecules encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface are reverse transcribed, thereby generating single-stranded DNA molecules encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface. The actual chemical reactions occur in solution in the compartments (e.g., droplets).

[0127] 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 differs from the complement 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 comprises three additional consecutive 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 comprises multiple consecutive 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 comprises multiple consecutive thymine residues. In some embodiments, the 5' end of the second nucleic acid molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues.

[0128] 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 (cellular) barcode as the first nucleic acid molecule to match the antigen to 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, in the 3' to 5' direction, a second sequence of three riboguanosine residues, a second TSO, a second UMI, a second surface barcode, and a second sequencing primer. In some embodiments where the 10X Genomics system is used, only the UMI is different for each of the nucleic acid molecules on the same surface. As long as the cellular barcode is the same, other components (e.g., the TSO and sequencing primer) may be different. In some embodiments, the second nucleic acid molecule attached to the solid surface is attached at the 5' end of the second nucleic acid molecule attached to the solid surface.

[0129] 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 starting from the 3' end of the three consecutive cytidine residues.

[0130] In some embodiments, the nucleic acid molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface comprises a second single-stranded DNA molecule encoding a region of a gene encoding an antigen-binding fragment of an antibody captured on the modified cell surface. In some embodiments, the 3' end of the portion of the single-stranded DNA molecule encoding a region of a gene encoding an antigen-binding fragment of a 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 starting from the 3' end of the portion of the single-stranded DNA molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface. In some embodiments, the single-stranded DNA molecule encoding a region of a gene is reverse transcribed starting from the 3' end of the second single-stranded DNA molecule attached to the solid surface.

[0131] In some embodiments, the antibody-secreting cells are placed in a compartment with a solid surface. In some embodiments, the compartment comprises a single antibody-secreting cell. In some embodiments, the compartment comprises a single plasma cell or plasmablast. In some embodiments, the solid surface comprises a bead. In some embodiments, the antibody-secreting cells in the compartment are lysed. In some embodiments, the solid surface (e.g., a bead) is lysed upon partitioning, and the surface-attached DNA molecules are released into the solution within the compartment. In some embodiments, the partition is oil. In some embodiments, the partition is in the form of a droplet.

[0132] In some embodiments, the barcoded nucleic acid molecule further comprises a third sequencing primer.

[0133] In some embodiments, each amplicon is generated from a library of first amplicons that contain genes encoding antigen-binding fragments of target antibodies captured on the modified cell surface.

[0134] In some embodiments, the library of first amplicons is sample demultiplexed, aligned, filtered, and UMI counted. In some embodiments, sequencing the library of first amplicons comprises next generation sequencing.

[0135] In some embodiments, each amplicon of the first amplicon library is mapped and aligned to 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.

[0136] 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; Erratum in: Nat. Biotechnol., 2016, 34, 888), each amplicon of the first amplicon library comprises a unique molecular identifier, and all amplicons of the first amplicon library that are mapped to annotated genes of the standard reference genome are binned and the binned amplicons are counted for each unique molecular identifier (UMI), thereby generating a single-cell count matrix that comprises mapped counts for each annotated gene and counts for each UMI.

[0137] In some embodiments, the single cell count matrix is ​​calculated by dividing the total number of annotated genes by the log of the total number of counted UMIs. 10The high quality cells and extensively profiled genes are filtered out by calculating the ratio divided by . In some embodiments, cells with a gene to UMI ratio below about 0.1 are filtered out. In some embodiments, cells with more than about 4 times the interquartile range of 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 more than about 4 times the interquartile range of 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 threshold can be set for the gene to UMI ratio and the mitochondrial threshold. These values ​​are highly dependent on the tissue used. Tissues such as intestine, lung, or skin have more dead / dying cells with high mitochondrial contamination, while more stable tissues such as blood and lymph nodes require less stringent thresholds.

[0138] In some embodiments, the single cell count matrix is ​​normalized such that the total number of UMIs counted is about 1,000, 5,000, 10,000, 20,000, 50,000, or 100,000. In some embodiments, the single cell count matrix is ​​normalized such that the total number of UMIs counted is about 10,000. In some embodiments, the single cell count matrix is ​​normalized such that the total number of UMIs counted is about 5,000. In some embodiments, the single cell count matrix is ​​normalized such that the total number of UMIs counted is about 20,000.

[0139] In some embodiments, principal component embeddings of the single cell count matrix are computed by principal component analysis (PCA) and used as input to compute a uniform manifold approximation and projection (UMAP). Alternatively, tSNE projections can be computed from the PCA embeddings (van der Maaten et al., J. Mach. Learning Res., 2008, 9, 2579-2605). Additional iterative clustering of major cell types can be performed to identify similarities across batches. In some embodiments, the UMAP is generated using the method disclosed in Korsunsky et al., Nat. Methods, 2019, 16, 1289-1296.

[0140] In some embodiments, the cluster-specific centroids are used to determine a per-cell linear adjustment function, which is then applied to correct for differences in the batches, thereby generating a batch-corrected embedding, and the batch-corrected embedding is used to generate a uniform manifold approximation and projection (UMAP) in two dimensions. This method is called harmonic (Korsunsky et al., Nat. Methods, 2019, 16, 1289-1296), although several alternatives exist, including the computation of integrated anchors from Seurat (Hao et al., Cell, 2021, 184, 3573-3587).

[0141] In some embodiments, a weighted k-nearest neighbor graph is determined and the Leiden algorithm is applied to the weighted k-nearest neighbor graph with resolution values ​​of about 0.1, about 0.2, about 0.5, and about 1.0 to determine cell type clusters in an unsupervised manner. In some embodiments, a weighted k-nearest neighbor graph is determined and the Leiden algorithm is applied to the weighted k-nearest neighbor graph with a resolution value of about 0.1 to determine cell type clusters in an unsupervised manner. In some embodiments, a weighted k-nearest neighbor graph is determined and the Leiden algorithm is applied to the weighted k-nearest neighbor graph with a resolution value of about 0.2 to determine cell type clusters in an unsupervised manner. In some embodiments, a weighted k-nearest neighbor graph is determined and the Leiden algorithm is applied to the weighted k-nearest neighbor graph with a resolution value of about 0.5 to determine cell type clusters in an unsupervised manner. In some embodiments, a weighted k-nearest neighbor graph is determined and the Leiden algorithm is applied to the weighted k-nearest neighbor graph with a resolution value of about 1.0 to determine cell type clusters in an unsupervised manner. Leiden is a community detection algorithm that performs clustering iteratively by splitting nodes of similar cells to generate well-connected clusters (Traag et al., Scientific Reports, 2019, 9, 5233). Alternative algorithms include Louvain, K-means, and NMF clustering.

[0142] In some embodiments, a pairwise Wilcox test is performed on all of the cells in one cluster to compare the results of the first pairwise Wilcox test, and another pairwise Wilcox test is performed on all of the cells in all other clusters to quantify the p-value and fold change for each gene in each cluster. In some embodiments, if 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 clustered subtype named by the top genes from the differential expression results. 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.

[0143] In some embodiments, sample demultiplexing is performed, de novo assembly of read pairs into contigs is achieved, and the contigs are aligned and annotated for a family of second amplicons to a germline segment V(D)J reference sequence.

[0144] In some embodiments, alignment of the contig to the germline segment V(D)J reference sequence comprises aligning to 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.

[0145] In some embodiments, the VDJ sequences are compared against sequences of a human germline reference database or a mouse germline reference database, thereby marking the VDJ sequences for quality alignment by confirming in-frame alignment, CDR3 length, and / or lack of stop codons in the variable region sequences, and the VDJ sequences are filtered based on the mark. In some embodiments, the VDJ sequences are compared against sequences of a human germline reference database or a mouse germline reference database, thereby marking the VDJ sequences for quality alignment by confirming in-frame alignment. In some embodiments, the VDJ sequences are compared against sequences of a human germline reference database or a mouse germline reference database, thereby marking the VDJ sequences for CDR3 length. In some embodiments, the VDJ sequences are compared against sequences of a human germline reference database or a mouse germline reference database, thereby marking the VDJ sequences for lack of stop codons in the variable region sequences. Alternatively, this step can be omitted and all possible VDJ sequences can be retained, which can be aligned with IgBlast to confirm in-frame, high quality sequences to either the human or mouse variable regions.

[0146] This step may not be possible in less well-studied organisms if no database of germline VDJ sequences is available.

[0147] In some embodiments, the VDJ sequence is mapped to a reference database of immunoglobulin chains (e.g., IMGT).

[0148] In some embodiments, the accuracy of the immunoglobulin isotype, variable region mapping, linking region mapping, diversity region mapping, full length variable region for both heavy and light chain sequences per cell is confirmed by alignment. In some embodiments, the immunoglobulin isotype is confirmed by alignment. In some embodiments, variable region mapping is confirmed by alignment. In some embodiments, linking region mapping is confirmed by alignment. In some embodiments, diversity region mapping is confirmed by alignment. In some embodiments, full length variable region for both heavy and light chain sequences per cell is confirmed by alignment. In some embodiments, VDJ sequences are mapped and annotated to germline by IgBlast.

[0149] 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.

[0150] In some embodiments, the library of third amplicons is mapped to a custom short read reference that includes a barcode nucleic acid molecule reference associated with each antigen. In some embodiments, the counts of each uniquely mapped barcode nucleic acid molecule are summed for each cell in the barcoded antigen single cell matrix.

[0151] In some embodiments, the barcode nucleic acid molecules across all cells are quantified and normalized by taking the centered log ratio of each barcode nucleic acid molecule of the plurality of barcode nucleic acid molecules across each sample capture. Alternatively, either centered log ratio or denoising and scaling by background (DSB) can be used (Mule et al., bioRxiv, 2020.02.24.963603).

[0152] In some embodiments, the background antigen signal is removed by DSB for each barcode nucleic acid molecule of the plurality of barcoded nucleic acid molecules. Alternatively, the median log ratio can be used.

[0153] In some embodiments, the antigen signal distribution is determined and if the antigen signal distribution has a well-separated bimodal distribution, the z-transformed values ​​are used to calculate the antigen specificity, if the antigen signal distribution does not have a well-separated bimodal distribution, quantiles are used. In some embodiments, ranks can be used instead of quantiles.

[0154] In some embodiments, analysis of the first amplicon library, the second amplicon library, and the third amplicon library is simultaneous to obtain at least one candidate sequence for the antigen positive target antibody captured on the modified cell surface.

[0155] In some embodiments, cells with effective antibody constant regions are subsetted using analysis of a first amplicon library and analysis of a second amplicon library. In some embodiments, the antibody constant region comprises an IgE constant region. In some embodiments, the subsetted antibody secreting cells contain detectable levels of IgE heavy chain and CD79a, but lack detectable levels of Ms4a1 and CD19, and the subsetted antibody secreting cells are IgE + They are plasma cells or plasmablasts.

[0156] In some embodiments, IgE + Differential expression of antibody secreting cells is assessed for IgA secreting cell isotypes, IgG secreting cell isotypes, and / or IgM secreting cell isotypes. In some embodiments, assessing differential expression includes assessing IgE secreting cell isotypes, IgG secreting cell isotypes, and / or IgM secreting cell isotypes. + The unique transcriptional signature of antibody-secreting cells is further characterized.

[0157] In some embodiments, IgE +The antigen specificity of the antibody secreting cells is evaluated. In some embodiments, the evaluating comprises IgE + comparing the antigen specificity of the antibody-secreting cells to a plurality of antigens and a control antigen, the plurality of antigens including the target antigen. In some embodiments, the comparing includes calculating an empirical score for each of the plurality of antigens and the control antigen by subtracting a quantile of the signal associated with the antigen (qT) from a quantile of the signal associated with the control antigen (qC) using a penalty factor (x) to determine an antigen specificity score, where 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.

[0158] In some embodiments, an antigen specificity score for each of the plurality of antigens and the control antigen is determined. In some embodiments, a control antigen specificity score is calculated and cells are selected where the antigen specificity score is high and the control antigen specificity score is low. In some embodiments, the VDJ region specifies an antigen-specific antibody.

[0159] In some embodiments, the antibody candidates are H :V L The antibody sequence is used to select from a ranked list of antigen signals.

[0160] In some embodiments, differential expression between isotypes, conditions, and / or antigen specificities is determined, where determining the differential expression between isotypes comprises performing a pairwise Wilcox test on all of the cells in one isotype and a pairwise Wilcox test on all of the cells in another isotype, thereby quantifying the p-value and fold change of each gene for each cluster, and determining the differential expression between antigen specificities comprises performing a pairwise Wilcox test on all of the cells in one isotype and a pairwise Wilcox test on all of the cells in another isotype, thereby quantifying the p-value and fold change of each gene for each cluster.

[0161] There are many models / statistics for differential expression analysis. Some examples include: 1) "Wilcox" (which uses the Wilcoxon rank sum test to identify differentially expressed genes between two cell populations) (described herein); 2) "bimod" (which is a likelihood ratio test of single-cell gene expression) (McDavid et al. al., Bioinformatics, 2013), 3) “roc” (identify markers of gene expression using ROC analysis, and for each gene, evaluate (using AUC) a classification index built based on that gene alone to classify between two cell groups, where an AUC value of 1 means that the expression value of this gene alone is able to perfectly classify the two groups (i.e., each of the cells in cell 1 exhibits a higher level than each of the cells in cell 2), an AUC value of 0 also means that there is perfect classification, but in the opposite direction, and a value of 0.5 means that the gene has no predictive power to classify the two groups, which returns a “predictive power” (abs(AUC-0.5)×2) ranking matrix of putative differentially expressed genes), 4) “t” (identify genes differentially expressed between two cell groups using Student’s t-test), 5) “ne” (identify genes differentially expressed between two cell groups using Student’s t-test), 'gbinom' (identifies differentially expressed genes between two cell groups using a negative binomial generalized linear model), 6) 'Poisson' (identifies differentially expressed genes between two cell groups using a Poisson generalized linear model), 7) 'LR' (determines differentially expressed genes using a logistic regression framework, builds a logistic regression model predicting group membership based on each feature separately, and compares this to a null model using a likelihood ratio test), 8) 'MAST' (identifies differentially expressed genes between two cell groups using a hurdle model adjusted to scRNA-seq data and performs DE tests using the MAST package in R), 9) 'DESeq2' (identifies differentially expressed genes between two cell groups based on a model using DESeq2 using a negative binomial distribution) (Love et al., Genome Biol., 2014), and 10) 'Detection Rate' (identifies differentially expressed genes between two cell groups based on the percent detection rate between cell groups).

[0162] In some embodiments, VDJ sequence similarities in antigen positive target cells are clustered, and clustering VDJ sequence similarities comprises sequence-based alignment, grouping similar variable regions by amino acid sequence.

[0163] In some embodiments, the antigen positive target cells are IgE + It is.

[0164] In some embodiments, redundant sequences are trimmed.

[0165] The disclosure also provides a method of identifying a region of a gene encoding an antigen-binding fragment of a target antibody, the target antibody being secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with a second member of a binding pair to allow the second member of the binding pair to bind to a cell surface, the population of antibody-secreting cells comprising antibody-secreting cells that secrete the target antibody; b) contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to the second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; c) contacting the population of antibody-secreting cells with a secondary anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the secondary anti-Ig antibody; and d) contacting the population of antibody-secreting cells with an antigen comprising a barcode nucleic acid molecule, whereby the antibody-secreting cells are bound to the second member of the binding pair. a) contacting said antibody-secreting cells with said antibody-secreting cells, said antibody being secreted and captured by said antibody capture complex, said antibody binding to said antigen; e) collecting said pool of antibody-secreting cells, said pool comprising antibody-secreting cells that secrete said target antibody; and f) separating said pool of antibody-secreting cells into single antibody-secreting cells, said single antibody-secreting cells comprising said ... 2) hybridizing a portion of the nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of a target antibody to a portion of a second nucleic acid molecule attached to the solid surface; 3) preparing a library of first amplicons of gene expression, a library of second amplicons of variable regions, diversity regions, and joining regions (VDJ), and a library of third amplicons of antigen barcode nucleic acid molecules.4) sequencing each of the three amplicon libraries; and g) identifying the region of the gene encoding the antigen-binding fragment of the target antibody.

[0166] In order that the subject matter disclosed herein may be more effectively understood, examples are provided below. It should be understood that these examples are for illustrative purposes and should not be construed as limiting the claimed subject matter in any manner. Throughout these examples, molecular cloning reactions and other standard recombinant DNA techniques were performed according to the methods described in Maniatis et al., Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, unless otherwise stated. EXAMPLES

[0167] Materials and Methods Reagents used [Table 5] [Table 6] [Table 7-1] [Table 7-2]

[0168] Cell surface biotinylation Cells were washed and resuspended in PBS containing 5% w / v BSA. Surface Fc was blocked with 1:10 diluted Fc block (eBioscience) for 15 min at 4° C. along with detection of surface BCR (for cell line assays). Cells were washed two more times with PBS, spun down at 500 g for 10 min, resuspended in 1 mL freshly prepared NHS-biotin (Sigma, 0.5 mg / mL in PBS) and incubated for 15 min at 37° C. Biotinylated cells were washed three times in cold PBS containing 5% w / v BSA, changing tubes after each washing step.

[0169] Preparation of secretory IgE capture reagent External FcεRIα was conjugated with streptavidin using the Lightning-Link® Streptavidin Conjugation Kit to form the secreted IgE capture reagent. Briefly, a 0.5 mg / mL solution of external FcεRIα containing 1 mg / mL streptavidin was prepared. 100 microliters of the modification reagent was added to the solution with gentle mixing. The cap was removed from the vial of streptavidin conjugation mix, and the mixture containing the modification reagent was added directly to the lyophilized material. The material was gently resuspended by using a pipette to remove and redispense the added suspension once or twice. The cap was replaced on the vial, and the vial was left to stand for 3 hours at 20-25°C in the dark. 100 μL of quenching reagent was then added with gentle mixing. In this way, the secreted IgE capture reagent was generated.

[0170] Incubation of cells bearing biotinylated cell surfaces with secreted IgE capture reagent The washed biotinylated cells were diluted at 1-3 × 10 in 1 mL of PBS containing 5 w / v% BSA and 30 μg / mL of purified secreted IgE capture reagent. 6The cells were resuspended at 1000 cells / mL and placed in a 6-well plate. In some embodiments, a volume of up to 3 mL in a 6-well plate can be used. The cells were incubated at 37° C. for 1 hour on a rotating platform to allow capture of secreted IgE. The cells were then washed twice in PBS containing 5% w / v BSA.

[0171] Preparation of lung cells Prior to obtaining lung samples, tubes and reagents were prepared. Ten milliliters of sterile water was added to 50 mg of Liberase® TH to have a final concentration of 26 collagenase Wunsch units / mL or 5.0 mg / mL collagenase. One milliliter aliquots were placed into individual tubes and stored at -20°C. Liberase® Mix (digestion buffer) was prepared as needed. [Table 8]

[0172] For each mouse, 1.5 mL of Liberase® mix was pipetted into a separate 5 mL flow cytometry tube. The tubes were kept on ice or in a cold rack until required for digestion.

[0173] For each mouse, the largest lobe of the right or whole lung was placed into a separate 2 mL Eppendorf® tube containing 1 mL of Liberase® mix. In these tubes, the lung material was cut into small pieces, typically about 2-3 mm cubes. This material was incubated at 37°C for 20 minutes. The digested material was added to a gentleMACS™ C tube and digestion was stopped by adding 0.5 M EDTA to a final concentration of 10 mM. Two milliliters of MACS® buffer were also added. The cap was tightly tightened and the capped tube was run on a gentleMACS™ Octo Dissociator using the preloaded program m_lung_02_01. The resulting cells were centrifuged at 400g for 4 minutes and the supernatant was decanted. Red blood cells in the pellet were lysed with 1 mL of red blood cell lysis buffer for 3 minutes at ambient temperature. The lysis buffer was then inactivated by adding 10 mL of DPBS. The cells were centrifuged at 400g for 4 minutes, the supernatant was decanted, and the pellet was resuspended in 1mL of DPBS. The suspension was filtered through a 100μm Millipore® plate filter into a 2mL deep-well plate. The cells were centrifuged at 400g for 4 minutes and resuspended in 500μL of DPBS. 50 microliters of each sample was pooled with 100μL of the other sample, and 150μL of the pooled sample was added to a round-bottom 96-well plate.

[0174] Preparation of draining lymph node cells 1-2 mL of RPMI medium was added to a 70 μm 12-well cell strainer plate. Draining lymph nodes were collected and added to the plate. PCR plates were prepared, and each well was punctured with a 20G needle and placed on a 2 mL deep well plate.

[0175] Draining lymph nodes were mashed on a 74 μm cell strainer in 2 mL of RPMI medium using the rear end of a 3 mL syringe. The preparation was filtered through a 100 μm Millipore® plate filter into a 2 mL deep well plate, and the filtered cells were centrifuged at 400×g for 4 min. The pellet was resuspended in 500 μL of DPBS and transferred to a 96-well deep bottom plate. The plate was centrifuged at 400×g for 4 min. The cells were resuspended in 200 μL of PBS. 200 μL of the suspension was plated in a 96-well round bottom tube. The plate was centrifuged at 400×g for 4 min, and the supernatant was flicked off. A typical cell count from a mouse administered 50 μg of HDM extract diluted in 20 μl of saline intranasally 3 times a week for 15 weeks was 230×10 6 It was a cell.

[0176] Preparation of bone marrow cells 200 μL of RPMI medium was added to a 48-well plate. A PCR plate was prepared, each well was punctured with a 20G needle, and placed on a 2 mL deep-well plate.

[0177] Mouse femurs were extracted and the bones were washed. Both ends of each bone were cut and placed in a PCR plate with holes. The PCR plate was placed on a 2 mL deep-well collection plate and centrifuged at 500 x g for 4 min. If BM remained within the bone, the centrifugation step was repeated. The pellet was resuspended in 500 μL of red blood cell lysis buffer and added. The suspension was incubated at ambient temperature for 3 min, after which 1–2 mL of PBS was added to inactivate the lysis buffer. The resulting cells were centrifuged at 400 x g for 4 min and the pellet was resuspended in 1 mL of DPBS. The suspension was filtered through a 100 μm Millipore plate filter into a 2 mL deep-well plate. From each sample, approximately 100–200 μL was removed for fluorescence minus one (FMO) along with a pool of samples used for FMO. The aliquoted and pooled samples were placed into 12 wells for FMO. The remaining samples were centrifuged at 400 x g for 4 min.

[0178] Bone marrow samples were resuspended in 200 μL of PBS. Half of the bone marrow samples were plated into 96-well round-bottom plates. Plates were centrifuged at 400×g for 4 min and the supernatant was flicked off. Typical cell counts from mice administered 50 μg of HDM extract diluted in 20 μl of saline intranasally three times a week for 15 weeks were 116×10 6 It was a cell.

[0179] Preparation of spleen cells Spleens were extracted from mice and homogenized in 5 mL of DPBS in gentleMACS™ tubes using preloaded program TBA. The homogenized spleens were centrifuged at 400×g for 4 minutes and the supernatant was discarded. The spleens were then resuspended in 4 mL of red blood cell lysis buffer and incubated at room temperature for 5 minutes, after which 10 mL of DPBS was added to neutralize the lysis buffer. The spleens were then centrifuged at 400×g for 4 minutes and the supernatant was discarded. The spleens were then resuspended in 10 mL of DPBS and filtered through a 70 μM filter for single cell suspension. Cell counts and viability were determined, and the spleens were then stained for antigen+B cells.

[0180] Isolation of pan B cells B cells were isolated using the EasySep® Mouse Pan B Cell Isolation Kit. Briefly, samples were diluted to 1 × 10 in 0.5-8 mL of PBS containing 5% w / v BSA. 8Cells were prepared. 50 microliters of rat serum was added per mL of sample. Samples were added to 14 mL polystyrene round-bottom tubes (catalog number 35205, lot number 00421123). Cells were mixed and incubated at ambient temperature for 5 minutes. 50 microliters of RapidSpheres® were added per milliliter of sample, vortexed for 30 seconds, and the mixture was incubated at 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 The Big Easy EasySep® magnet (StemCell catalog number 18001) for 2.5 minutes at ambient temperature. The concentrated cell suspension was poured into a new tube and counted. The procedure is outlined below. [Table 9]

[0181] Example 1: Development of capture complexes To pair secreted antibodies with the ASCs that secrete them, we obtained immunoglobulin capture complexes that can locally capture secreted antibodies on the surface of ASCs (Figure 1). To achieve this, the cell surface was biotinylated using NHS-biotin, which has a hydrophilic active group that binds to cell surface primary amines (-NH2). An affinity matrix was then assembled around the cells using streptavidin coupled to an Fc binding reagent (αIgk for IgG and FcεRIα extracellular domain for IgE) to capture the immunoglobulins secreted from ASCs (Figure 1). To mark the antigen specificity of PCs, we combined this method with barcoded antigen staining and / or fluorescently tagged antigen staining (Figure 1). Single cell transcriptomes (scRNA-seq), barcoded antigen signals, and VH:VL antibody sequences were generated using 10X Genomics reagents.

[0182] To determine the functionality, sensitivity, and specificity of the IgG capture and IgE capture complexes, ARH77, an IgG (kappa light chain) secreting cell line, and U266, an IgE (lambda light chain) secreting myeloma line, were used in the demonstration of the main experiment (Figure 4A, Figure 4B, Figure 4D, and Figure 4E). The lack of BCR surface expression on both cell lines was first confirmed (Figure 16A and Figure 16B). ARH77 and U266 cells were biotinylated using NHS-biotin, and capture complexes were assembled using streptavidin coupled to anti-human Igk in the case of IgG (StAv-aIgk) and FcεRIα, the extracellular domain of the high affinity IgE receptor, in the case of IgE (StAv-FcεRIα). The sensitivity of capture was determined by staining for IgG and IgE (Figure 4A and Figure 4B). Although ARH77 and U266 cells lacking any part of the affinity matrix lacked IgG and IgE on their surface, successful assembly of all components of the secretory capture complex allowed specific detection of secreted IgG and IgE on the surface of the cells secreting them (Figure 4A and Figure 4B). To further determine the minimal concentrations of StAv-αIgk and StAv-FcεRIα required for binding of secreted antibodies, we performed a dose titration of the capture complex and observed that StAv-FcεRIα was saturated at 15-30 μg / mL, while StAv-αIgk was saturated at 3.75 μg / mL (Figure 4D and Figure 4E).

[0183] To test the specificity of the capture complex, IgM-expressing Ramos cells were mixed with U266 cells at 1:1 (Figure 4F) or 100:1 (Figure S16C) and IgE-secreted capture complexes were assembled on the mixed population. No binding of secreted IgE was observed on the surface of Ramos cells, suggesting minimal or no crosstalk with neighboring cells under the dilutions and culture conditions tested (Figure 4F, Figure S16C).

[0184] Example 2: Proof of concept of secreted IgE capture Capture of secreted IgE: Experimental procedure An approach was undertaken to pair a given secreted IgE antibody with the PC that secreted it. Figure 2 (step 1) shows the surface of a PC biotinylated with NHS-biotin, as the N-hydroxysuccinimide group of NHS-biotin has a hydrophilic active group that reacts non-specifically with cell surface primary amines (-NH2). Step 2 shows the secreted IgE capture reagent bound to the biotinylated PC. Figure 2 (step 2) also shows the external FceRIa of the secreted IgE capture reagent that captures the PC secreted IgE. Figure 2 (step 3) shows the captured IgE bound to the barcoded antigen and bound by an anti-IgE antibody.

[0185] The secretory IgE capture reagent successfully captured secretory IgE on the surface of U266 cells. IgE-secreting U266 cells were surface biotinylated as described herein, washed three times in cold PBS with 5% w / v BSA, changing tubes for each washing step, and incubated with secreted IgE capture reagent as described herein. Cells were then washed and stained with Brilliant™ Violet 711-labeled anti-IgE (BD) in PBS with 5% w / v BSA, 10 mg / mL dextran sulfate, and 1:50 Fc block for 30 minutes at 4° C. Cells were then washed with 10× Brilliant™ Violet buffer. Cells were then washed twice in PBS and fixed with BD Cytofix™. Flow cytometry data was then acquired.

[0186] It was found that the secretory IgE capture reagent specifically captured secretory IgE antibodies on the surface of U266 cells (Figure 4B). Importantly, control cells that were not biotinylated, control cells that were not incubated with the secretory IgE capture reagent, or Brilliant IgE antibodies were not biotinylated. (商標)No cell surface IgE antibodies were detected in control cells that were not contacted with Violet 711-labeled anti-IgE. This confirmed that each step was necessary to detect captured secreted IgE. Furthermore, IgE was not detected on U266 cells as BCR. This confirmed that captured secreted IgE antibodies, and not 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, which secrete human IgM, in a 1:1 ratio. For U266 cells only, the mixed cells were incubated with the secretion IgE capture reagent. Then, for U266 cells only, the cells were washed in PBS and fixed with Cytofix™. Flow cytometry data was acquired again. It was found that the secretion IgE capture reagent captured secreted IgE antibodies on the surface of U266 cells, but did not capture IgM secreted by Ramos cells. This demonstrated the specificity of the secreted IgE capture reagent (FIG. 4C).

[0187] Example 3: Capture of secreted IgE on the surface of primary plasma cells IgE Venus Reporter mice and Blimp-1 mCherryReporter mice were used to determine whether secretory IgE capture reagents could capture secretory IgE on the surface of IgE-producing primary plasma cells. Specifically, a Venus reporter inserted downstream of the membrane IgE locus identified IgE-secreting cells, while an mCherry reporter inserted downstream of Blimp-1 identified PCs (Asrat et al., Sci. Immunol., 2020, 10, eaav8402). Mice were challenged with 50 μg of HDM extract diluted in 20 μl of saline administered intranasally three times a week for 15 weeks. Mice were anesthetized and sacrificed as described herein. BM cells were prepared as described herein and LDLN were prepared as described herein. BM from mice were pooled together and separately, dLN from mice were pooled together. Pan-B cells were isolated and stained with an antibody mix containing Brilliant™ Violet 711-labeled anti-IgE (BD) in PBS with 5% w / v BSA for 30 min at 4° C. to detect secreted IgE. Labeled cells were washed in PBS with 5% w / v BSA, fixed with Cytofix™, and sorted on a BD FACSymphony® S6 flow cytometer (FIGS. 7A and 7B). Plasma cells were sorted in the following order: cells, single cells, live cells, DUMP. - cells, CD138 + Blimp-1 + The cells were gated on the Blimp1 cells (Figure 7A and Figure 7B). Dump is a collection of markers in the same channel, including CD3, CD11b, IgD, IgM, Ly6g, etc. Any cells that expressed these markers were excluded. Anti-CD138 antibody was used to detect CD138 and plasma cells were classified as Blimp1 cells. + CD138 + IgE-secreting PCs were gated as IgE Venus+ The cells were sorted (Figure 7C).

[0188] Example 4: Single-cell library preparation and sequencing of antigen-specific IgE-expressing plasma cells FIG. 7A shows a representative overview of the steps of Examples 3-5, and FIG. 9 shows the steps from binding of barcoded antigen to secreted IgE captured on the surface of PCs through library selection and sequencing.

[0189] Plasma cells were prepared as described in Example 3. Single PCs were suspended in PBS with 0.04 w / v% BSA and loaded onto a Chromium® Controller device (10x Genomics) at 15,000 cells per lane. Partitions of PCs in barcoded beads were formed and PCs were dissolved. Reverse transcription was performed and then partitions were destroyed. RNA-seq, feature barcode, and V(D)J libraries were prepared using the Chromium® Next GEM Single Cell 5' Kit, version 2 (10x Genomics). After library amplification, cDNA was split into separate RNA-seq, feature barcode, and V(D)J aliquots. To enrich the V(D)J aliquots for BCR sequences, the Chromium® Automated Single Cell Mouse and Human BCR Amplification & Library Construction Kit (10x Genomics) was used. Feature barcode libraries of DNA barcoded antigens or DNA barcoded cell surface proteins were prepared using the Chromium® 5' Feature Barcode Library Construction kit (10x Genomics). Paired-end sequencing was performed on the RNA-seq library on an Illumina NovaSeq® 6000 sequencing system, where read 1 was 26 bp for the unique molecular identifiers (UMIs) and cell barcodes, read 2 was 80 bp for the transcript reads, 10 bp for the i7 reads, and 10 bp for the i5 reads. Paired-end sequencing was also performed on the feature barcode library, where read 1 was 26 bp for the UMIs and cell barcodes, read 2 was 35 bp for the feature barcode reads, 10 bp for the i7, and 10 bp for the i5 reads. Paired-end sequencing was also performed on the V(D)J library, where read 1 was 150 bp, 10 bp for the i7, 10 bp for the i5, and read 2 was 150 bp.For RNA-seq and feature barcode libraries, Cell Ranger Single-Cell Software Suite, version 6.1.1 (10x Genomics) was used for sample demultiplexing, alignment, filtering, and UMI counting. Human GRCh38 and mouse mm10 genome assemblies and RefSeq gene models for human and mouse, respectively, were used for alignment. For V(D)J libraries, Cell Ranger software was used to perform sample demultiplexing, de novo assembly of read pairs into contigs, and alignment and annotation of contigs against all V(D)J germline reference sequences from the germline mouse IMGT reference database.

[0190] Example 5: Single-cell analysis of antigen-specific IgE-expressing plasma cells Figure 10 shows a representative flow chart of the steps described in this example. After sequencing, the single-cell gene expression library, the secreted antibody V(D)J region library, and the barcoded antigen library were simultaneously analyzed to obtain candidate antigen-positive secreted IgE antibody sequences.

[0191] The single-cell gene expression library was mapped and aligned to the standard mouse reference genome Mm10. The unique mapped count of each gene was then determined by CellRanger software. The reads were mapped to the standard reference genome using the single-cell alignment software STAR, where each read contains a unique molecular identifier that maps to an annotated gene in the standard Mm10 reference genome. The mapped reads were binned and counted for each unique molecular identifier (UMI), thereby generating a single-cell count matrix that comprises the mapped count of each annotated gene and the count of each UMI. The single-cell count matrix was then calculated by multiplying the total number of annotated genes by the log of the total number of counted UMIs. 10The counts were filtered out by calculating the ratio of genes to UMIs divided by the number of UMIs. Cells with a gene to UMI ratio below about 0.1 were filtered out. Cells with more than 4 times the interquartile range of the total number of UMIs were filtered out. Cells with more than about 80% of reads mapped to mitochondrial genes were filtered out. High quality cells with a gene to UMI ratio above about 0.1, less than about 4 times the interquartile range of the total number of UMIs counted, and less than 80% of reads mapped to mitochondrial genes were retained. Count matrices from each individual sample capture were normalized to a total count of 10,000 and batch corrected using the Harmony algorithm to generate a combined uniform manifold approximation and projection (UMAP). Unsupervised clustering with the Leiden algorithm was then used to determine cell type clusters, and Wilcox tests were used to identify specific cell type and cluster marker genes.

[0192] The IgBlast sequence analysis tool (Ye et al., Nucleic Acids Res., 2013, 41, W34-W40) was performed to align the V(D)J sequences against the germline mouse IMGT reference database. Valid V(D)J sequences were then filtered by productive in-frame alignment, full-length CDR3, and lack of stop codons in the sequence. The IgBlast tool confirmed gene mapping to immunoglobulin isotypes, variable regions, diverse regions, and joining regions, as well as accurate full-length variable regions for both heavy and light chain sequences per cell.

[0193] The barcoded antigen library was mapped by CellRanger software to a custom short read reference containing DNA tag sequences for four barcoded antigens, namely Der p 1, Der p 2, Der f 1, and Ole e 1. These tag sequences were quantified across all cells and normalized by taking the central log ratio (CLR) per barcoded antigen across each sample capture. In addition, background antigen signals for each barcoded antigen were removed by DSB (background denoising and scaling). Both CLR and DSB normalized values ​​were used to quantify the target antigen signals, i.e., Der p 1, Der p 2, and Der f 1, relative to the control signal, i.e., Ole e 1.

[0194] The antigen specificity score (AgSS) of IgG was calculated by subtracting the quantile of the hIL-6Rα barcoded UMI counts multiplied by a penalty factor (x) from the quantile of the hIL-4Rα barcoded UMI counts (AgSS=Q hIL-4Rα - Q hIL-6Rα x For IgE, AgSS was calculated using a similar formula for mouse and human single-cell data. For IgE scores, the target antigen was either Der p1, Der p2, or Der f1, and the control was always Ole e1.

[0195] Ig BCR sequences were clustered using scirpy v0.10 using the function scirpy.pp.ir_dist based on amino acid sequence with a Hamming distance of less than 4. Clustered BCRs were clonotyped using the function scirpy.tl.define_clonotype based on heavy chain CDR3 amino acid sequence based on the previous criterion of a Hamming distance of less than 4. The scirpy documentation can be found on the World Wide Web at "scverse.org / scirpy / latest / api.html".

[0196] After processing the three single cell libraries, gene expression and V(D)J region sequencing data were used to subset PCs with effective IgE constant regions and detectable expression of IgE and CD79a, but lacking expression of Ms4a1 and CD19, typically found in non-PC B cells. IgE-expressing PCs were then assessed for differential expression to additional IgA, IgG, and IgM plasma cell isotypes to further characterize the unique transcriptional signature of IgE-secreting PCs. IgE-secreting PCs were then assessed for antigen specificity by comparing the normalized levels of the target antigens, i.e., Der p 1, Der p 2, or Der f 1, to the normalized levels of the control antigen Ole e 1. This was done by calculating an empirical score for each target antigen and subtracting the quantile rank of the target antigen from the quantile rank of the control antigen using a penalty factor. The formula used to do this is described below.

[0197] Antigen specificity = qT-qC x 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. An antigen specificity score (AgSS) was determined for each cell for each of the three target antigens and used to prioritize cells with the strongest signal of the target antigen while minimizing the signal of the control antigen. Antibody candidates were selected from the ranked list of target antigen signals using the paired VH:VL antibody sequences of the cell in question. Figure 11 shows the visualization of antigen specificity.

[0198] Example 6: IgE binding ELISA After selection of allergen-specific antibodies, the relative binding to Der p 1, Der p 2, and Der f 1 was quantified by ELISA. Figure 12 shows a table of IgE candidates based on antigen signal. The data was analyzed using 1,069 IgE +Cells were obtained. Specifically, binding microtiter plates were coated overnight at 4° C. with 100 μL of native Der p 1, Der p 2, Der f 1, and Ole e 1 at 2 μg / mL in PBS. IgE antibodies were prepared at a concentration of 1 μg / mL and serially diluted 3-fold in PBS with 0.5 w / v% BSA. After washing with 0.05% v / v Tween® 20 in PBS and blocking in PBS with 0.5 w / v% BSA, 100 μL of diluted IgE antibodies were transferred to the plate and incubated for 1 hour at ambient temperature. Then, 100 μL of anti-mouse IgE antibodies conjugated to horseradish peroxidase (HRP) diluted 1:5000 were added. The plate was then incubated for 1 hour at ambient temperature and washed with 0.05% v / v Tween® 20 in PBS. The HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) was then added. The reaction was stopped (with 100 μL of sulfuric acid 2.0 N) and the optical density values ​​were determined using a plate reader at 450 nm. IgE mAb1, mAb3, and mAb6 were found to bind efficiently to Der p 1 and Der f 1 (Figure 13A). However, these antibodies did not bind to the negative control allergen Ole e 1 (Figure 13B). Compared to IgE mAb1, mAb4, and mAb6, the other IgE mAbs bound less efficiently to Der p 1 and Der f 1. Overall, these data show that the three IgE antibodies captured by the secreted IgE capture reagent and whose V(D)J regions were determined bound specifically to Der p 1 and Der f 1.

[0199] Example 7: In vivo capture and profiling of IgE plasma cells To validate in vivo IgE capture, we used the previously described IgE assay, which was exposed to house dust mite (HDM) extract for 15 weeks. Venus Reporter mice and Blimp-1 mCherrySecreted IgE was captured using Stav-FcεRIα extracellular domain as described in reporter mice, FIG. 1 (FIG. 7A). Cells were stained with an antibody mix including PC marker, αIgE for secreted IgE, and oligonucleotide barcoded HDM allergens including Der p1, Der p2, Der f1, and olive allergen (Ole e1) as a negative control. PCs were gated using Blimp-1 signal (FIG. 7B and FIG. 7C) and IgE PCs were sorted and compared with non-IgE PCs (FIG. 7B and FIG. 7C). Consistent with what was observed with U266 and Ramos (FIG. 4F), secreted IgE was only detected in IgE PCs and not other isotypes (non-IgE PCs), confirming the specificity of the trap in mixed cell populations (FIG. 7B and FIG. 7C).

[0200] Example 8: IgE plasma cells sequenced from bone marrow and lymph nodes of HDM-exposed mice To elucidate the biology of tissue-resident IgE PCs, we merged single-cell transcriptome profiles from lung dLN and BM, clustered cells, and compared IgE PCs and non-IgE PCs. We obtained 12,646 IgG+ cells, 17,524 IgA+ cells, 3,503 IgM+ cells, and 1,703 IgE+ cells (Figure 7D). From the single-cell transcriptome, we confirmed immunoglobulin expression and identified specific gene expression programs for each isotype. IgE PCs expressed high levels of Ighe, Slpi, Sdf2l1, and Pdia4. IgG PCs expressed Ighg1 and slightly higher Cd74 and B2m, while IgA PCs expressed Igha and higher levels of Cd79a and Cd69. IgM PCs expressed Ighm and higher Slc3a2, Ctss, and Ghg (Fig. 7E).

[0201] Using barcoded HDM allergens, it was possible to directly map the IgE PCs to their specificity for Der p1, Der p2, Der f1 (HDM allergens) and Ole e1 (olive allergen, negative control) and compare their AgSS calculated as described in Example 4. The antigens were Der p1, Der p2, and Der f1, and all controls were Ole e1 (olive antigen) (Figure 7F).

[0202] Several allergen-specific IgE antibodies were selected based on their reactivity profiles (see Table 3) and their relative binding to Der p1, Der p2, and Der f1, as well as to the olive allergen, Ole e1, was tested by ELISA. Two IgE antibodies (IgE mAb12_2 and mAb 3_1) bound to Der p1 (Figure 6F, Figure 19A), three IgEs (IgE mAb12_2, mAb8_2, and mAb21_2) bound to Der p2, and six IgEs (IgE mAb12_2, mAb1_2, mAb 1_1, mAb3_1, mAb4_1, and mAb 6_1) bound to Der f1. Ab12_2 bound to Ole e1 in addition to Der p1, Der p2, and Der f1 (FIG. 7G), but showed no binding to Fel d1 (FIG. 19B).

[0203] To determine whether IgE that showed HDM allergen binding had anaphylactic potential, a passive cutaneous anaphylaxis (PCA) assay was performed. Mice were intradermally sensitized in the left ear with a cocktail of Der p1 or Der p2 specific IgE (Figure 7G and Figure 19A) and in the right ear with a cocktail of irrelevant DNP-IgE as a negative control. After 24 hours, mice were intravenously administered Evans Blue containing Der p1 (for group A) or Der p2 (for group B) (Figure 7H). Mast cell degranulation, measured by Evans Blue dye leakage, was observed only in the ear (left ear) sensitized with Der p1 or Der p2 specific IgE (Figure 7H), indicating that the cocktail of IgE antibodies used induced cross-linking of FcεRIα and mast cell degranulation.

[0204] Example 9: Human primary cells: Identification and profiling of IgE PCs from bone marrow of allergic donors To determine whether IgE secretion capture could be extended to human primary cells, we performed IgE secretion capture on the BM of self-reported allergic donors. As previously described in Figure 1, B lineage enriched cells were coated with NHS-biotin and secreted IgE was captured using the StAv-FcεRIα extracellular domain. PCs were gated as CD38 high / CD20 low (Figure 20A) and the specificity and functionality of StAv-FcεRIα secretion capture was confirmed in non-allergic BM with complete biotin-StAv-FcεRIα assembly (Figure 8A) as well as in allergic BM lacking StAv-FcεRIα (Figure 8A). To perform a complete validation of human IgE secretion capture, a donor with confirmed cat, timothy grass, and HDM reactive IgE in serum was selected (Figure 8B, Figure 20B), IgE secretion capture was performed, and all IgE PCs were sorted for comparison along with non-IgE PCs from BM.

[0205] After sorting, scRNA-seq retrieved the complete transcriptome and BCR sequences of 5,559 bone marrow PCs from cat-, grass-, and dust-allergic individuals. 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 no B cell markers such as MS4A1 and CD19 were observed (Figure 8C). Ten IgE PCs were identified by VH:VL sequencing and alignment to a human germline database using IgBlast. These IgE PCs correlated very strongly with IGHE transcript expression with a small fraction of cells from IgA and IgG isotypes that also expressed IGHE, possibly reflecting continuous class switching between these isotypes or sterile IGHE transcription in some IgA and IgG cells (Figures 8C and 8D). This suggests that the disclosed capture method allows for the isolation and profiling of IgE PCs despite their rarity in human bone marrow.

[0206] 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 three amino acid mismatches, deletions, or insertions. Ten IgE PCs uniquely clustered into five clonotypes, defined as groups of PCs with highly similar heavy chain amino acid CDR3 (see Table 4). Two clonotypes, designated 753 (n=4 cells) and 696 (n=3 cells), were generated exclusively from IgE cells, and each clonotype displayed identical CDR3 sequences, respectively (Figure 8E). Two other clonotypes, designated 1330 and 576, were single cell clones derived from individual IgE cells (Figure 8E). Interestingly, clonotype 21 contained 14 IgG cells and a single IgE (Figure 8E). Closer inspection of the CDR3 nucleotide sequence of clonotype 21 revealed that the isolated IgE sequence contained three point mutations within the CDR3 region, while all IgG members of clonotype 21 had identical CDR3 regions (Figure 8F). In addition, the IgE sequence had somatic hypermutations in the upstream V locus compared to the IgG clonotype members (Figure 8F), suggesting an additional round of affinity maturation.

[0207] After selecting IgE clones, we tested the binding of purified IgE and a control (DNP-IgE) by ELISA to relevant allergens to which the donors were allergic (Fel d1 for cat, Der p1 for dust or Can e1 for dog as controls) (Figure 8G). One of the purified IgE mAbs bound to Fel d1 but not to Can e1 or Der p1 (Figure 8G), suggesting that the disclosed IgE capture method can be used to successfully isolate human IgE antibodies from allergic BM.

[0208] Example 10: Proof of concept of secreted IgG capture Preparation of secreted IgG capture reagent Anti-mouse IgK antibody was conjugated with streptavidin using the Lightning-Link® Streptavidin Conjugation Kit to form the secreted IgG capture reagent. Briefly, a solution of anti-mouse IgK antibody containing 1 mg / mL streptavidin was prepared. 100 microliters of the modification reagent was added to the solution with gentle mixing. The cap was removed from the vial of streptavidin conjugation mix, and the mixture containing the modification reagent was added directly to the lyophilized material. The material was gently resuspended by using a pipette to remove and redispense the added suspension once or twice. The cap was replaced on the vial, and the vial was left to stand in the dark at 20-25°C for 3 hours. 100 μL of the quench reagent was then added with gentle mixing. In this way, the secreted IgK capture reagent was generated.

[0209] Capture of secreted IgG: Experimental procedure Figure 3 (Step 1) shows a PC surface biotinylated with NHS-biotin. Figure 3 (Step 2) shows the Secreted IgG capture reagent bound to the biotinylated PC. Figure 3 (Step 2) also shows the anti-mouse IgK antibody of the Secreted IgG capture reagent capturing the PC secreted IgG. Figure 3 (Step 3) shows the captured IgG bound to the barcoded antigen and bound by the anti-IgG antibody.

[0210] Capture of secreted IgG on the surface of primary plasma cells To detect antigen-specific IgG-secreting PCs, VI3 / VelocImmune mice were immunized in the footpad with immunogen twice a week for 4 weeks. Mice were rested for 1 month and then boosted with immunogen 4 days later, anesthetized, sacrificed, and BM, spleen, and draining lymph nodes were obtained. Cells from bone marrow, spleen, and draining lymph nodes were prepared as described herein. Single cell preparations from spleen and lymph nodes were pooled and surface Fc receptors were blocked with Fc block diluted 1:10 for 15 min at 4°C. The pooled preparations were then stained using an antibody cocktail to detect antigen-specific IgG. +Gating was performed on 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 were obtained commercially. The surface of the PC was biotinylated as described herein, washed three times in cold PBS with 5% w / v BSA, changing tubes after each washing step, and incubated with secreted IgG capture reagent as described herein in the presence of secreted IgG capture reagent and in the absence of secreted IgE capture reagent. Cells were incubated for 1 hour at 37°C on a rotating platform to allow capture of secreted Ig antibodies and stained for 30 minutes at 4°C with an antibody mix containing fluorescein isothiocyanate-labeled anti-IgG (BD Biosciences and Southern Biotech) in PBS with 5% BSA to detect captured IgG and barcoded phycoerythrin-labeled immunogen. Labeled PCs were sorted on a BD FACSSymphony® S6 flow cytometer to identify PCs with captured secreted IgG (Figure 14B). Controls were used to detect antigen-specific populations with captured secreted IgG on their cell surface (Figure 14C).

[0211] Example 11: Preparation of single cell libraries and sequencing of plasma cells with secreted IgG captured on their surface For IgG-secreting PCs with captured IgG, gene expression libraries, V(D)J regions of secreted IgG, and barcoded antigens were prepared and sequenced in the manner described in Example 3. Individual libraries were also mapped, quantified, normalized, and processed in the manner described in Example 4. Due to the humanized nature of the V(D)J regions of the VelocImmune® mice, the V(D)J libraries were mapped to a standard human germline IMGT reference. After processing the three single cell libraries, gene expression and V(D)J region sequencing data were used to subset PCs with valid IgG constant regions and detectable expression of IgG and CD79a, but lacking expression of Ms4a1 and CD19, typically found in non-PC B cells. To select antigen-specific IgG-expressing PCs, antigen specificity scores were calculated for the target immunogen against a control antigen (hIL-6Ra) as described in Example 4. Results are shown in Figure 15, UMAP of bone marrow in Figures 15A and 15B, and spleen in Figures 15C and 15D. The x-axis of Figures 15B and 15D represents the median log ratio of the immunogen antigen signal, and the y-axis represents the median log ratio of the hIL-6Rα antigen signal. Figure 15D shows only plasma cells that lack expression of CD19 and MS4A1, but are determined by expression of CD79A. Cells are colored by plasma cell isotype, as determined from VDJ library sequencing.

[0212] Example 12: IgG and Ag+ ELISA To test antigen specificity and IgG expression of BCR sequences from IgG secretion traps, hIL-4Rα antigen or anti-human IgG (Jackson Labs) were plated at 1 ug / mL or 2 ug / mL, respectively, overnight at +4°C. The next day, plates were washed 3 times in PBS supplemented with 0.05% Tween® 20 (PBS-T) and blocked with PBS with 0.5% BSA for 1 hour at room temperature. Plates were then washed 3 times with PBS-T and diluted supernatants from Expi293F cells transfected with individual BCRs from BMPC, PB / PC-like, or splenic B cell fractions were plated for 1 hour at room temperature. Plates were then washed 3 times with PBS-T and incubated with anti-human IgG-HRP antibody (Jackson Labs) at 1:10,000 for 1 hour at room temperature. Plates were washed 3 times in PBS-T and developed using TMB substrate, stopped using 1N sulfuric acid, and read at 450 nm.

[0213] Example 13: Identification and isolation of antigen-specific IgG antibody-secreting cells from immunized mice The αIgk antibody was conjugated to streptavidin to capture secreted antibodies using the kappa light chain as described in Figure 1. The StAv-αIgk capture complex was then used on NHS-biotin coated ASCs from VelocImmune® mice immunized with human IL-4Ra (hIL-4Ra). + / IgG + Detection of secreting plasma cells was achieved by staining cells with anti-mouse IgG and barcoded hIL-4Ra antigen. Barcoded hIL-6Ra was used as a negative control (Figure 5A).

[0214] As shown in Figs. 5 and 17, Ag + / IgG +To detect ASCs, mice were immunized with hIL-4Ra monomer protein via the footpad (Figures 5A and 17A). Single cell suspensions of BM cells were generated and B lineage cells were enriched by negative selection (Figure 5A). As described herein, cells were coated with NHS-biotin and secreted IgG was captured with StAv-aIgk (Figure 1). After assembling the biotin-StAv-aIgk complex on the surface, enriched B cells / ASCs were stained with an antibody mix including 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 pooled and stained using a similar antibody cocktail to compare VH:VL sequences from Ag+ B cells and plasmablasts (PB) to BMPCs (Figures 17B and 17C). BM-derived Ag + / IgG + PCs were sorted using staining controls along with spleen / dLN-derived B cells expressing surface BCR and ASCs (PB / PC) (Figure 5B, and Figures 17B, 17C, and 17D).

[0215] 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. Barcoded antigen counts for hIL-4Ra and the negative control antigen hIL-6Ra revealed Ag + / IgG +The cells were validated (Figure 5C). To prioritize specificity and minimize polyreactivity, an antigen specificity score (AgSS) was determined to select cells with strong specificity for hIL-4Ra and minimal hIL-6Ra binding. Cells with high AgSS were prioritized for hIL-4Ra, and cells with minimal barcoded UMI numbers were prioritized for the control antigen hIL-6Ra (Figure 5C). Combined transcriptional analysis and unbiased clustering showed that either spleen- or BM-derived plasma cells clustered together and expressed classical PC markers such as Slamf7, Xbp1, Sdc1, Prdm1, and Slc3a2 (Figure 5D, Figure 18A). However, splenic B cells clustered very differently from plasma cells and expressed typical B cell markers such as Ms4a1 and Cd79a (Figure 18A). In addition, Ag+ cells with high AgSS (>0.5) from both BM and spleen generally clustered together (Figure 5D). Specifically, in BM PCs, cells with high AgSS clustered together at the bottom of the UMAP (Figure 5D).

[0216] Example 14: Avidity of antigen-specific IgG plasma cells isolated from bone marrow and spleen / dLN B cells Three distinct cell populations were obtained by scRNA-seq: bone marrow plasma cells (BMPC), spleen / dLN plasmablasts / plasmacytoids (Sp / dLN PB / PC), and spleen / dLN B cells (Sp / dLN B cells). From each of these populations, 20 unique VH:VL sequences from cells with the highest hIL-4Rα barcode signal were selected for cloning and expression analysis (Figure 18B and Figure 18C). Twenty heavy and light chain variable sequences from BMPC, 19 from Sp / dLN B cells, and 19 from Sp / dLN PB / PC were successfully cloned into vectors expressing human IgG4 and human Igk constants (see Table 1).

[0217] Consistent IgG expression was observed across all three populations, but anti-hIL-4Rα activity was observed in 80% (16 / 20) of BMPCs compared to 32% (6 / 19) and 53% (10 / 19) of Sp / dLN PB / PCs and Sp / dLN B cells, respectively (Figure 5E, Table 1). Considering the various levels of IgG in the supernatants, BMPCs also had a higher median ratio of hIL-4Ra to total IgG binding, indicating that these sequences would have higher affinity (Figure 5E). This was confirmed by Biacore analysis of supernatants and purified antibodies, with BMPC-derived antibodies having the highest affinity antibodies as measured by the negative log10 of the binding Kd (Figure 5F and Figure 18D).

[0218] Example 15: scRNA-seq of captured IgG cells Ag + After cell validation, we investigated the unique BCR sequences obtained by scRNA-seq of the VDJ region to identify the Ag + BMPCs were compared to spleen / dLN-derived B and PB / PCs. BCR sequences from all cells were mapped to specific V, D, and J gene segments by IgBlast, and the degree of somatic hypermutation was measured by percent similarity to germline V regions. Ags in all three compartments were + Ag+ cells showed a lower percent similarity to germline V regions than non-Ag+ cells, and therefore more frequent mutations than non-Ag+ cells (Figure 6A). The 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 identical CDR3 amino acid sequences in both heavy and light chains (Table 2). While 10 unique CDR3 sequence pairs were clonally shared between all three compartments, the majority of each BCR repertoire was specific to each compartment (Table 2).

[0219] In addition to BCR sequence analysis, we also compared the transcriptomes of Ag+ and non-Ag+ cells in each of the three compartments to identify gene signatures associated with hIL-4Ra specificity. In BMPCs, several gene expression programs differed based on hIL-4Ra specificity (Figures 6C and 6D). Tmsbx4, Cd24a, Tmsb10, and Fkbp11 all showed significant upregulation in hIL-4Ra-specific BMPCs. Non-Ag+ cells showed increases in Ly6d, Prg2, Tmem176a, and Tmem176b (Figures 6C and 6D). Subclustering of BMPCs showed that Cd24a, Ppib, Fkbp11, and Ssr2 transcripts were all highly expressed toward the bottom of the UMAP projection (Figure 6E), while the gene program was marked by Tmem176a, Tmem176b, Ly6d, and Cd74 expression clusters in the central and upper regions of the UMAP projection (Figure 6E). However, despite significant transcriptional differences, BCR sequences cloned from either transcriptionally distinct region yielded high affinity hIL-4Ra-specific antibodies as measured by the negative log10 of the binding Kd (Figure 6F).

[0220] Example 16: Trap-N-Seq expanded in human bone marrow to characterize short-lived vs long-lived vaccine-specific IgG plasma cells. In this example, vaccine-specific IgG+ plasma cells (PCs) were isolated from human bone marrow using TRAPnSeq. After enrichment for B cells and antibody-secreting cells (ABCs), cells were biotinylated with NHS-biotin and an affinity matrix was assembled using streptavidin coupled to anti-human Igk (StAv-αIgk) to capture secreted IgG. After assembling the biotin-StAv-αIgk complex on the surface, enriched B cells / ASCs were stained with an antibody mix containing PC-specific markers, αIgG for detection of secreted IgG, and fluorescently tagged antigens (listed in the table below) for detection of Ag+ cells.

[0221] After gating on PCs as CD20-CD38++ (Figure 21A), we validated the use of the secretion trap to isolate vaccine-specific IgG+ PCs from human bone marrow. The components required for detection of secreted IgG on the surface of PCs are shown in Figure 21B, and the components required for detection of antigen-specific IgG are shown in Figure 21C. Complete assembly of all components of the secretion trap allowed the detection of secreted IgG antibodies (Figure 21D, left) as well as antibodies that were vaccine specific (Figure 21D, right). [Table 10]

[0222] Example 17: Secretion trapping combined with antigen tracking The workflow of secretion trapping combined with antigen tracking is shown in Figure 22.

[0223] Biotinylated human IL-4 receptor a (hIL-4Ra) extracellular domain was conjugated to PE-labeled streptavidin (SA-PE) tetramer overnight at 4° C. Separately, trap antibody (mouse anti-human IgG (BD catalog no. 555784, clone no. G18-145)) was conjugated to streptavidin overnight using a commercially available kit (abcam catalog no. ab102921).

[0224] A25 cells were suspended in NHS-biotin (Sigma catalog number 203112) at a final concentration of 0.5 mg / mL in DPBS / FBS for 15 minutes at 37° C. A25 cells were derived from the commercially available A20 line by knocking out the endogenous surface BCR. A25 cells are therefore mouse B cells that do not have a surface or secreted BCR.

[0225] After incubation, cells were washed three times in DPBS / FBS and transferred to a new conical tube after each wash to remove residual biotin.

[0226] Biotinylated cells were incubated at 10 million cells / mL in DPBS / FBS with or without streptavidin-conjugated trap antibody (or "SA-anti-huIgG") for approximately 15 minutes at 4°C (performed at a 1:100 dilution). The 1:100 cell to trap antibody ratio was a ratio that ensured that the trap reagent bound to all biotinylated cells before adding to the antibody in the next step. After incubation, cells were washed twice in DPBS / FBS and then resuspended in DPBS / FBS.

[0227] Four antibodies with different affinities were used: Each individual antibody was added to the cell suspension at approximately 1 mg of antibody per 2 million cells; after incubation, cells were washed twice in DPBS / FBS. [Table 11]

[0228] The cells were then incubated with 5 nM hIL-4Ra labeled with AlexaFluor 647 ("AF647") in DPBS / FBS for 30 minutes at room temperature (RT). After incubation, the cells were washed twice in DPBS / FBS.

[0229] The cells were then incubated with pre-conjugated hIL-4Ra Biotin-StAv-PE in DPBS / FBS at room temperature for 45 min twice (i.e., 2 x 45 min). The concentration of the antigen hIL-4Ra was 20 nM. After incubation, the cells were washed twice in DPBS / FBS and analyzed by flow cytometry. As shown in Figure 23C, the four antibodies show a clear separation by affinity.

[0230] As a control, after chase antigen incubation (hIL-4Ra-biotin-StAv-PE), cells were washed twice in DPBS / FBS and then stained with anti-huIgK-PE-Cy7 antibody to detect antibody on the surface of the cells. The Ab 4 / negative control did not stain the antigen but was positive for IgK, indicating that the antibody was still captured on the cell surface.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

[0231] The present disclosure provides the following exemplary embodiments. 1. An antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule; the first member of the binding pair is capable of binding to the second member of the binding pair; the antibody capture molecule is capable of binding to a target antibody, the target antibody being secreted by an antibody-secreting cell; An antibody capture complex, wherein the antibody capture molecule does not bind to the kappa light chain of the target antibody when the second member of the binding pair is biotin and the first member of the binding pair is streptavidin. 2. The first member of the binding pair comprises avidin, streptavidin, or anti-biotin, and the second member of the binding pair comprises biotin; or the first member of the binding pair comprises one of jun and fos and the second member of the binding pair comprises the other of jun and fos; the first member of the binding pair comprises one of mad and max and the second member of the binding pair comprises the other of mad and max; or the first member of the binding pair comprises one of myc and max and the second member of the binding pair comprises the other of myc and max; or 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 2. The antibody capture complex of embodiment 1, wherein the first member of the binding pair comprises one of the azide and the phosphine, and the second member of the binding pair comprises the other of the azide and the phosphine. 3. The antibody capture complex of embodiment 1, wherein the first member of the binding pair comprises avidin or streptavidin and the second member of the binding pair comprises biotin. 4. The antibody capture complex of embodiment 1, wherein the second member of the binding pair comprises a surface marker of an antibody-secreting cell and the first member of the binding pair comprises an antibody that binds to the surface marker. 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). 6. The antibody capture complex of embodiment 1, wherein the second member of the binding pair comprises an interleukin-6 receptor (IL-6R) and the first member of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6). 7. The antibody capture complex of embodiment 1, wherein the second member of the binding pair comprises CD27 and the first member of the binding pair comprises an anti-CD27 antibody or CD70. 8. The second member of the binding pair comprises fluorescein isothiocyanate (FITC) and the first member of the binding pair comprises an anti-FITC antibody; or the second member of the binding pair comprises phycoerythrin (PE) and the first member of the binding pair comprises an anti-PE antibody; or the second member of the binding pair comprises an APC and the first member of the binding pair comprises an anti-APC antibody; or the second member of the binding pair comprises BV421 and the first member of the binding pair comprises an anti-BV421 antibody; or the second member of the binding pair comprises BV510 and the first member of the binding pair comprises an anti-BV510 antibody; or the second member of the binding pair comprises BV605 and the first member of the binding pair comprises an anti-BV605 antibody; or the second member of the binding pair comprises BV650 and the first member of the binding pair comprises an anti-BV650 antibody; or the second member of the binding pair comprises BV711 and the first member of the binding pair comprises an anti-BV711 antibody; or 2. The antibody capture complex of embodiment 1, wherein the second member of the binding pair comprises BV786 and the first member of the binding pair comprises an anti-BV786 antibody. 9. The antibody capture complex of any one of embodiments 1 to 8, wherein the antibody capture molecule comprises a capture antibody. 10. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain kappa antibody, or an anti-light chain lambda antibody. 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. 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. 13. The antibody capture complex of embodiment 11, wherein the anti-Fcα antibody comprises an anti-FcαRI antibody. 14. The antibody capture complex of embodiment 11, wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody. 15. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-IgM antibody. 16. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-IgG antibody. 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. 18. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-IgA antibody. 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. 20. The antibody capture complex of embodiment 9, wherein the capture antibody comprises an anti-IgE antibody. 21. The antibody capture complex of any one of embodiments 1 to 8, wherein the antibody capture molecule comprises an Fc receptor or an ectodomain of an Fc receptor. 22. The antibody capture complex of embodiment 21, wherein the Fc receptor comprises an Fcγ receptor, an Fcα receptor, or an Fcε receptor. 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. 24. The antibody capture complex of embodiment 22, wherein the Fcα receptor comprises an FcαRI receptor or an Fcα / μR receptor. 25. The antibody capture complex of embodiment 22, wherein the Fcε receptor comprises an FcεRI receptor or an FcεRII receptor. 26. An antibody capture complex according to any one of embodiments 1 to 8, wherein the antibody capture molecule comprises Protein A or Protein G. 27. An antibody capture complex according to any one of embodiments 1 to 8, wherein the antibody capture molecule comprises protein L. 28. The antibody capture complex of embodiment 1, wherein the antibody capture complex comprises avidin or streptavidin linked to the ectodomain 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 the population of antibody-secreting cells with a second member of the binding pair to allow the second member of the binding pair to bind to the cell surface of the population of antibody-secreting cells, the population of antibody-secreting cells comprising antibody-secreting cells that secrete the target antibody; contacting a population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to a second member of the binding pair on the cell surface of the antibody-secreting cells, whereby the antibody capture molecule captures target antibodies secreted by the antibody-secreting cells; contacting a population of antibody-secreting cells with an antigen, whereby a target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the antigen. 30. The first member of the binding pair comprises avidin, streptavidin, or anti-biotin, and the second member of the binding pair comprises biotin; or the first member of the binding pair comprises one of jun and fos and the second member of the binding pair comprises the other of jun and fos; the first member of the binding pair comprises one of mad and max and the second member of the binding pair comprises the other of mad and max; or the first member of the binding pair comprises one of myc and max and the second member of the binding pair comprises the other of myc and max; or 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 30. The antibody capture complex of embodiment 29, wherein the first member of the binding pair comprises one of the azide and the phosphine, and the second member of the binding pair comprises the other of the azide and the phosphine. 31. The method of embodiment 29, wherein the first member of the binding pair comprises avidin or streptavidin and the second member of the binding pair comprises biotin. 32. The second member of the binding pair comprises fluorescein isothiocyanate (FITC) and the first member of the binding pair comprises an anti-FITC antibody; or the second member of the binding pair comprises phycoerythrin (PE) and the first member of the binding pair comprises an anti-PE antibody; or the second member of the binding pair comprises an APC and the first member of the binding pair comprises an anti-APC antibody; or the second member of the binding pair comprises BV421 and the first member of the binding pair comprises an anti-BV421 antibody; or the second member of the binding pair comprises BV510 and the first member of the binding pair comprises an anti-BV510 antibody; or the second member of the binding pair comprises BV605 and the first member of the binding pair comprises an anti-BV605 antibody; or the second member of the binding pair comprises BV650 and the first member of the binding pair comprises an anti-BV650 antibody; or the second member of the binding pair comprises BV711 and the first member of the binding pair comprises an anti-BV711 antibody; or 30. The antibody capture complex of embodiment 29, wherein the second member of the binding pair comprises BV786 and the first member 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 with an antibody capture complex, the population of antibody-secreting cells comprising antibody-secreting cells that secrete a target antibody, the antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to a second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; contacting a population of antibody-secreting cells with an antigen, whereby a target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the antigen. 34. The method of embodiment 33, wherein the second member of the binding pair comprises a surface marker of a population of antibody-secreting cells, and the first member of the binding pair is an antibody that binds to the surface marker. 35. The method of embodiment 34, wherein the surface markers include 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 embodiment 33, wherein the second member of the binding pair comprises an interleukin-6 receptor (IL-6R) and the first member of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6). 37. The method of embodiment 33, wherein the second member of the binding pair comprises CD27 and the first member of the binding pair comprises an anti-CD27 antibody or CD70. 38. The method of any one of embodiments 29 to 37, wherein the antibody capture molecule comprises a capture antibody. 39. The method of embodiment 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 embodiment 39, wherein the anti-Fc antibody comprises an anti-Fcγ antibody, an anti-Fcα antibody, or an anti-Fcε antibody. 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. 42. The method of embodiment 40, wherein the anti-Fcα antibody comprises an anti-FcαRI antibody. 43. The method of embodiment 40, wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody. 44. The method of embodiment 38, wherein the capture antibody comprises an anti-IgM antibody. 45. The method of embodiment 38, wherein the capture antibody comprises an anti-IgG antibody. 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. 47. The method of embodiment 38, wherein the capture antibody comprises an anti-IgA antibody. 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. 49. The method of embodiment 38, wherein the capture antibody comprises an anti-IgE antibody. 50. The method of any one of embodiments 29 to 37, wherein the antibody capture molecule comprises an Fc receptor or an ectodomain of an Fc receptor. 51. The method of embodiment 50, wherein the Fc receptor comprises an Fcγ receptor, an Fcα receptor, or an Fcε receptor. 52. The method of 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. 53. The method of embodiment 51, wherein the Fcα receptor comprises an FcαRI receptor or an Fcα / μR receptor. 54. The method of embodiment 51, wherein the Fcε receptor comprises an FcεRI receptor or an FcεRII receptor. 55. The method of any one of embodiments 29 to 37, wherein the antibody capture molecule comprises Protein A or Protein G. 56. The method of any one of embodiments 29 to 37, wherein the antibody capture molecule comprises protein L. 57. The method of any one of embodiments 29-56, further comprising contacting the population of antibody-secreting cells with a secondary anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the secondary anti-Ig antibody. 58. The method of embodiment 57, wherein the secondary anti-Ig antibody is anti-IgE or anti-IgG. 59. The method of embodiment 57 or 58, wherein the secondary anti-Ig antibody is detectably labeled. 60. Secondary anti-Ig antibodies 70. The method of claim 69, which is labeled with a radioactive compound, a fluorescent compound, or an enzyme. 61. A radioactive compound is 3 H, 14 C. 19 F, 35 S, 125 I, 131 I, 111 In, or 99 61. The method of embodiment 60, comprising Tc. 62. The method of embodiment 60, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or a fluorescent protein. 63. The method of embodiment 60, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase. 64. The method of any one of embodiments 29 to 63, wherein in the step of contacting the population of antibody-secreting cells with an antigen, the antigen is a barcoded antigen. 65. The method of any one of embodiments 29 to 64, wherein in the step of contacting the population of antibody-secreting cells with an antigen, the population of antibody-secreting cells is contacted with a plurality of barcoded antigens, each barcoded antigen of the plurality of barcoded antigens comprising a unique antigen linked to a unique nucleic acid molecule. 66. The method of embodiment 65, wherein the unique nucleic acid molecule comprises DNA. 67. The method of embodiment 65, wherein the unique nucleic acid molecule comprises RNA. 68. The method of any one of embodiments 65-67, wherein each unique nucleic acid molecule comprises from about 10 nucleotides to about 500 nucleotides. 69. The method of any one of embodiments 65 to 68, wherein each barcoded antigen is detectably labeled. 70. Each barcoded antigen is 70. The method of claim 69, which is labeled with a radioactive compound, a fluorescent compound, or an enzyme. 71. A radioactive compound is 3 H, 14 C. 19 F, 35 S, 125 I, 131 I, 111 In, or 99 71. The method of embodiment 70, comprising Tc. 72. The method of embodiment 70, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or a fluorescent protein. 73. The method of embodiment 70, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase. 74. The method of any one of embodiments 64-73, wherein the barcoded antigen comprises an allergen. 75. The method of any one of embodiments 29 to 74, wherein the antibody-secreting cells are plasma cells or plasmablasts. 76. The method according to any one of embodiments 29 to 75, wherein the population of antibody-secreting cells is obtained by enriching a population of cells obtained from a human for immune cells. 77. The method of embodiment 76, wherein the population of cells obtained from a human is enriched for plasma cells or plasmablasts. 78. The method of embodiment 77, wherein the enriched population of immune cells is enriched for plasma cells or plasmablasts using pan-B cell enrichment. 79. The method of any one of embodiments 29 to 78, wherein the population of antibody-secreting cells is obtained from human lymph nodes, lungs, bone marrow, or blood. 80. The method of any one of embodiments 29-79, further comprising detecting the antigen bound by the target antibody. 81. The method of any one of embodiments 29 to 80, further comprising sorting the population of antibody-secreting cells to collect a pool of antibody-secreting cells, wherein each of the antibody-secreting cells in the pool secretes an antibody that is captured by the antibody capture molecule and bound by the antigen, and wherein the collected pool of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody. 82. The method according to embodiment 81, wherein the pool of collected antibody-secreting cells is separated into single cells and the antibody-secreting cells secreting the target antibody are isolated. 83. The method of any one of embodiments 57 to 80, further comprising detecting a secondary anti-Ig antibody. 84. The method of embodiment 83, further comprising sorting the population of antibody-secreting cells to collect a pool of antibody-secreting cells, wherein each antibody-secreting cell in the pool secretes an antibody that is captured by the antibody capture molecule and bound by the antigen and the secondary anti-Ig antibody, and wherein the collected pool of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody bound by the antigen and the secondary anti-Ig antibody. 85. The method of embodiment 84, further comprising separating the collected pool of antibody-secreting cells into single cells and isolating the antibody-secreting cells that secrete the target antibody. 86. The method of any one of embodiments 29 to 85, further comprising contacting the population of antibody-secreting cells with a second member of the binding pair after contacting the population of antibody-secreting cells with the Fc block. 87. A method for capturing IgE antibodies secreted by antibody-secreting cells, the IgE antibodies being directed against an allergen, the method comprising: contacting a population of antibody-secreting cells with NHS-biotin to allow binding of biotin to the cell surface, the population of antibody-secreting cells comprising antibody-secreting cells that secrete IgE antibodies; contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising streptavidin linked to the ectodomain of the high affinity IgE receptor (FcεRIa), whereby the antibody capture molecule binds to IgE antibodies secreted by the antibody-secreting cells; contacting the population of antibody-secreting cells with an antigen, where the antigen is an antigenic portion of an allergen or a mixture of antigenic portions, whereby the IgE antibodies captured by the antibody capture complex bind to the antigen. 88. The method of embodiment 87, wherein the antigen comprises a plurality of bar-coded antigens, each of the plurality of bar-coded antigens being a different antigenic portion of an allergen. 89. The method of embodiment 87 or 88, further comprising sorting the population of antibody-secreting cells to collect a pool of antibody-secreting cells, wherein each of the antibody-secreting cells in the pool secretes an IgE antibody that is captured by the antibody capture complex and bound by the antigen, and wherein the collected pool of antibody-secreting cells comprises antibody-secreting cells that secrete IgE antibodies. 90. The method of embodiment 87 or 88, further comprising contacting the population of antibody-secreting cells with an anti-IgE antibody to allow the IgE antibody secreted by the antibody-secreting cells and captured by the antibody capture complex to bind to the anti-IgE antibody. 91. The method of embodiment 90, further comprising sorting the population of antibody-secreting cells to collect a pool of antibody-secreting cells, wherein each of the antibody-secreting cells in the pool secretes an IgE antibody that is captured by the antibody capture complex and bound by the antigen and the anti-IgE antibody, and wherein the collected pool of antibody-secreting cells comprises antibody-secreting cells that secrete IgE antibodies. 92. The method of embodiment 89 or 91, further comprising separating the collected pool of antibody-secreting cells into single cells and isolating antibody-secreting cells that secrete IgE antibodies. 93. 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 an antigen such that the antigen can bind to antibodies, including target antibodies, captured on the cell surface of the population of antibody-secreting cells, where the antigen in the first labeled form is conjugated to a first detectable label; (b) washing the population of antibody-secreting cells to remove unbound antigen; (c) isolating the population of antibody-secreting cells; (i) an unlabeled form of the antigen; (ii) a second, labeled form of the antigen; or (iii) contacting with 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 the 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 a target antibody, and 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. 94. The method of embodiment 93, wherein the first labeled form of the antigen is at a concentration of 0.001 nM to 1 uM. 95. The method of embodiment 93, wherein the first labeled form of the antigen is at a concentration of 0.1 to 7.5 nM. 96. The method of any one of embodiments 93 to 95, wherein the first detectable label is a first fluorescent label. 97. The method of any one of embodiments 93 to 96, wherein the antigen is a protein in monomeric form. 98. The method of any one of embodiments 93 to 96, wherein the antigen is a protein in multimeric form. 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. 100. The method of any one of embodiments 97-99, wherein the first labeled form of the antigen is a monovalent form of the antigen. 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. 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. 103. The method of embodiment 102, wherein the multivalent form of the antigen is provided by a multivalent molecule to which the antigen is attached. 104. The method of 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 trimerizing molecule, such as a Foldon. 105. The method of any one of embodiments 97-104, wherein the second labeled form of the antigen is a monovalent form of the antigen. 106. The method of any one of embodiments 99-104, wherein the second labeled form of the antigen is a multivalent form of the antigen. 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. 108. The method of 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 trimerizing molecule, such as a Foldon. 109. The method of any of embodiments 106-108, wherein the multivalent form of the antigen is labeled with a second detectable label. 110. The method of embodiment 109, wherein the second detectable label is a second fluorescent label. 111. The method of embodiment 100, wherein in step (c), the population of antibody-secreting cells is contacted with an unlabeled form of the antigen. 112. The method of embodiment 111, wherein the unlabeled form of the antigen is a monovalent form of the antigen. 113. The method according to embodiment 111 or 112, wherein the unlabeled form of the antigen is in at least a 2- to 4-fold molar ratio relative to the first labeled form of the antigen used in step (a). 114. The method of embodiment 100, wherein in step (c), the population of antibody-secreting cells is contacted with a second, labeled form of the antigen. 115. The method of embodiment 114, wherein the second labeled form of the antigen is a multivalent form of the antigen. 116. The method of embodiment 114 or 115, wherein the second labeled form of the antigen is in at least a 2- to 4-fold molar ratio relative to the first labeled form of the antigen used in step (a). 117. The method according to any one of embodiments 111 to 116, wherein the contacting in step (c) and the washing in step (d) are repeated at least once before harvesting the cells in step (e). 118. The method of embodiment 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 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. 120. The method of 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. 121. The method of 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. 122. The method of embodiment 118, wherein the antigen is a protein that exists in both monomeric and multimeric forms, the unlabeled form being a monovalent form of the antigen, and the second, labeled form of the antigen being a multivalent form of the antigen. 123. The method of any one of embodiments 118-122, wherein the cell is contacted simultaneously with an unlabeled form of the antigen and a second labeled form of the antigen. 124. The method according to any one of embodiments 118 to 123, wherein the unlabeled form of the antigen is in at least a 2- to 4-fold molar ratio relative to the first labeled form of the antigen used in step (a). 125. The method of any of embodiments 93-124, wherein the first detectable label is a fluorescent label and fluorescence-activated cell sorting is used to collect cells that remain bound to the first labeled form of the antigen. 126. The method of any one of embodiments 111-124, 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, and two-dimensional fluorescence-activated cell sorting is performed to collect a pool of antibody-secreting cells that remain bound to the first labeled form of the antigen, and the collected pool of antibody-secreting cells comprises antibody-secreting cells that secrete a target antibody that remains bound to the first labeled form of the antigen. 127. The method of embodiments 93 to 126, wherein the targeting antibody has an affinity in the range of about 0.1 pM to about 25 nM (KD). 128. The method of embodiment 127, wherein the affinity is less than about 10 nM (KD). 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. 130. The method of embodiment 82, 85, 92 or 129, further comprising isolating nucleic acid encoding the antibody from isolated antibody-secreting cells that secrete the target antibody. 131. A method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, the target antibody being secreted by a cell comprising a modified cell surface, the target antibody being subsequently captured on the modified cell surface, the method comprising: a) providing an antibody-secreting cell comprising a modified cell surface, the antibody-secreting cell secreting a target antibody, which is then captured on the modified cell surface, the target antibody captured on the modified cell surface being bound to an antigen, the antigen being linked to a barcode nucleic acid molecule, the antibody-secreting cell further comprising 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) 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 a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface with a portion of a second nucleic acid molecule attached to a solid surface; d) preparing a first library of amplicons of gene expression in antibody-secreting cells, a second library of amplicons of variable, diversity and joining (VDJ) regions in antibody-secreting cells, and a third library of amplicons of antigen barcode nucleic acid molecules; e) sequencing each of the three amplicon libraries; f) identifying the region of the gene encoding the antigen-binding fragment of the target antibody that is captured on the modified cell surface. 132. The method of embodiment 131, wherein a portion of the barcode nucleic acid molecule is complementary to the first template switch oligonucleotide (TSO), and the portion of the barcode nucleic acid molecule terminates at the 3' end of the barcode nucleic acid molecule. 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 oligo (TSO). 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. 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). 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. 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. 138. The method of any of embodiments 131 to 137, wherein the first nucleic acid molecule attached to the solid surface comprises, in the 5' to 3' direction, a first sequence of three riboguanosine residues, a first TSO, a first UMI, a first surface barcode, and a first sequencing primer. 139. The method of any one of embodiments 131 to 138, wherein the first nucleic acid molecule attached to the solid surface is attached at the 3' end of the first nucleic acid molecule attached to the solid surface. 140. The method of any one of embodiments 131-139, 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 embodiment 140, wherein the first DNA molecule attached to the solid surface comprises a first single-stranded DNA molecule attached to the solid surface. 142. The method of embodiment 141, wherein the portion of the first single-stranded DNA molecule attached to the solid surface comprises a first TSO, and the portion of the barcode nucleic acid molecule hybridized to the first single-stranded DNA molecule is complementary to the 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. 143. The method of any one of embodiments 131-142, wherein the barcode nucleic acid molecule is a single-stranded DNA barcode nucleic acid molecule, 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 embodiments 131 to 143, wherein the 3' end of a portion of the barcode nucleic acid molecule comprises three consecutive cytidine or ribocytidine residues. 145. The method of embodiment 144, further comprising reverse transcribing an mRNA molecule encoding a region of the gene encoding the antigen-binding fragment of the target antibody captured on the modified cell surface, thereby generating a single-stranded cDNA molecule encoding a 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 complement 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 embodiment 145, wherein the 3' end of an mRNA molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface comprises multiple consecutive adenine residues. 147. The method of embodiment 145 or embodiment 146, wherein the 5' end of the single-stranded DNA molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface comprises multiple consecutive thymine residues. 148. The method of any one of embodiments 131-147, 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 embodiments 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 embodiments 131-149, wherein the second nucleic acid molecule attached to the solid surface further comprises a second surface barcode, and the nucleotide sequences of the first and second surface barcode nucleic acid molecules are the same. 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. 152. The method of any of embodiments 131-151, wherein the second nucleic acid molecule attached to the solid surface comprises, in the 5' to 3' direction, a second sequence of three riboguanosine residues, a second TSO, a second UMI, a second surface barcode, and a second sequencing primer, and the nucleotide sequences of the first and second surface barcode nucleic acid molecules are identical. 153. The method of any one of embodiments 131-152, wherein the second nucleic acid molecule attached to the solid surface is attached at the 3' end of the second nucleic acid molecule attached to the solid surface. 154. The method of any one of embodiments 131-153, 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 embodiment 154, wherein the second DNA molecule attached to the solid surface comprises a second single-stranded DNA molecule attached to the solid surface. 156. The method of embodiment 155, wherein the 5' end of a portion of the second single-stranded DNA molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues, and the 3' end of a portion of the nucleic acid molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface comprises three consecutive cytidine or ribocytidine residues, and 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 embodiments 131 to 156, wherein the nucleic acid molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface comprises a second single-stranded DNA molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface. 158. The method of embodiment 156, wherein the 3' end of a portion of the single-stranded DNA molecule encoding a region of the gene encoding an 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 a portion of the second single-stranded DNA molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues, and 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 a region of the gene encoding an antigen-binding fragment of the target antibody captured on the modified cell surface. 159. The method of any one of embodiments 131 to 158, wherein the antibody-secreting cells are placed in a compartment with a solid surface. 160. The method of any one of embodiments 131-159, wherein the solid surface comprises beads. 161. The method of embodiment 159 or 160, further comprising lysing antibody-secreting cells within the compartment. 162. The method of any one of embodiments 131 to 161, wherein the barcoded nucleic acid molecule further comprises a third sequencing primer, the third sequencing primer starting at the 5' end of the barcoded nucleic acid molecule. 163. The method of any one of embodiments 131 to 162, wherein each amplicon of the library of first amplicons comprises a gene, and each amplicon does not comprise a nucleic acid molecule encoding a region of the gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface. 164. The method of embodiment 163, wherein the method further comprises sample demultiplexing, aligning, filtering, and UMI counting the library of first amplicons. 165. The method of any one of embodiments 131 to 164, wherein sequencing the library of first amplicons comprises next-generation sequencing. 166. The method of any one of embodiments 131 to 165, wherein the method further comprises mapping and aligning each amplicon of the library of first amplicons to a standard reference genome. 167. The method of embodiment 166, wherein the standard reference genome comprises a human standard reference genome. 168. The method of embodiment 167, wherein the human standard reference genome is GRCh38. 169. The method of embodiment 166, wherein the standard reference genome comprises a mouse standard reference genome. 170. The method of embodiment 169, wherein the mouse standard reference genome is mm10. 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 a standard reference genome using a splice-aware aligner (STAR), each amplicon of the first amplicon library comprises a unique molecular identifier, and all amplicons of the first amplicon library that are mapped to annotated genes of the standard reference genome are binned, and the binned 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. 172. The method is to use the logarithm of the total number of UMIs counted over the total number of annotated genes. 10 172. The method of embodiment 171, further comprising filtering the single cell count matrix for high quality cells and extensively profiled genes by calculating a ratio divided by 1:1, where cells with a ratio below about 0.1 are filtered out, cells with more than about 4 times the interquartile range of the total number of UMIs counted are filtered out, and cells with more than about 80% of the reads mapped to mitochondrial genes are filtered out. 173. The method of embodiment 172, wherein the method further comprises normalizing the single cell count matrix so that the total number of counted UMIs is approximately 10,000. 174. The method of embodiment 172 or 173, wherein the method further comprises calculating a PCA embedding of the single cell count matrix and performing iterative clustering of major cell types to identify similarities across batches, thereby generating a uniform manifold approximation and projection (UMAP). 175. A method according to any of embodiments 172 to 174, wherein the method further comprises: using the cluster-specific centroids to determine a per-cell linear adjustment function; applying the per-cell linear adjustment function to correct for differences in the batch, thereby generating a batch-corrected embedding; and using the batch-corrected embedding to generate a uniform manifold approximation and projection (UMAP) in two dimensions. 176. The method of embodiment 174 or 175, wherein the method further comprises determining a weighted k-nearest neighbor graph and applying the Leiden algorithm to the weighted k-nearest neighbor graph with resolution values ​​of about 0.1, about 0.2, about 0.5, and about 1.0 to determine cell type clusters in an unsupervised manner. 177. The method of embodiment 176, wherein the method further comprises performing a pairwise Wilcox test on all of the cells in one cluster to compare the results of the first pairwise Wilcox test, and performing another pairwise Wilcox test on all of the cells in all other clusters to quantify the p-value and fold change for each gene in each cluster, where if the 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 clustered subtype named by the top genes from the differential expression results. 178. The method of any of embodiments 131 to 177, further comprising sample demultiplexing, de novo assembly of read pairs into contigs, and aligning and annotating the contigs to a germline segment V(D)J reference sequence for the second family of amplicons. 179. The method of embodiment 178, wherein alignment of the contig to the germline segment V(D)J reference sequence comprises aligning to a human germline reference database or a mouse germline reference database. 180. The method of embodiment 179, wherein the human germline reference database comprises the IMGT database of human germline immunoglobulin sequences. 181. The method of embodiment 179, wherein the mouse germline reference database comprises the IMGT germline reference database. 182. The method of any one of embodiments 179 to 181, wherein the method further comprises: labeling the VDJ sequences for quality alignment by comparing the VDJ sequences against sequences in a human germline reference database or a mouse germline reference database, thereby confirming in-frame alignment, CDR3 length, and the absence of stop codons in the variable region sequences, and filtering the VDJ sequences based on the labels. 183. The method of embodiment 182, wherein the method further comprises mapping the VDJ sequence to a reference database of immunoglobulin chains. 184. The method of embodiment 179, wherein the accuracy of immunoglobulin isotype, variable region mapping, linkage region mapping, diversity region mapping, full length variable region for both heavy and light chain sequences per cell is confirmed by alignment. 185. The method of any of embodiments 131 to 184, wherein the method further comprises sample demultiplexing, aligning, filtering, and UMI counting the third amplicon library. 186. The method of any one of embodiments 131 to 185, wherein the method further comprises mapping the library of third amplicons to a custom short read reference comprising a barcoded nucleic acid molecule reference associated with each antigen, and a count of each uniquely mapped barcoded nucleic acid molecule is summed for each cell in the barcoded antigen single cell matrix. 187. The method of embodiment 186, wherein the method further comprises quantifying the barcode nucleic acid molecules across all cells and normalizing the quantification by taking the centered log ratio of each barcode nucleic acid molecule of the plurality of barcode nucleic acid molecules across each sample capture. 188. The method of embodiment 187, wherein the method further comprises, for each barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules, removing background antigen signals by background denoising and scaling (DSB). 189. The method of embodiment 188, wherein the method further comprises determining an antigen signal distribution, and if the antigen signal distribution has a well-separated bimodal distribution, the z-transformed values ​​are used to calculate the antigen specificity, and if the antigen signal distribution does not have a well-separated bimodal distribution, quantile values ​​are used. 190. The method of embodiment 189, wherein the method further comprises statistically modeling the target antigen and negative control antigen distributions. 191. The method of any one of embodiments 131 to 190, wherein the analysis of the first amplicon library, the second amplicon library, and the third amplicon library are analyzed simultaneously to obtain at least one candidate sequence for the antigen-positive target antibody captured on the modified cell surface. 192. The method of embodiment 191, wherein the method further comprises using analysis of the first amplicon library and analysis of the second amplicon library to subset cells that have a valid antibody constant region. 193. The method of embodiment 192, wherein the antibody constant region comprises an IgE constant region. 194. A subset of antibody-secreting cells contains detectable levels of IgE heavy chain and CD79a, but lacks detectable levels of Ms4a1 and CD19, and the subset of antibody-secreting cells is IgE + 194. The method of embodiment 192 or 193, wherein the cell is an antibody-secreting cell. 195. The method comprises: detecting IgE against IgA-secreting cell isotypes, IgG-secreting cell isotypes, and / or IgM-secreting cell isotypes. +195. The method of embodiment 194, further comprising assessing differential expression of antibody secreting cells. 196. Evaluating differential expression of IgE + 196. The method of embodiment 195, further characterizing the unique transcriptional signature of the antibody-secreting cells. 197. Method: IgE + 202. The method of embodiment 196, further comprising assessing the antigen specificity of antibody-secreting cells. 198. Evaluating IgE + 200. The method of embodiment 197, comprising comparing the antigen specificity of the antibody-secreting cells to a plurality of antigens and a control antigen, wherein the plurality of antigens comprises the target antigen. 199. The comparing step includes calculating an empirical score for each of the plurality of antigens and the control antigen by subtracting a quantile value (qT) of the signal associated with the antigen from a quantile value (qC) of the signal associated with the control antigen using a penalty coefficient (x) to determine an antigen specificity score, where antigen specificity score = qT - qC. x 199. The method of embodiment 198, wherein 200. The method of embodiment 199, wherein the penalty coefficient is the same for all antigens. 201. The method of embodiment 199, wherein the penalty coefficient is different for different antibody isotypes. 202. The method of embodiment 199, wherein the penalty coefficient is different when other antigens are used. 203. The method of embodiment 199, wherein the method further comprises determining an antigen specificity score for each of the plurality of antigens and the control antigen. 204. The method of embodiment 203, wherein the method further comprises calculating a control antigen specificity score, and selecting cells if the antigen specificity score is high and the control antigen specificity score is low. 205. The method of embodiment 204, wherein the VDJ region specifies an antigen-specific antibody. 206. A method for determining if a pair of cells H :V L205. The method of embodiment 204, further comprising using the antibody sequences to select antibody candidates from the ranked list of antigen signals. 207. The method of any one of embodiments 191-206, wherein the method further comprises determining differential expression between isotypes, conditions, and / or antigen specificities, wherein determining differential expression between isotypes comprises performing a pairwise Wilcox test on all of the cells in one isotype and a pairwise Wilcox test on all of the cells in another isotype, thereby quantifying the p-value and fold change of each gene for each cluster, and determining differential expression between antigen specificities comprises performing a pairwise Wilcox test on all of the cells in one isotype and a pairwise Wilcox test on all of the cells in another isotype, thereby quantifying the p-value and fold change of each gene for each cluster. 208. The method of any one of embodiments 191 to 207, wherein the method further comprises clustering VDJ sequence similarities in antigen-positive target cells, and clustering VDJ sequence similarities comprises sequence-based alignment, grouping similar variable regions by amino acid sequence. 209. Antigen-positive target cells are IgE + The method of any one of embodiments 191 to 208, wherein 210. The method of any one of embodiments 191 to 209, wherein the method further comprises trimming redundant sequences. 211. A method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, the target antibody being secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with a second member of a binding pair, the population of antibody-secreting cells comprising antibody-secreting cells that secrete a target antibody, whereby the second member of the binding pair binds to the cell surface of the population of antibody-secreting cells; b) contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to a second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures target antibodies secreted by the antibody-secreting cells; c) contacting the population of antibody-secreting cells with a second anti-Ig antibody to allow target antibodies secreted by the antibody-secreting cells and captured by the antibody capture complex to bind to the second anti-Ig antibody; d) contacting the population of antibody-secreting cells with an antigen comprising a barcode nucleic acid molecule to allow target antibodies secreted by the antibody-secreting cells and captured by the antibody capture complex to bind to the antigen; e) harvesting a pool of antibody-secreting cells, where each cell in the pool secretes an antibody captured by the antibody capture complex and bound by the secondary anti-Ig antibody and by the antigen comprising the barcode nucleic acid molecule, whereby the pool of antibody-secreting cells comprises antibody-secreting cells that secrete the target antibody; f) 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 that includes a region of a gene encoding an antigen-binding fragment of a target antibody with a portion of a second nucleic acid molecule attached to a solid surface; 3) preparing a library of first amplicons of gene expression, a library of second amplicons of variable, diversity and joining regions (VDJ), and a library of third amplicons of antigen barcode nucleic acid molecules; 4) sequencing each of the three amplicon libraries; and g) identifying a region of the gene that encodes an antigen-binding fragment of the target antibody. 212. The first member of the binding pair comprises avidin, streptavidin, or anti-biotin, and the second member of the binding pair comprises biotin; or the first member of the binding pair comprises one of jun and fos and the second member of the binding pair comprises the other of jun and fos; the first member of the binding pair comprises one of mad and max and the second member of the binding pair comprises the other of mad and max; or the first member of the binding pair comprises one of myc and max and the second member of the binding pair comprises the other of myc and max; or 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 212. The antibody capture complex of embodiment 211, wherein the first member of the binding pair comprises one of the azide and the phosphine, and the second member of the binding pair comprises the other of the azide and the phosphine. 213. The method of embodiment 211, wherein the first member of the binding pair comprises avidin or streptavidin, and the second member of the binding pair comprises biotin. 214. The second member of the binding pair comprises fluorescein isothiocyanate (FITC) and the first member of the binding pair comprises an anti-FITC antibody; or the second member of the binding pair comprises phycoerythrin (PE) and the first member of the binding pair comprises an anti-PE antibody; or the second member of the binding pair comprises an APC and the first member of the binding pair comprises an anti-APC antibody; or the second member of the binding pair comprises BV421 and the first member of the binding pair comprises an anti-BV421 antibody; or the second member of the binding pair comprises BV510 and the first member of the binding pair comprises an anti-BV510 antibody; or the second member of the binding pair comprises BV605 and the first member of the binding pair comprises an anti-BV605 antibody; or the second member of the binding pair comprises BV650 and the first member of the binding pair comprises an anti-BV650 antibody; or the second member of the binding pair comprises BV711 and the first member of the binding pair comprises an anti-BV711 antibody; or 212. The antibody capture complex of embodiment 211, wherein the second member of the binding pair comprises BV786 and the first member 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, the target antibody being secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with an antibody capture complex, the population of antibody-secreting cells comprising antibody-secreting cells that secrete a target antibody, the antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to a second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; b) contacting the population of antibody-secreting cells with a second anti-Ig antibody to allow target antibodies secreted by the antibody-secreting cells and captured by the antibody capture complex to bind to the second anti-Ig antibody; c) contacting the population of antibody-secreting cells with an antigen comprising a barcode nucleic acid molecule, whereby target antibodies secreted by the antibody-secreting cells and captured by the antibody capture complex bind to the antigen; d) harvesting a pool of antibody-secreting cells, where each cell in the pool secretes an antibody captured by the antibody capture complex and bound by the secondary anti-Ig antibody and by the antigen comprising the barcode nucleic acid molecule, whereby 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 a target antibody with a portion of a second nucleic acid molecule attached to a solid surface; 3) preparing a first amplicon library of gene expression in antibody-secreting cells, a second amplicon library of variable, diversity, and joining (VDJ) regions in antibody-secreting cells, and a third amplicon library of antigen barcode nucleic acid molecules; 4) sequencing each of the three amplicon libraries; and f) identifying a region of the gene that encodes an antigen-binding fragment of the target antibody. 216. The method of embodiment 215, wherein the second member of the binding pair comprises a surface marker of an antibody-secreting cell, and the first member of the binding pair is an antibody that binds to the surface marker. 217. The method of embodiment 216, wherein the surface markers include 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 embodiment 215, wherein the second member of the binding pair comprises interleukin-6 receptor (IL-6R) and the first member of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6). 219. The method of embodiment 215, wherein the second member of the binding pair comprises CD27 and the first member of the binding pair comprises an anti-CD27 antibody or CD70. 220. The method of any one of embodiments 211 to 219, wherein the antibody capture molecule comprises a capture antibody. 221. The method of embodiment 220, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain kappa antibody, or an anti-light chain lambda antibody. 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. 223. The method of 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. 224. The method of embodiment 222, wherein the anti-Fcα antibody comprises an anti-FcαRI antibody. 225. The method of embodiment 222, wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody. 226. The method of embodiment 220, wherein the capture antibody comprises an anti-IgM antibody. 227. The method of embodiment 220, wherein the capture antibody comprises an anti-IgG antibody. 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. 229. The method of embodiment 220, wherein the capture antibody comprises an anti-IgA antibody. 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. 231. The method of embodiment 220, wherein the capture antibody comprises an anti-IgE antibody. 232. The method of any one of embodiments 211 to 219, wherein the antibody capture molecule comprises an Fc receptor or an ectodomain of an Fc receptor. 233. The method of embodiment 232, wherein the Fc receptor comprises an Fcγ receptor, an Fcα receptor, or an Fcε receptor. 234. The method of 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. 235. The method of embodiment 233, wherein the Fcα receptor comprises an FcαRI receptor or an Fcα / μR receptor. 236. The method of embodiment 233, wherein the Fcε receptor comprises an FcεRI receptor or an FcεRII receptor. 237. The method of any one of embodiments 211 to 219, wherein the antibody capture molecule comprises Protein A or Protein G. 238. The method of any one of embodiments 211 to 219, wherein the antibody capture molecule comprises protein L. 239. The method of any one of embodiments 211 to 238, wherein the secondary anti-Ig antibody is anti-IgE or anti-IgG. 240. The method of any one of embodiments 211-239, wherein the secondary anti-Ig antibody is detectably labeled. 241. The method of embodiment 240, wherein the secondary anti-Ig antibody is labeled with a radioactive compound, a fluorescent compound, or an enzyme. 242. A radioactive compound is 3 H, 14 C. 19 F, 35 S, 125 I, 131 I, 111 In, or 99 242. The method of embodiment 241, comprising Tc. 243. The method of embodiment 241, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or a fluorescent protein. 244. The method of embodiment 241, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase. 245. The method of any one of embodiments 211-244, wherein the antibody-secreting cell is contacted with a plurality of barcoded antigens, each barcoded antigen of the plurality of barcoded antigens comprising a unique antigen linked to a unique nucleic acid molecule. 246. The method of embodiment 245, wherein the unique nucleic acid molecule comprises DNA. 247. The method of embodiment 245, wherein the unique nucleic acid molecule comprises RNA. 248. The method of any one of embodiments 245-247, wherein each unique nucleic acid molecule comprises from about 10 nucleotides to about 500 nucleotides. 249. The method of any one of embodiments 245 to 248, wherein each barcoded antigen is detectably labeled. 250. Each barcoded antigen is 70. The method of claim 69, which is labeled with a radioactive compound, a fluorescent compound, or an enzyme. 251. A radioactive compound is 3 H, 14 C. 19 F, 35 S, 125 I, 131 I, 111 In, or 99 251. The method of embodiment 250, comprising Tc. 252. The method of embodiment 250, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or a fluorescent protein. 253. The method of embodiment 250, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase. 254. The method of any one of embodiments 211-253, wherein the barcoded antigen comprises an allergen. 255. The method of any one of embodiments 211-254, wherein the antibody-secreting cells are plasma cells or plasmablasts. 256. The method of any one of embodiments 211 to 255, wherein the method further comprises, after enriching the population of cells obtained from the human for immune cells, contacting the surface of the population of antibody-secreting cells with a second member of the binding pair or contacting the surface of the population of antibody-secreting cells with an antibody capture complex. 257. The method of embodiment 256, wherein the population of cells obtained from a human is enriched for plasma cells or plasmablasts. 258. The method of embodiment 257, wherein the enriched population of immune cells is enriched for plasma cells or plasmablasts using pan-B cell enrichment. 259. The method of any one of embodiments 211-258, wherein the population of antibody-secreting cells is obtained from human lymph nodes, lungs, bone marrow, or blood. 260. The method of any one of embodiments 211-259, wherein the method further comprises detecting one or more of a secondary anti-Ig antibody and / or a barcoded antigen. 261. The method of any one of embodiments 211 to 260, wherein the method further comprises sorting the population of antibody-secreting cells to obtain a pool of antibody-secreting cells bound by one or more of a secondary anti-Ig antibody and / or a barcoded antigen. 262. The method of any one of embodiments 211 to 261, wherein the method further comprises contacting the population of antibody-secreting cells with a second member of the binding pair after contacting the population of antibody-secreting cells with the Fc block. 263. The method of any one of embodiments 211-262, wherein a portion of the barcode nucleic acid molecule is complementary to a first template switch oligonucleotide (TSO). 264. The method of any one of embodiments 211-263, wherein the portion of the first nucleic acid molecule attached to the solid surface comprises a first template switch oligo (TSO). 265. The method of any one of embodiments 211-264, 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 embodiments 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 embodiments 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 embodiments 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 of embodiments 211 to 268, wherein the first nucleic acid molecule attached to the solid surface comprises, in the 5' to 3' direction, a first sequence of three riboguanosine residues, a first TSO, a first UMI, a first surface barcode, and a first sequencing primer. 270. The method of any one of embodiments 211-269, wherein the first nucleic acid molecule attached to the solid surface is attached at the 3' end of the first nucleic acid molecule attached to the solid surface. 271. The method of any one of embodiments 211-270, 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 embodiment 271, wherein the first DNA molecule attached to the solid surface comprises a first single-stranded DNA molecule attached to the solid surface. 273. The method of 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. 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, 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 3' end of the first TSO. 275. The method of any one of embodiments 211 to 274, wherein the 3' end of a portion of the barcode nucleic acid molecule comprises three consecutive cytidine residues. 276. The method of embodiment 275, further comprising reverse transcribing an mRNA molecule encoding a region of the gene encoding an antigen-binding fragment of the target antibody, thereby generating a single-stranded DNA molecule encoding a region of the gene encoding an antigen-binding fragment of the target antibody, 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 complement 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 embodiment 276, wherein the 3' end of an mRNA molecule encoding a region of a gene encoding an antigen-binding fragment of a target antibody comprises multiple consecutive adenine residues. 278. The method of embodiment 276 or embodiment 277, wherein the 5' end of the single-stranded DNA molecule encoding a region of the gene encoding an antigen-binding fragment of the target antibody comprises multiple consecutive thymine residues. 279. The method of any one of embodiments 211-278, 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 embodiments 211-279, wherein the second nucleic acid molecule attached to the solid surface further comprises a second UMI. 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. 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. 283. The method of any of embodiments 211-282, wherein the second nucleic acid molecule attached to the solid surface comprises, in the 5' to 3' direction, a second sequence of three riboguanosine residues, a second TSO, a second UMI, a second surface barcode, and a second sequencing primer. 284. The method of any one of embodiments 211-283, wherein the second nucleic acid molecule attached to the solid surface is attached at the 3' end of the second nucleic acid molecule attached to the solid surface. 285. The method of any one of embodiments 211-284, 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 embodiment 285, wherein the second DNA molecule attached to the solid surface comprises a second single-stranded DNA molecule attached to the solid surface. 287. The method of embodiment 286, 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 the 3' end of the portion of the nucleic acid molecule encoding a region of the gene encoding an antigen-binding fragment of the target antibody comprises three consecutive cytidine residues, and the method further comprises reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting at the 3' end of the three consecutive cytidine residues. 288. The method of any one of embodiments 211 to 287, wherein the nucleic acid molecule encoding a region of the gene encoding an antigen-binding fragment of the target antibody comprises a second single-stranded DNA molecule encoding a region of the gene encoding an antigen-binding fragment of the target antibody. 289. The method of embodiment 287, wherein the 3' end of a portion of the single-stranded DNA molecule encoding a region of the gene encoding an antigen-binding fragment of the target antibody comprises three consecutive cytidine residues and the 5' end of a portion of the second single-stranded DNA molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues, and 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 a region of the gene encoding an antigen-binding fragment of the target antibody. 290. The method of any one of embodiments 211-289, wherein each single antibody-secreting cell is placed within a compartment with a solid surface. 291. The method of any one of embodiments 211-290, wherein the solid surface comprises beads. 292. The method of embodiment 290 or embodiment 291, further comprising lysing a single antibody-secreting cell within the compartment. 293. The method of any one of embodiments 211 to 292, wherein the barcoded nucleic acid molecule further comprises a third sequencing primer. 294. The method of any one of embodiments 211 to 293, wherein each amplicon of the library of first amplicons comprises a gene, and each amplicon does not comprise a nucleic acid molecule comprising a region of the gene encoding an antigen-binding fragment of a target antibody. 295. The method of embodiment 294, wherein the method further comprises sample demultiplexing, aligning, filtering, and UMI counting the library of first amplicons. 296. The method of any one of embodiments 211 to 295, wherein sequencing the library of first amplicons comprises next-generation sequencing. 297. The method of any one of embodiments 211 to 296, wherein the method further comprises mapping and aligning each amplicon of the third library of amplicons to a standard reference genome. 298. The method of embodiment 297, wherein the standard reference genome comprises a human standard reference genome. 299. The method of embodiment 298, wherein the human standard reference genome is GRCh38. 300. The method of embodiment 297, wherein the standard reference genome comprises a mouse standard reference genome. 301. The method of embodiment 300, wherein the mouse standard reference genome is mm10. 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 a standard reference genome using a splice-aware aligner (STAR), each amplicon of the first amplicon library comprises a unique molecular identifier, and all amplicons of the first amplicon library that are mapped to annotated genes of the standard reference genome are binned, and the binned 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. 303. The method is to use the logarithm of the total number of UMIs counted for the total number of annotated genes. 10 303. The method of embodiment 302, further comprising filtering the single cell count matrix for high quality single antibody-secreting cells and extensively profiled genes by calculating a ratio divided by 0.1, filtering out single antibody-secreting cells with a ratio below about 0.1, filtering out single antibody-secreting cells that are greater than about 4 times the interquartile range of the total number of UMIs counted, and filtering out single antibody-secreting cells with greater than about 80% of reads mapped to mitochondrial genes. 304. The method of embodiment 303, wherein the method further comprises normalizing the single cell count matrix so that the total number of counted UMIs is approximately 10,000. 305. The method of embodiment 303 or embodiment 304, wherein the method further comprises calculating a PCA embedding of the single cell count matrix and performing iterative clustering of major cell types to identify similarities across batches, thereby generating a uniform manifold approximation and projection (UMAP). 306. A method according to any of embodiments 303 to 305, wherein the method further comprises: using the cluster-specific centroids to determine a per-cell linear adjustment function; applying the per-cell linear adjustment function to correct for differences in the batch, thereby generating a batch-corrected embedding; and using the batch-corrected embedding to generate a uniform manifold approximation and projection (UMAP) in two dimensions. 307. The method of embodiment 305 or embodiment 306, wherein the method further comprises determining a weighted k-nearest neighbor graph and applying the Leiden algorithm to the weighted k-nearest neighbor graph with 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 embodiment 305, wherein the method further comprises: performing a pairwise Wilcox test on all of the single antibody-secreting cells in one cluster to compare the results of the first pairwise Wilcox test; and performing another pairwise Wilcox test on all of the single antibody-secreting cells in all other clusters to quantify the p-value and fold change for each gene in each cluster; where if a gene has a low p-value and a high fold change, the single antibody-secreting cells are labeled by common markers of plasma cells, plasmablasts, or B cells; and the single antibody-secreting cells are labeled by clustered subtypes named by the top genes from the differential expression results. 309. The method of any of embodiments 211 to 308, wherein the method further comprises sample demultiplexing, de novo assembly of read pairs into contigs, and aligning and annotating the contigs to a germline segment V(D)J reference sequence for the second family of amplicons. 310. The method of embodiment 309, wherein the alignment of the contig to the germline segment V(D)J reference sequence comprises aligning to a human germline reference database or a mouse germline reference database. 311. The method of embodiment 310, wherein the human germline reference database comprises the IMGT database of human germline immunoglobulin sequences. 312. The method of embodiment 310, wherein the mouse germline reference database comprises the IMGT germline reference database. 313. The method of any one of embodiments 310 to 312, wherein the method further comprises: labeling the VDJ sequences for quality alignment by comparing the VDJ sequences against sequences in a human germline reference database or a mouse germline reference database, thereby confirming in-frame alignment, CDR3 length, and the absence of stop codons in the variable region sequences; and filtering the VDJ sequences based on the labels. 314. The method of embodiment 313, wherein the method further comprises mapping the VDJ sequence to a reference database of immunoglobulin chains. 315. The method of embodiment 310, wherein the accuracy of immunoglobulin isotype, variable region mapping, linkage region mapping, diversity region mapping, full-length variable region for both heavy and light chain sequences per cell is confirmed by alignment. 316. The method of any of embodiments 211 to 315, wherein the method further comprises sample demultiplexing, aligning, filtering, and UMI counting the third amplicon library. 317. The method of any one of embodiments 211 to 316, wherein the method further comprises mapping the library of third amplicons to a custom short read reference comprising a barcoded nucleic acid molecule reference associated with each antigen, and a count of each uniquely mapped barcoded nucleic acid molecule is summed for each cell in the barcoded antigen single cell matrix. 318. The method of embodiment 317, wherein the method further comprises quantifying the barcoded nucleic acid molecules across all single antibody-secreting cells and normalizing the quantification by taking the centered log ratio of each barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules across each sample capture. 319. The method of embodiment 318, wherein the method further comprises, for each barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules, removing background antigen signals by background denoising and scaling (DSB). 320. The method of embodiment 319, wherein the method further comprises determining an antigen signal distribution, and if the antigen signal distribution has a well-separated bimodal distribution, the z-transformed values ​​are used to calculate the antigen specificity, and if the antigen signal distribution does not have a well-separated bimodal distribution, quantile values ​​are used. 321. The method of embodiment 320, wherein the method further comprises statistically modeling the target antigen and negative control antigen distributions. 322. The method of any one of embodiments 211 to 321, wherein the analysis of the first amplicon library, the second amplicon library, and the third amplicon library are analyzed simultaneously to obtain at least one candidate sequence for the antigen-positive target antibody. 323. The method of embodiment 322, wherein the method further comprises using analysis of the first amplicon library and analysis of the second amplicon library to subset plasma cells or plasmablasts that have a valid antibody constant region. 324. The method of embodiment 323, wherein the antibody constant region comprises an IgE constant region. 325. A subset of plasma cells or plasmablasts contains detectable levels of IgE heavy chain and CD79a but lacks detectable levels of Ms4a1 and CD19, and the subset of plasma cells or plasmablasts is IgE + The method of embodiment 323 or embodiment 324, wherein the cells are plasma cells or plasmablasts. 326. The method comprises: detecting IgE against IgA-secreting plasma cells or plasmablasts isotypes, IgG-secreting plasma cells or plasmablasts isotypes, and / or IgM-secreting plasma cells or plasmablasts isotypes. + 326. The method of embodiment 325, further comprising assessing differential expression of plasma cells or plasmablasts. 327. Evaluating differential expression of IgE + The method of embodiment 326, further characterizing the unique transcriptional signature of the plasma cells or plasmablasts. 328. Method: IgE + 328. The method of embodiment 327, further comprising assessing the antigen specificity of the plasma cells or plasmablasts. 329. Evaluate IgE + 329. The method of embodiment 328, comprising comparing the antigen specificity of the plasma cells or plasmablasts to a plurality of antigens and a control antigen, wherein the plurality of antigens comprises a target antigen. 330. The comparing step includes calculating an empirical score for each of the plurality of antigens and the control antigen by subtracting a quantile value (qT) of the signal associated with the antigen from a quantile value (qC) of the signal associated with the control antigen using a penalty coefficient (x) to determine an antigen specificity score, where antigen specificity score = qT - qC. x 330. The method of embodiment 329, wherein 331. The method of embodiment 330, wherein the penalty coefficient is the same for all antigens. 332. The method of embodiment 330, wherein the penalty coefficient is different for different antibody isotypes. 333. The method of embodiment 330, wherein the penalty coefficient is different when other antigens are used. 334. The method of embodiment 331, wherein the method further comprises determining an antigen specificity score for each of the plurality of antigens and the control antigen. 335. The method of embodiment 334, wherein the method further comprises calculating a control antigen specificity score, and selecting cells if the antigen specificity score is high and the control antigen specificity score is low. 336. The method of embodiment 335, wherein the VDJ region specifies an antigen-specific antibody. 337. A method for determining the relationship between paired V H :V L 336. The method of embodiment 335, further comprising using the antibody sequences to select antibody candidates from the ranked list of antigen signals. 338. The method of any one of embodiments 322-337, wherein the method further comprises determining differential expression between isotypes, conditions, and / or antigen specificities, wherein determining differential expression between isotypes comprises performing a pairwise Wilcox test on all of the single antibody secreting cells in one isotype and a pairwise Wilcox test on all of the single antibody secreting cells in another isotype, thereby quantifying the p-value and fold change of each gene for each cluster, and determining differential expression between antigen specificities comprises performing a pairwise Wilcox test on all of the single antibody secreting cells in one isotype and a pairwise Wilcox test on all of the single antibody secreting cells in another isotype, thereby quantifying the p-value and fold change of each gene for each cluster. 339. The method of any one of embodiments 320 to 338, wherein the method further comprises clustering VDJ sequence similarities in antigen-positive target cells, and clustering VDJ sequence similarities comprises sequence-based alignment, grouping similar variable regions by amino acid sequence. 340. Antigen-positive target cells are IgE + The method of any one of embodiments 320 to 339, wherein 341. The method of any one of embodiments 320 to 340, wherein the method further comprises trimming redundant sequences. Various modifications of the described subject matter, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description, and such modifications are also intended to fall within the scope of the appended claims.

Claims

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

2. the first member of the binding pair comprises avidin, streptavidin, or anti-biotin, and the second member of the binding pair comprises biotin; or the first member of the binding pair comprises one of jun and fos, and the second member of the binding pair comprises the other of jun and fos; the first member of the binding pair comprises one of mad and max, and the second member of the binding pair comprises the other of mad and max; or the first member of the binding pair comprises one of myc and max, and the second member of the binding pair comprises the other of myc and max; or 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 212. The method of claim 211, wherein 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.

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

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

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

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

8. the second member of the binding pair comprises fluorescein isothiocyanate (FITC) and the first member of the binding pair comprises an anti-FITC antibody; or the second member of the binding pair comprises phycoerythrin (PE) and the first member of the binding pair comprises an anti-PE antibody; or the second member of the binding pair comprises an APC and the first member of the binding pair comprises an anti-APC antibody; or the second member of the binding pair comprises BV421 and the first member of the binding pair comprises an anti-BV421 antibody; or the second member of the binding pair comprises BV510 and the first member of the binding pair comprises an anti-BV510 antibody; or the second member of the binding pair comprises BV605 and the first member of the binding pair comprises an anti-BV605 antibody; or the second member of the binding pair comprises BV650 and the first member of the binding pair comprises an anti-BV650 antibody; or the second member of the binding pair comprises BV711 and the first member of the binding pair comprises an anti-BV711 antibody; or 212. The antibody capture complex of embodiment 211, wherein the second member of the binding pair comprises BV786 and the first member 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. 10. The antibody capture complex of claim 9, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain kappa antibody, or an anti-light chain lambda antibody.

11. 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.

14. 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. 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. 20. 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 ectodomain of the Fc receptor.

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

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

25. 23. 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. 2. The antibody capture complex of claim 1, wherein the antibody capture complex comprises avidin or streptavidin linked to the ectodomain of the high affinity IgE receptor (FcεRIa).

29. 1. 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 member of a binding pair such that said second member of said binding pair can bind to a cell surface of said population of antibody-secreting cells, said population of antibody-secreting cells comprising said antibody-secreting cells that secrete said target antibody; contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising a first member of the binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to the second member of the binding pair on the cell surface of the antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; contacting the population of antibody-secreting cells 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 first member of the binding pair comprises avidin, streptavidin, or anti-biotin, and the second member of the binding pair comprises biotin; or the first member of the binding pair comprises one of jun and fos, and the second member of the binding pair comprises the other of jun and fos; the first member of the binding pair comprises one of mad and max, and the second member of the binding pair comprises the other of mad and max; or the first member of the binding pair comprises one of myc and max, and the second member of the binding pair comprises the other of myc and max; or 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 212. The method of claim 211, wherein 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.

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

32. the second member of the binding pair comprises fluorescein isothiocyanate (FITC) and the first member of the binding pair comprises an anti-FITC antibody; or the second member of the binding pair comprises phycoerythrin (PE) and the first member of the binding pair comprises an anti-PE antibody; or the second member of the binding pair comprises an APC and the first member of the binding pair comprises an anti-APC antibody; or the second member of the binding pair comprises BV421 and the first member of the binding pair comprises an anti-BV421 antibody; or the second member of the binding pair comprises BV510 and the first member of the binding pair comprises an anti-BV510 antibody; or the second member of the binding pair comprises BV605 and the first member of the binding pair comprises an anti-BV605 antibody; or the second member of the binding pair comprises BV650 and the first member of the binding pair comprises an anti-BV650 antibody; or the second member of the binding pair comprises BV711 and the first member of the binding pair comprises an anti-BV711 antibody; or 30. The antibody capture complex of claim 29, wherein the second member of the binding pair comprises BV786 and the first member of the binding pair comprises an anti-BV786 antibody.

33. 1. A method for capturing a target antibody secreted by an antibody-secreting cell, the method comprising: contacting a population of antibody-secreting cells with an antibody capture complex, said population of antibody-secreting cells comprising said antibody-secreting cells that secrete said target antibody, said antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby said first member of said binding pair binds to a second member of said binding pair on the cell surface of said population of antibody-secreting cells, whereby said antibody capture molecule captures the target antibody secreted by said antibody-secreting cells; contacting the population of antibody-secreting cells 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. 34. The method of claim 33, wherein the second member of the binding pair comprises a surface marker of the population of antibody-secreting cells and the first member of the binding pair is an antibody that binds to the surface marker.

35. 35. The method of claim 34, wherein the surface markers include 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. 34. The method of claim 33, wherein the second member of the binding pair comprises interleukin-6 receptor (IL-6R) and the first member of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6).

37. 34. The method of claim 33, wherein the second member of the binding pair comprises CD27 and the first member 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. 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. 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. 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-Fcα antibody comprises an anti-FcαRI antibody.

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

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

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

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

48. 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. 39. 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 ectodomain of the Fc receptor.

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

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

54. 52. 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. 57. The method of any one of claims 29-56, further comprising contacting the population of antibody-secreting cells with a secondary anti-Ig antibody, whereby the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex binds to the secondary anti-Ig antibody.

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

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

60. The secondary anti-Ig antibody is 60. The method of claim 59, which is labeled with a radioactive compound, a fluorescent compound, or an enzyme.

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

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

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

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

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

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

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

68. 68. The method of any one of claims 65-67, wherein each unique nucleic acid molecule comprises from 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. Each barcoded antigen is 70. The method of claim 69, which is labeled with a radioactive compound, a fluorescent compound, or an enzyme.

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

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

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

74. 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 cells are plasma cells or plasmablasts.

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

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

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

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

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

81. 81. The method of any one of claims 29 to 80, further comprising sorting the population of antibody-secreting cells to collect a pool of antibody-secreting cells, 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 wherein the collected pool of antibody-secreting cells comprises the antibody-secreting cells that secrete the target antibody.

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

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

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

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

86. 86. The method of any one of claims 29 to 85, further comprising contacting the population of antibody-secreting cells with the second member of the binding pair after contacting the population of antibody-secreting cells with an Fc block.

87. 1. A method for capturing IgE antibodies secreted by antibody-secreting cells, the IgE antibodies being directed against an allergen, the method comprising: contacting a population of antibody-secreting cells with NHS-biotin to allow biotin to bind to the cell surface, said population of antibody-secreting cells comprising said antibody-secreting cells that secrete said IgE antibodies; contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising streptavidin linked to the ectodomain of the high affinity IgE receptor (FcεRIa), whereby the antibody capture molecule binds to the IgE antibodies secreted by the antibody-secreting cells; contacting the population of antibody-secreting cells with an antigen, the antigen being an antigenic portion or a mixture of antigenic portions of the allergen, whereby the IgE antibodies captured by the antibody capture complex bind to the antigen.

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

89. 89. The method of claim 87 or 88, further comprising sorting the population of antibody-secreting cells to collect a pool of antibody-secreting cells, wherein each of the antibody-secreting cells in the pool secretes an IgE antibody that is captured by the antibody capture complex and bound by the antigen, and wherein the collected pool of antibody-secreting cells comprises the antibody-secreting cells that secrete the IgE antibody.

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

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

92. 92. The method of claim 89 or 91, further comprising separating the collected pool of antibody-secreting cells into single cells and isolating the antibody-secreting cells that secrete the IgE antibodies.

93. contacting the population of antibody-secreting cells with an antigen, (a) contacting the population of antibody-secreting cells with a first labeled form of the antigen such that the antigen can bind to antibodies, including target antibodies, captured on the cell surface of the population of antibody-secreting cells, wherein the antigen in the first labeled form is conjugated to a first detectable label; (b) washing the population of antibody-secreting cells to remove unbound antigen; (c) isolating said population of antibody-secreting cells by (i) an unlabeled form of the antigen; (ii) a second, labeled form of the antigen; or (iii) contacting with 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; (e) collecting the 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 the 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.

94. 94. The method of claim 93, wherein the first labeled form of the antigen is at a concentration of 0.001 nM to 1 uM.

95. 94. The method of claim 93, wherein the first labeled form of the antigen is at a concentration of 0.1 to 7.5 nM.

96. 96. The method of any one of claims 93 to 95, wherein the first detectable label is a first fluorescent label.

97. 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 multimeric form.

99. 97. 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. 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. 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. 101. 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. 103. The method of claim 102, wherein the multivalent form of the antigen is provided by a multivalent molecule to which the antigen is attached.

104. 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 trimerizing molecule, such as Foldon.

105. 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. 105. The method of any one of claims 99 to 104, wherein the second labeled form of the antigen is a multivalent form of the antigen.

107. 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. 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 trimerizing molecule such as Foldon.

109. The method of any of claims 106 to 108, wherein the multivalent form of the antigen is labeled with a second detectable label.

110. 110. The method of claim 109, wherein the second detectable label is a second fluorescent label.

111. 101. The method of claim 100, wherein in step (c), the population of antibody-secreting cells is contacted with an unlabeled form of the antigen.

112. 112. The method of claim 111, wherein the unlabeled form of the antigen is a monovalent form of the antigen.

113. 113. The method of claim 111 or 112, wherein the unlabeled form of the antigen is in at least a 2-4 fold molar ratio to the first labeled form of the antigen used in step (a).

114. 101. The method of claim 100, wherein in step (c), the population of antibody-secreting cells is contacted with the second, labeled form of the antigen.

115. 115. The method of claim 114, wherein the second labeled form of the antigen is a multivalent form of the antigen.

116. 116. The method of claim 114 or 115, wherein the antigen in the second labeled form is in at least a 2-4 fold molar ratio to the first labeled form of the antigen used in step (a).

117. 117. The method of claims 111-116, wherein the contacting of step (c) and the washing of step (d) are repeated at least once prior to harvesting the cells in step (e).

118. 101. 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. 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. 119. 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. 119. 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. 119. 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. 123. The method of any one of claims 118-122, wherein the cell is contacted with the unlabeled form of the antigen and the second labeled form of the antigen simultaneously.

124. 124. The method of any one of claims 118 to 123, wherein the unlabelled form of the antigen is in at least a 2- to 4-fold molar ratio to the first labelled form of the antigen used in step (a).

125. 125. The method of any of claims 93-124, wherein the first detectable label is a fluorescent label and fluorescence activated cell sorting is used to collect cells that remain bound to the first labeled form of the antigen.

126. 125. The method of any one of claims 111 to 124, 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, and wherein two-dimensional fluorescence activated cell sorting is performed to collect 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 the antibody-secreting cells that secrete the target antibody that remains bound to the first labeled form of the antigen.

127. The method of claims 93-126, wherein the targeting antibody has an affinity in the range of about 0.1 pM to about 25 nM (KD).

128. 128. The method of claim 127, wherein the affinity is less than about 10 nM (KD).

129. 129. The method of any one of claims 93-128, 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. 130. The method of claim 82, 85, 92 or 129, further comprising isolating antibody-encoding nucleic acid from said isolated antibody-secreting cells that secrete said target antibody.

131. 1. A method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, the target antibody being secreted by a cell comprising a modified cell surface, the target antibody being subsequently captured on the modified cell surface, the method comprising: a) providing an antibody-secreting cell comprising a modified cell surface, said antibody-secreting cell secreting a target antibody, which is then captured on said modified cell surface, said target antibody captured on said modified cell surface being bound to an antigen, said antigen being linked to a barcode nucleic acid molecule, said antibody-secreting cell further comprising a nucleic acid molecule comprising said region of the gene encoding the antigen-binding fragment of the target antibody captured on said modified cell surface; 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 a nucleic acid molecule comprising a region of a gene encoding an antigen-binding fragment of a target antibody captured on the modified cell surface with a portion of a second nucleic acid molecule attached to a solid surface; d) preparing a first amplicon library of gene expression in antibody-secreting cells, a second amplicon library of variable, diversity and joining (VDJ) regions in antibody-secreting cells, and a third amplicon library of antigen barcode nucleic acid molecules; e) sequencing each of the three amplicon libraries; f) identifying a region of the gene encoding the antigen-binding fragment of the target antibody that is captured on the modified cell surface.

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

133. 133. The method of Claim 131 or 132, wherein said portion of the first nucleic acid molecule attached to the solid surface comprises a first template switch oligo (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. 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. 136. The method of any one of claims 131-135, wherein the first nucleic acid molecule attached to the solid surface further comprises a first surface barcode.

137. 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. 138. The method of any of claims 131-137, wherein the first nucleic acid molecule attached to the solid surface comprises, in a 5' to 3' direction, the first sequence of three riboguanosine residues, the first TSO, the first UMI, the first surface barcode, and the first sequencing primer.

139. 139. The method of any one of claims 131 to 138, wherein the first nucleic acid molecule attached to the solid surface is attached at a 3' end of the first nucleic acid molecule attached to the solid surface.

140. 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. 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. 142. The method of Claim 141, wherein a portion of the first single-stranded DNA molecule attached to the solid surface comprises a first TSO, and the portion of the barcode nucleic acid molecule hybridized to the first single-stranded DNA molecule is complementary to the first TSO, and the method further comprises reverse transcribing the first single-stranded DNA molecule attached to the solid surface starting at a 3' end of the portion of the barcode nucleic acid molecule that is complementary to the first TSO.

143. 143. The method of any one of claims 131-142, wherein the barcode nucleic acid molecule is a single stranded DNA barcode nucleic acid molecule, 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 at the 5' end of the first TSO.

144. 144. The method of any one of claims 131-143, wherein the 3' end of the portion of the barcode nucleic acid molecule comprises three consecutive cytidine or ribocytidine residues.

145. 145. The method of 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 generating 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 complement 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 comprises 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 multiple consecutive adenine residues.

147. The method of claim 145 or 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 multiple consecutive thymine residues.

148. 148. The method of any one of claims 131-147, 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. 150. The method of any one of claims 131-149, wherein the second nucleic acid molecule attached to the solid surface further comprises a second surface barcode, and wherein the nucleotide sequences of the first and second surface barcode nucleic acid molecules are the same.

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

152. 152. The method of any of claims 131-151, wherein the second nucleic acid molecule attached to the solid surface comprises, in a 5' to 3' direction, the second sequence of three riboguanosine residues, a second TSO, the second UMI, the second surface barcode, and the second sequencing primer, and wherein the nucleotide sequences of the first and second surface barcode nucleic acid molecules are the same.

153. 153. The method of any one of claims 131 to 152, wherein the second nucleic acid molecule attached to the solid surface is attached at a 3' end of the second nucleic acid molecule attached to the solid surface.

154. 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. 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. 156. The method of claim 155, wherein a 5' end of a portion of the second single-stranded DNA molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues and a 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, the method further comprising reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting at the 3' end of the three consecutive cytidine or ribocytidine residues.

157. 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. 157. The method of claim 156, wherein a 3' end of a 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 a 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, the method further comprising 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.

159. The method of any one of claims 131 to 158, wherein the antibody-secreting cells are disposed within a compartment with the solid surface.

160. 160. The method of any one of claims 131 to 159, wherein the solid surface comprises a bead.

161. 161. The method of claim 159 or 160, further comprising lysing the antibody-secreting cells within the compartment.

162. 162. The method of any one of claims 131-161, wherein the barcoded nucleic acid molecule further comprises a third sequencing primer, said third sequencing primer beginning at the 5' end of the barcoded nucleic acid molecule.

163. 163. The method of any one of claims 131-162, wherein each amplicon of the first library of amplicons comprises a gene, and each amplicon does not comprise a nucleic acid molecule encoding the region of the gene that encodes the antigen-binding fragment of the target antibody captured on the modified cell surface.

164. 164. The method of claim 163, wherein the method further comprises sample demultiplexing, aligning, filtering, and UMI counting the first amplicon library.

165. 165. The method of any one of claims 131 to 164, wherein sequencing the first library of amplicons comprises next generation sequencing.

166. 166. The method of any one of claims 131 to 165, wherein the method further comprises mapping and aligning each amplicon of the first library of amplicons to a standard reference genome.

167. 167. The method of claim 166, wherein the standard reference genome comprises a human standard reference genome.

168. 168. The method of claim 167, wherein the human standard reference genome is GRCh38.

169. 167. The method of claim 166, wherein the standard reference genome comprises a mouse standard reference genome.

170. 170. The method of claim 169, wherein the mouse standard reference genome is mm10.

171. 171. The method of any one of claims 166-170, wherein said mapping and aligning comprises mapping each amplicon of said first amplicon library to said standard reference genome using a splice-aware aligner (STAR), wherein each amplicon of said first amplicon library comprises a unique molecular identifier, and all amplicons of said first amplicon library that are mapped to annotated genes of said standard reference genome are binned and said binned 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 further comprises: subtracting the logarithm of the total number of UMIs counted from the total number of annotated genes; １０ 172. The method of claim 171, further comprising filtering the single cell count matrix for high quality cells and extensively profiled genes by calculating a ratio divided by 0.1, wherein cells with a ratio below about 0.1 are filtered out, cells with more than about 4 times the interquartile range of the total number of UMIs counted are filtered out, and cells with more than about 80% of the reads mapping to mitochondrial genes are filtered out.

173. 173. The method of claim 172, wherein the method further comprises normalizing the single cell count matrix so that the total number of counted UMIs is about 10,000.

174. The method of claim 172 or 173, wherein the method further comprises calculating a PCA embedding of the single cell count matrix and performing an iterative clustering of major cell types to identify similarities across batches, thereby generating a Uniform Manifold Approximation and Projection (UMAP).

175. 175. The method of any of claims 172-174, further comprising: using cluster-specific centroids to determine a per-cell linear adjustment function; applying the per-cell linear adjustment function to correct for differences in the batch, thereby generating a batch-corrected embedding; and using the batch-corrected embedding to generate a uniform manifold approximation and projection (UMAP) in two dimensions.

176. 176. The method of claim 174 or 175, wherein the method further comprises determining a weighted k-nearest neighbor graph and applying the Leiden algorithm to the weighted k-nearest neighbor graph at resolution values ​​of about 0.1, about 0.2, about 0.5, and about 1.0 to determine cell type clusters in an unsupervised manner.

177. 177. The method of claim 176, wherein the method further comprises performing a pairwise Wilcox test on all of the cells in one cluster to compare the results of the first pairwise Wilcox test, and performing another pairwise Wilcox test on all of the cells in all other clusters to quantify the p-value and fold change for each gene in each cluster, where if a gene has a low p-value and a high fold change then the cell is labeled with a common marker of antibody secreting cells and the cell is labeled with a clustered subtype named by the top genes from the differential expression results.

178. 178. The method of any of claims 131-177, wherein the method further comprises performing sample demultiplexing, de novo assembly of read pairs into contigs, and aligning and annotating the contigs to a germline segment V(D)J reference sequence for a second family of amplicons.

179. The method of claim 178, wherein aligning the contig to a germline segment V(D)J reference sequence comprises aligning to a human germline reference database or a mouse germline reference database.

180. 180. The method of claim 179, wherein the human germline reference database comprises the IMGT database of human germline immunoglobulin sequences.

181. 180. The method of claim 179, wherein the mouse germline reference database comprises an IMGT germline reference database.

182. 182. The method of any one of claims 179-181, wherein the method further comprises: marking the VDJ sequences for quality alignment by comparing VDJ sequences against sequences of the human germline reference database or the mouse germline reference database, thereby verifying in-frame alignment, CDR3 length, and absence of stop codons in variable region sequences, and filtering the VDJ sequences based on the marking.

183. The method of claim 182, wherein the method further comprises mapping the VDJ sequence to a reference database of immunoglobulin chains.

184. The method of claim 179, wherein the accuracy of the immunoglobulin isotype, the variable region mapping, the joining region mapping, the diversity region mapping, and the full length variable region for both heavy and light chain sequences per cell is confirmed by the alignment.

185. The method of any of claims 131-184, wherein the method further comprises sample demultiplexing, aligning, filtering, and UMI counting the third library of amplicons.

186. 186. The method of any one of claims 131-185, wherein the method further comprises mapping the third library of amplicons to a custom short read reference comprising a 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.

187. 187. The method of claim 186, wherein said method further comprises quantifying the barcode nucleic acid molecules across all cells and normalizing the quantification by taking the centered log ratio of each barcode nucleic acid molecule of a plurality of barcode nucleic acid molecules across each sample capture.

188. 188. The method of claim 187, wherein the method further comprises, for each barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules, removing background antigen signal by background denoising and scaling (DSB).

189. The method of claim 188, wherein the method further comprises determining an antigen signal distribution, and if the antigen signal distribution has a well-separated bimodal distribution, z-transformed values ​​are used to calculate antigen specificity, and if the antigen signal distribution does not have a well-separated bimodal distribution, quantile values ​​are used.

190. 190. The method of claim 189, wherein the method further comprises statistically modeling the target antigen and negative control antigen distributions.

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

192. 192. The method of claim 191, wherein the method further comprises using analysis of the first amplicon library and analysis of the second amplicon library to subset cells having effective antibody constant regions.

193. The method of claim 192, wherein the antibody constant region comprises an IgE constant region.

194. The subset of antibody-secreting cells contains detectable levels of IgE heavy chain and CD79a, but lacks detectable levels of Ms4a1 and CD19, and the subset of antibody-secreting cells is ＋ 194. The method of claim 192 or 193, which is an antibody-secreting cell.

195. The method further comprises: detecting the IgE against an IgA-secreting cell isotype, an IgG-secreting cell isotype, and / or an IgM-secreting cell isotype. ＋ 195. The method of claim 194, further comprising assessing differential expression of antibody secreting cells.

196. Evaluating the differential expression of the IgE ＋ 196. The method of claim 195, further characterizing the unique transcriptional signature of antibody-secreting cells.

197. The method further comprises: ＋ 200. The method of claim 196, further comprising evaluating the antigen specificity of the antibody-secreting cells.

198. The evaluating step comprises: ＋ 200. The method of claim 197, comprising comparing the antigen specificity of antibody-secreting cells to a plurality of antigens and a control antigen, wherein the plurality of antigens includes the target antigen.

199. The comparing includes calculating an empirical score for each of the plurality of antigens and the control antigen by subtracting a quantile of signals associated with an antigen (qT) from a quantile of signals associated with the control antigen (qC) using a penalty factor (x) to determine an antigen specificity score, where the antigen specificity score=qT−qC ｘ The method of claim 198, wherein:

200. 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. 200. The method of claim 199, wherein the penalty factor is different when other antigens are used.

203. 200. The method of claim 199, wherein the method further comprises determining the antigen specificity score for each of the plurality of antigens and the control antigen.

204. 204. The method of claim 203, wherein the method further comprises calculating a control antigen specificity score, and selecting the cell if the antigen specificity score is high and the control antigen specificity score is low.

205. The method of claim 204, wherein the VDJ region specifies an antigen-specific antibody.

206. The method further comprises: Ｈ :V Ｌ 205. The method of claim 204, further comprising using the antibody sequences to select antibody candidates from the ranked list of antigen signals.

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

208. 208. The method of any one of claims 191 to 207, wherein the method further comprises clustering VDJ sequence similarities in antigen positive target cells, and wherein the clustering VDJ sequence similarities comprises sequence-based alignment, grouping similar variable regions by amino acid sequence.

209. The antigen-positive target cells are ＋ The method according to any one of claims 191 to 208, wherein

210. 210. The method of any one of claims 191 to 209, wherein the method further comprises trimming redundant sequences.

211. 1. A method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, the target antibody being secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with a second member of a binding pair, said population of antibody-secreting cells comprising said antibody-secreting cells that secrete said target antibody, whereby said second member of said binding pair binds to the cell surface of said population of antibody-secreting cells; b) contacting the population of antibody-secreting cells with an antibody capture complex, the antibody capture complex comprising a first member of the binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to the second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; c) contacting the population of antibody-secreting cells with a secondary anti-Ig antibody to allow the target antibodies secreted by the antibody-secreting cells and captured by the antibody capture complex to bind to the secondary anti-Ig antibody; d) contacting the population of antibody-secreting cells with an antigen comprising a barcode nucleic acid molecule to allow the target antibody secreted by the antibody-secreting cells and captured by the antibody capture complex to bind to the antigen; e) harvesting a pool of antibody-secreting cells, each cell in the pool secreting an antibody captured by the antibody capture complex and bound by the secondary anti-Ig antibody and by the antigen comprising the barcode nucleic acid molecule, the pool of antibody-secreting cells comprising the antibody-secreting cells secreting the target antibody; f) 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 comprising a region of a gene encoding an antigen-binding fragment of a target antibody with a portion of a second nucleic acid molecule attached to a solid surface; 3) preparing a library of first amplicons of gene expression, a library of second amplicons of variable, diversity and joining regions (VDJ), and a library of third amplicons of antigen barcode nucleic acid molecules; 4) sequencing each of the three amplicon libraries; and g) identifying a region of the gene that encodes an antigen-binding fragment of the target antibody.

212. the first member of the binding pair comprises avidin, streptavidin, or anti-biotin, and the second member of the binding pair comprises biotin; or the first member of the binding pair comprises one of jun and fos, and the second member of the binding pair comprises the other of jun and fos; the first member of the binding pair comprises one of mad and max, and the second member of the binding pair comprises the other of mad and max; or the first member of the binding pair comprises one of myc and max, and the second member of the binding pair comprises the other of myc and max; or 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 212. The method of claim 211, wherein 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.

213. 212. The method of claim 211, wherein the first member of the binding pair comprises avidin or streptavidin and the second member of the binding pair comprises biotin.

214. the second member of the binding pair comprises fluorescein isothiocyanate (FITC) and the first member of the binding pair comprises an anti-FITC antibody; or the second member of the binding pair comprises phycoerythrin (PE) and the first member of the binding pair comprises an anti-PE antibody; or the second member of the binding pair comprises an APC and the first member of the binding pair comprises an anti-APC antibody; or the second member of the binding pair comprises BV421 and the first member of the binding pair comprises an anti-BV421 antibody; or the second member of the binding pair comprises BV510 and the first member of the binding pair comprises an anti-BV510 antibody; or the second member of the binding pair comprises BV605 and the first member of the binding pair comprises an anti-BV605 antibody; or the second member of the binding pair comprises BV650 and the first member of the binding pair comprises an anti-BV650 antibody; or the second member of the binding pair comprises BV711 and the first member of the binding pair comprises an anti-BV711 antibody; or 212. The antibody capture complex of embodiment 211, wherein the second member of the binding pair comprises BV786 and the first member of the binding pair comprises an anti-BV786 antibody.

215. 1. A method for identifying a region of a gene encoding an antigen-binding fragment of a target antibody, the target antibody being secreted by an antibody-secreting cell, the method comprising: a) contacting a population of antibody-secreting cells with an antibody capture complex, the population of antibody-secreting cells comprising the antibody-secreting cells that secrete the target antibody, the antibody capture complex comprising a first member of a binding pair linked to an antibody capture molecule, whereby the first member of the binding pair binds to a second member of the binding pair on the cell surface of the population of antibody-secreting cells, whereby the antibody capture molecule captures the target antibody secreted by the antibody-secreting cells; b) contacting the population of antibody-secreting cells with a second anti-Ig antibody to allow the target antibodies secreted by the antibody-secreting cells and captured by the antibody capture complex to bind 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 cells and captured by the antibody capture complex binds to the antigen; d) collecting a pool of antibody-secreting cells, each cell in the pool secreting an antibody captured by the antibody capture complex and bound by the secondary anti-Ig antibody and by the antigen comprising the barcode nucleic acid molecule, the pool of antibody-secreting cells comprising the antibody-secreting cells secreting 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 a target antibody with a portion of a second nucleic acid molecule attached to a solid surface; 3) preparing a first amplicon library of gene expression in antibody-secreting cells, a second amplicon library of variable, diversity and joining regions (VDJ) in antibody-secreting cells, and a third amplicon library of antigen barcode nucleic acid molecules; 4) sequencing each of the three amplicon libraries; and f) identifying a region of the gene that encodes an antigen-binding fragment of the target antibody.

216. 216. The method of claim 215, wherein the second member of the binding pair comprises a surface marker of the antibody-secreting cell and the first member of the binding pair is an antibody that binds to the surface marker.

217. 217. The method of claim 216, wherein the surface markers include 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. 216. The method of claim 215, wherein the second member of the binding pair comprises interleukin-6 receptor (IL-6R) and the first member of the binding pair comprises an anti-IL-6R antibody or interleukin-6 (IL-6).

219. 216. The method of claim 215, wherein the second member of the binding pair comprises CD27 and the first member 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. 221. The method of claim 220, wherein the capture antibody comprises an anti-Fc antibody, an anti-light chain kappa antibody, or an anti-light chain lambda 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-Fcα antibody comprises an anti-FcαRI antibody.

225. The method of claim 222, wherein the anti-Fcε antibody comprises an anti-FcεRI antibody or an anti-FcεRII antibody.

226. 221. 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. 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. 221. The method of claim 220, wherein the capture antibody comprises an anti-IgA antibody.

230. 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. 221. 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 ectodomain 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. 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. 220. 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 secondary anti-Ig antibody is anti-IgE or anti-IgG.

240. The method of any one of claims 211 to 239, wherein the secondary anti-Ig antibody is detectably labeled.

241. The method of claim 240, wherein the secondary anti-Ig antibody is labeled with a radioactive compound, a fluorescent compound, or an enzyme.

242. The radioactive compound is ３ H. １４ C. １９ F. ３５ S. １２５ I, １３１ I, １１１ In, or ９９ The method of claim 241, comprising Tc.

243. 242. The method of claim 241, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or a fluorescent protein.

244. 242. The method of claim 241, wherein the enzyme comprises alkaline phosphatase, horseradish peroxidase, luciferase, or glucose oxidase.

245. 245. The method of any one of claims 211-244, wherein the antibody-secreting cell is contacted with a plurality of barcoded antigens, each barcoded antigen of the plurality comprising 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. 248. The method of any one of claims 245-247, wherein each unique nucleic acid molecule comprises from about 10 nucleotides to about 500 nucleotides.

249. The method of any one of claims 245-248, wherein each barcoded antigen is detectably labeled.

250. Each barcoded antigen is 250. The method of claim 249, which is labeled with a radioactive compound, a fluorescent compound, or an enzyme.

251. The radioactive compound is ３ H. １４ C. １９ F. ３５ S. １２５ I, １３１ I, １１１ In, or ９９ The method of claim 250, comprising Tc.

252. 251. The method of claim 250, wherein the fluorescent compound comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, or a fluorescent protein.

253. 251. 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 211-253, wherein the barcoded antigen comprises an allergen.

255. The method of any one of claims 211 to 254, wherein the antibody secreting cells are plasma cells or plasmablasts.

256. 256. The method of any one of claims 211 to 255, wherein the method further comprises, after enriching a population of cells obtained from a human for immune cells, contacting a surface of the population of antibody-secreting cells with the second member of a binding pair or contacting a surface of the population of antibody-secreting cells with the antibody capture complex.

257. The method of claim 256, wherein the population of cells obtained from the human is enriched for plasma cells or plasmablasts.

258. The method of claim 257, wherein the enriched population of immune cells is enriched for plasma cells or plasmablasts using pan-B cell enrichment.

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

260. 260. The method of any one of claims 211-259, wherein the method further comprises detecting one or more of the secondary anti-Ig antibody and / or the barcoded antigen.

261. 261. The method of any one of claims 211-260, wherein the method further comprises sorting the population of antibody-secreting cells to obtain a pool of antibody-secreting cells bound by one or more of a secondary anti-Ig antibody and / or the barcoded antigen.

262. 262. The method of any one of claims 211-261, wherein the method further comprises contacting the population of antibody secreting cells with the second member of the binding pair after contacting the population of antibody secreting cells with an Fc block.

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

264. 264. The method of any one of Claims 211-263, wherein said portion of said first nucleic acid molecule attached to said solid surface comprises a first template switch oligo (TSO).

265. The method of any one of claims 211-264, 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. 266. The method of any one of claims 211-265, wherein the first nucleic acid molecule attached to the solid surface further comprises a first unique molecular identifier (UMI).

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

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

269. 269. The method of any of claims 211-268, wherein the first nucleic acid molecule attached to the solid surface comprises, in a 5' to 3' direction, the first sequence of three riboguanosine residues, the first TSO, the first UMI, the first surface barcode, and the first sequencing primer.

270. 270. The method of any one of claims 211 to 269, wherein the first nucleic acid molecule attached to the solid surface is attached at a 3' end of the first nucleic acid molecule attached to the solid surface.

271. 271. The method of any one of claims 211-270, wherein the first nucleic acid molecule attached to the solid surface comprises a first DNA molecule attached to the solid surface.

272. 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. 273. The method of claim 272, wherein a portion of the first single-stranded DNA molecule attached to the solid surface comprises a first TSO, the method further comprising reverse transcribing the first single-stranded DNA molecule attached to the solid surface starting at a 3' end of the portion of the barcode nucleic acid molecule that is complementary to the first TSO.

274. 274. The method of any one of claims 211-273, wherein the barcode nucleic acid molecule is a single stranded DNA barcode nucleic acid molecule, 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 at the 3' end of the first TSO.

275. The method of any one of claims 211-274, wherein the 3' end of the portion of the barcode nucleic acid molecule comprises three consecutive cytidine residues.

276. 276. The method of claim 275, further comprising reverse transcribing an mRNA molecule encoding said region of said gene encoding said antigen binding fragment of the target antibody, thereby generating a single stranded DNA molecule encoding said region of said gene encoding said antigen binding fragment of the target antibody, wherein the nucleotide sequence of said single stranded DNA molecule encoding said region of said gene encoding said antigen binding fragment of the target antibody differs from the complement of the mRNA molecule encoding said region of said gene encoding said antigen binding fragment of the target antibody in that the 3' end of said single stranded DNA molecule encoding said region of said gene encoding said 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 multiple consecutive adenine residues.

278. The method of claim 276 or 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 multiple consecutive thymine residues.

279. The method of any one of claims 211-278, 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. 280. The method of any one of claims 211-279, wherein the second nucleic acid molecule attached to the solid surface further comprises a second UMI.

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

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

283. 283. The method of any of claims 211-282, wherein the second nucleic acid molecule attached to the solid surface comprises, in a 5' to 3' direction, the second sequence of three riboguanosine residues, the second TSO, the second UMI, the second surface barcode, and the second sequencing primer.

284. 284. The method of any one of claims 211 to 283, wherein the second nucleic acid molecule attached to the solid surface is attached at a 3' end of the second nucleic acid molecule attached to the solid surface.

285. 285. The method of any one of claims 211-284, wherein the second nucleic acid molecule attached to the solid surface comprises a second DNA molecule attached to the solid surface.

286. 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. 287. The method of claim 286, wherein a 5' end of a portion of the second single-stranded DNA molecule attached to the solid surface comprises a second sequence of three consecutive riboguanosine residues and a 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 comprises three consecutive cytidine residues, the method further comprising reverse transcribing the second single-stranded DNA molecule attached to the solid surface starting at the 3' end of the three consecutive cytidine residues.

288. 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. 288. The method of claim 287, wherein a 3' end of a portion of the single stranded DNA molecule encoding the region of the gene encoding the antigen binding fragment of the target antibody comprises three consecutive cytidine residues and a 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, the method further comprising reverse transcribing the second single stranded DNA molecule attached to the solid surface starting at 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. 290. The method of any one of claims 211-289, wherein each said single antibody-secreting cell is disposed within a compartment with a solid surface.

291. 291. The method of any one of claims 211 to 290, wherein the solid surface comprises a bead.

292. 292. The method of claim 290 or 291, further comprising lysing the single antibody-secreting cell within the compartment.

293. 293. The method of any one of claims 211-292, wherein the barcoded nucleic acid molecule further comprises a third sequencing primer.

294. 30. The method of any one of claims 211-293, wherein each amplicon of the first library of amplicons comprises a gene, and wherein each amplicon does not comprise a nucleic acid molecule comprising the region of the gene that encodes the antigen-binding fragment of the target antibody.

295. 300. The method of claim 294, wherein the method further comprises sample demultiplexing, aligning, filtering, and UMI counting the first amplicon library.

296. 300. The method of any one of claims 211-295, wherein sequencing the library of first amplicons comprises next generation sequencing.

297. 300. The method of any one of claims 211 to 296, wherein said method further comprises mapping and aligning each amplicon of said third library of amplicons to a standard reference genome.

298. 300. The method of claim 297, wherein the standard reference genome comprises a human standard reference genome.

299. 300. The method of claim 298, wherein the human standard reference genome is GRCh38.

300. 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. 302. The method of any one of claims 297-301, wherein said mapping and aligning comprises mapping each amplicon of said first amplicon library to said standard reference genome using a splice-aware aligner (STAR), wherein each amplicon of said first amplicon library comprises a unique molecular identifier, and all amplicons of said first amplicon library that are mapped to annotated genes of said standard reference genome are binned and said binned 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.

303. The method further comprises: subtracting the logarithm of the total number of UMIs counted from the total number of annotated genes; １０ 303. The method of claim 302, further comprising filtering the single cell count matrix for high quality single antibody-secreting cells and extensively profiled genes by calculating a ratio divided by 0.1, wherein single antibody-secreting cells with a ratio below about 0.1 are filtered out, single antibody-secreting cells that are greater than about 4 times the interquartile range of the total number of UMIs counted are filtered out, and single antibody-secreting cells with greater than about 80% of reads mapped to mitochondrial genes are filtered out.

304. The method of claim 303, wherein the method further comprises normalizing the single cell count matrix so that the total number of counted UMIs is approximately 10,000.

305. The method of claim 303 or 304, wherein the method further comprises computing a PCA embedding of the single cell count matrix and performing iterative clustering of major cell types to identify similarities across batches, thereby generating a Uniform Manifold Approximation and Projection (UMAP).

306. 306. The method of any of claims 303-305, further comprising: using cluster-specific centroids to determine a per-cell linear adjustment function; applying the per-cell linear adjustment function to correct for differences in the batch, thereby generating a batch-corrected embedding; and using the batch-corrected embedding to generate a uniform manifold approximation and projection (UMAP) in two dimensions.

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

308. 306. The method of claim 305, wherein said method further comprises performing a pairwise Wilcox test on all of said single antibody secreting cells in one cluster to compare the results of said first pairwise Wilcox test, and performing another pairwise Wilcox test on all of said single antibody secreting cells in all other clusters to quantify the p-value and fold change for each gene in each cluster, where if a gene has a low p-value and high fold change then said single antibody secreting cells are marked by common markers of plasma cells, plasmablasts or B cells and said single antibody secreting cells are marked by clustered subtypes named by top genes from differential expression results.

309. The method of any of claims 211-308, wherein the method further comprises sample demultiplexing, de novo assembly of read pairs into contigs, and aligning and annotating the contigs to a germline segment V(D)J reference sequence for a second family of amplicons.

310. The method of claim 309, wherein aligning the contig to a germline segment V(D)J reference sequence comprises aligning 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 an IMGT germline reference database.

313. 313. The method of any one of claims 310 to 312, wherein the method further comprises: marking the VDJ sequences for quality alignment by comparing VDJ sequences against sequences of the human germline reference database or the mouse germline reference database, thereby verifying in-frame alignment, CDR3 length, and absence of stop codons in variable region sequences, and filtering the VDJ sequences based on the marking.

314. The method of claim 313, wherein the method further comprises mapping the VDJ sequence to a reference database of immunoglobulin chains.

315. The method of claim 310, wherein the accuracy of the immunoglobulin isotype, the variable region mapping, the linking region mapping, the diversity region mapping, and the full length variable region for both heavy and light chain sequences per cell is confirmed by the alignment.

316. The method of any of claims 211-315, wherein the method further comprises sample demultiplexing, aligning, filtering, and UMI counting the third library of amplicons.

317. 317. The method of any one of claims 211-316, wherein the method further comprises mapping the third library of amplicons to a custom short read reference comprising a 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, wherein said method further comprises quantifying said barcoded nucleic acid molecules across all single antibody-secreting cells and normalizing the quantification by taking the median log ratio of each barcoded nucleic acid molecule of a plurality of barcoded nucleic acid molecules across each sample capture.

319. 319. The method of claim 318, wherein the method further comprises removing background antigen signals by background denoising and scaling (DSB) for each barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules.

320. The method of claim 319, wherein the method further comprises determining an antigen signal distribution, and if the antigen signal distribution has a well-separated bimodal distribution, z-transformed values ​​are used to calculate antigen specificity, and if the antigen signal distribution does not have a well-separated bimodal distribution, quantile values ​​are used.

321. 321. The method of claim 320, wherein the method further comprises statistically modeling the target antigen and negative control antigen distributions.

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

323. 323. The method of claim 322, wherein said method further comprises using analysis of said first amplicon library and analysis of said second amplicon library to subset plasma cells or plasmablasts having effective antibody constant regions.

324. The method of claim 323, wherein the antibody constant region comprises an IgE constant region.

325. The subset of plasma cells or plasmablasts contains detectable levels of IgE heavy chain and CD79a, but lacks detectable levels of Ms4a1 and CD19, and the subset of plasma cells or plasmablasts is IgE ＋ The method of claim 323 or 324, which is a plasma cell or a plasmablast.

326. The method comprises: detecting the IgE to IgA-secreting plasma cells or plasmablasts isotypes, IgG-secreting plasma cells or plasmablasts isotypes, and / or IgM-secreting plasma cells or plasmablasts isotypes. ＋ The method of claim 325, further comprising assessing differential expression of plasma cells or plasmablasts.

327. Evaluating the differential expression of the IgE ＋ The method of claim 326, further characterizing a unique transcriptional signature of the plasma cell or plasmablast.

328. The method further comprises: ＋ The method of claim 327, further comprising assessing the antigen specificity of the plasma cells or plasmablasts.

329. The evaluating step comprises: ＋ 329. The method of claim 328, comprising comparing the antigen specificity of plasma cells or plasmablasts to a plurality of antigens and a control antigen, wherein the plurality of antigens includes the target antigen.

330. The comparing includes calculating an empirical score for each of the plurality of antigens and the control antigen by subtracting a quantile of signals associated with an antigen (qT) from a quantile of signals associated with the control antigen (qC) using a penalty factor (x) to determine an antigen specificity score, where the antigen specificity score=qT−qC ｘ The method of claim 329,

331. The method of claim 330, wherein the penalty factor is the same for all antigens.

332. The method of claim 330, wherein the penalty coefficient is different for different antibody isotypes.

333. The method of claim 330, wherein the penalty factor is different when other antigens are used.

334. 332. The method of claim 331, wherein the method further comprises determining the antigen specificity score for each of the plurality of antigens and the control antigen.

335. 335. The method of claim 334, wherein said method further comprises calculating a control antigen specificity score, and selecting said cell if said antigen specificity score is high and said control antigen specificity score is low.

336. The method of claim 335, wherein the VDJ region specifies an antigen-specific antibody.

337. The method further comprises: Ｈ :V Ｌ The method of claim 335, further comprising using the antibody sequences to select antibody candidates from the ranked list of antigen signals.

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

339. 339. The method of any one of claims 320-338, wherein the method further comprises clustering VDJ sequence similarities in antigen positive target cells, and wherein the clustering VDJ sequence similarities comprises sequence based alignment, grouping similar variable regions by amino acid sequence.

340. The antigen-positive target cells are ＋ The method of any one of claims 320 to 339, wherein

341. 341. The method of any one of claims 320 to 340, wherein the method further comprises trimming redundant sequences.

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