Detection composition and application thereof

Abstract

Provided is a composition comprising an antibody linked to an oligonucleotide by a disulfide bond wherein the oligonucleotide is specific for the antibody. Also provided is a kit comprising the composition, and a method of detecting one or more targets in a sample by the composition.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a composition for detecting a target and a use thereof. Background Art

[0002] Accurate and sensitive detection of target molecules in biological samples is key to biomedical research and clinical diagnosis. Immunoassays are currently the standard and most popular method for biomolecule detection because they are easy to use, rapid and cost-effective. These methods use a primary antibody that selectively binds to the target molecule (antigen), and this antibody-antigen interaction can be visualized by a coupled reporter on the primary antibody. More commonly, a labeled secondary antibody (which can recognize a primary antibody from the same host species) is used to react with the primary antibody-epitope complex to generate a signal. In classical immunoassays, fluorescence or luminescence is typically used to indicate the abundance and location of the target molecule. Due to the overlap of the antibody host species and the reporter spectrum, this method only allows low multiplexing of the target molecule. The use of light detection as a signal collection mechanism also limits the throughput of immunoassays.

[0003] DNA barcoding represents a promising strategy to overcome these limitations of immunoassays. Instead of fluorescent or luminescent signaling molecules, primary antibodies can be labeled with DNA molecules containing orthogonal barcode sequences. This allows the use of a combination of two or more DNA-labeled primary antibodies on the same sample to simultaneously detect two or more target molecules. Each individual antibody-antigen interaction can be examined by barcode readout. Given the high diversity of DNA sequences, DNA barcoding offers higher multiplexing that goes far beyond the scope of traditional fluorescent or luminescent signaling molecules. Importantly, careful design of DNA barcode sequences also allows for joint analysis of antibody DNA barcodes in parallel reactions using high-throughput sequencing, which greatly increases assay throughput. However, coupling antibodies to DNA by current methods often results in reduced signal and reduced antibody affinity and specificity. Moreover, the need to tailor primary antibodies for each application is laborious and expensive, thus severely limiting the scalability of this approach. Despite its great potential, these technical challenges have hindered the widespread adoption of DNA barcoding in immunoassays.

[0004] Therefore, there is an unmet need to develop a system that can use DNA barcoding in immunoassays in a more efficient manner. Summary of the invention

[0005] In order to overcome at least one of the above technical problems, the present disclosure provides a Multiplexed and Modular Barcoding of Antibodies (MaMBA) strategy, which is a simple and cost-effective strategy for barcoding antibodies with single-stranded DNA by using nanobodies as linkers. The MaMBA strategy is generally applicable to IgG that does not require chemical modification, enabling it to be quickly applied to a large number of readily available antibodies.

[0006] According to one aspect, the invention provides a composition comprising an antibody linked to an oligonucleotide via a disulfide bond, wherein the oligonucleotide is specific for the antibody.

[0007] According to some embodiments, the disulfide bond may be cleavable. According to some embodiments, the disulfide bond may be cleaved by a reducing agent. According to some embodiments, the disulfide bond may be cleaved by reductive cleavage, such as by TCEP (tris(2-carboxyethyl)phosphine), DTT (dithiothreitol), BME (β-mercaptoethanol), etc., to remove the antibody-specific oligonucleotide from the antibody.

[0008] According to some embodiments, the antibody-specific oligonucleotide may comprise a hybridization chain reaction (HCR) initiator.

[0009] According to some embodiments, an antibody-specific oligonucleotide may comprise a first barcode sequence that is specific for the antibody with which it is associated.

[0010] According to some embodiments, the barcode sequence may comprise a length of 5 to 15 nucleotides. According to some specific embodiments, the barcode sequence may comprise a length of 5 to 10 nucleotides, such as 5, 6, 7, 8, 9 or 10 nucleotides.

[0011] According to some embodiments, the oligonucleotide may further comprise a unique molecular identifier (UMI) sequence. According to some embodiments, the unique molecular identifier sequence may comprise a length of 10 to 20 nucleotides. According to some embodiments, the unique molecular identifier sequence may comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0012] According to some embodiments, the oligonucleotide may further comprise a second barcode sequence. The second barcode sequence may be specific to any information related to the sample to be detected, such as specific to the well where the sample is located.

[0013] According to some embodiments, the oligonucleotide may also include an amplification region for polymerase chain reaction (PCR) amplification reaction. According to some embodiments, the oligonucleotide may also include a pair of primer sequences for polymerase chain reaction (PCR), qPCR or sequencing.

[0014] According to some embodiments, the antibody connected with the oligonucleotide can be a secondary antibody. According to some embodiments, the antibody connected with the oligonucleotide can be selected from anti-IgE antibody, anti-IgM antibody, anti-IgG antibody, anti-IgA antibody and anti-IgD antibody. According to some embodiments, the antibody connected with the oligonucleotide can be a nanobody. According to some embodiments, the nanobody can specifically bind to the Fc region of anti-IgE, anti-IgM, anti-IgG, anti-IgA or anti-IgD antibody.

[0015] According to some embodiments, the antibody connected to the oligonucleotide can be obtained by reacting the antibody with a disulfide (-SS-) coupled oligonucleotide. According to some embodiments, the antibody can be modified with an azide moiety. According to some embodiments, the oligonucleotide can also be modified with a dibenzocyclooctyne group (DBCO).

[0016] According to another aspect, the present disclosure provides a kit for detecting one or more targets, comprising the above composition.

[0017] According to some embodiments, the test kit may include antibodies connected to one or more oligonucleotides, and the antibodies connected to each oligonucleotide include antibodies connected to specific HCR trigger chains and one or more amplification chains (amplifier). One or more amplification chains can trigger hybridization chain reactions. According to some embodiments, each amplification chain can be coupled to a detectable label, and the detectable label may include but is not limited to chemical groups, such as biotin, digoxigenin, acrylamide, amine, succinimidyl ester, thiol, azide, TCO, tetrazine, alkyne and DBCO; and fluorophores, such as FITC, cyanine dyes, Dylight fluorescent dyes, Atto dyes, Janelia Fluor dyes, Alexa Fluor dyes (e.g., Alexa Fluro 546, Alexa Fluro 488 and AlexaFluor 647).

[0018] According to some embodiments, the kit may include one or more pairs of primers for specifically amplifying antibody-specific oligonucleotides.

[0019] According to some embodiments, the kit may include one or more oligonucleotide-linked antibodies, each antibody comprising an antibody linked to an oligonucleotide, wherein the oligonucleotide comprises a barcode sequence and a region for an amplification reaction. According to some embodiments, the kit may include one or more pairs of primers for performing an amplification reaction.

[0020] According to yet another aspect, the present disclosure provides a method for detecting one or more targets in a sample by using the above composition or the above kit.

[0021] According to some embodiments, the method may include the following steps: a) incubating a first primary antibody with the above-mentioned composition to form a complex with the first primary antibody associated with the composition.

[0022] According to some embodiments, the antibody of the composition is a secondary antibody, and the complex is formed by the interaction between the first primary antibody and the secondary antibody.

[0023] According to some embodiments, the composition may comprise a secondary antibody linked to an oligonucleotide via a disulfide bond, wherein the oligonucleotide is specific for the antibody. According to some embodiments, the disulfide bond may be cleavable by a reducing agent.

[0024] According to some embodiments, the method may include the following steps: a) incubating a first primary antibody with the above-mentioned composition to form a complex having the first primary antibody associated with the composition; b) contacting the sample with the complex; c) performing an amplification reaction to produce a detectable amplification product; and optionally, d) adding a reducing agent and incubating to cleave disulfide bonds, and repeating steps a) to c).

[0025] According to some embodiments, one or more primary antibodies can be incubated with one or more secondary antibodies, respectively, to form one or more complexes, each of which can include a primary antibody and a secondary antibody associated with a specific oligonucleotide. According to some embodiments, each secondary antibody can be associated with a specific oligonucleotide that can be used as a barcode. Therefore, each of the complexes formed by the primary and secondary antibodies can be coupled to a specific barcode.

[0026] According to some embodiments, the specific oligonucleotide may include an HCR priming strand. According to some embodiments, the method of the present disclosure may include the following steps: a) incubating the first primary antibody with the above-mentioned composition to form a complex with the first primary antibody associated with the composition; b) contacting the sample with the complex; c) performing an HCR reaction to produce a detectable amplification product; and optionally, d) adding a reducing agent and incubating to cleave disulfide bonds, repeating steps a) to c). According to some embodiments, in step c), an HCR amplification strand is added.

[0027] According to some embodiments, the method can be used for in situ assays or ex vivo assays.

[0028] According to some embodiments, the specific oligonucleotide may include a first barcode sequence. According to some embodiments, the specific oligonucleotide may also include a unique molecular identifier (UMI) sequence. Both the first barcode sequence and the unique identifier sequence may be specific to the antibody associated therewith. According to some embodiments, the specific oligonucleotide may also include a second barcode sequence. The second barcode sequence may be specific to any information associated with the sample to be detected, such as the well in which the sample is located.

[0029] According to some embodiments, the method of the present disclosure may include the following steps: a) incubating a first primary antibody with the above-mentioned composition to form a complex having the first primary antibody associated with the composition; b) contacting the sample with the complex; c) adding a reducing agent and incubating to cleave disulfide bonds; d) collecting specific oligonucleotides and performing an amplification reaction to detect one or more targets; and optionally, e) repeating steps a) to d).

[0030] According to some embodiments, the methods of the present disclosure may be used to semi-quantitatively or quantitatively measure one or more targets in a sample.

[0031] According to some embodiments, the methods of the present disclosure can be used to sequence one or more targets.

[0032] According to some embodiments, before step b), the method may include a step of incubating the sample with a carrier associated with a target-specific binding substrate to capture the target in the sample. According to some embodiments, the target-specific binding substrate may be a second primary antibody. According to some embodiments, the target-specific binding substrate may be an antigen and the target to be detected may be an antibody.

[0033] According to some embodiments, the carrier may be selected from a microplate and magnetic beads.

[0034] According to some embodiments, in step a), one or more first antibodies may be incubated with one or more compositions, each of which is linked to an oligonucleotide specific for the antibody to which it is linked.

[0035] According to some embodiments, the methods of the present disclosure can be used for high-throughput sequencing.

[0036] According to some embodiments, washing and / or blocking steps may be required after each step.

[0037] According to some embodiments, the method may include a step of blocking using a protein-free blocking buffer and sheared salmon sperm DNA (ssDNA).

[0038] According to some embodiments, prior to step a), the method may include the step of contacting the sample with a plurality of target-specific binding substrates to capture the plurality of targets in the sample.

[0039] According to some embodiments, the target-specific binding substrate may be an antigen, and the target to be detected may be an antibody.

[0040] According to some embodiments, the target-specific binding substrate may be a primary antibody, and the target to be detected may be an antigen recognized by the primary antibody.

[0041] The present disclosure first combines MaMBA with a fluorescence signal amplification method (immune signal hybridization chain reaction, isHCR) and establishes a highly multiplexed and sensitive immunohistochemistry method for spatial protein profiling (multiplexed isHCR, misHCR). In addition, the cleavable MaMBA is used to increase the multiplicity of misHCR and a multi-round version of misHCR is developed (misHCR n Finally, the strategy has been demonstrated to be adaptable to conventional enzyme-linked immunosorbent assay (ELISA), and a barcoded combined immunosorbent assay (BLISA) has been developed for multiplexed and high-throughput protein detection coupled with DNA sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Design of MaMBA. A. Overview of DNA barcode module production. Nanobodies (Nb) targeting the Fc region of IgG are coupled to an azide (N3) group through an OaAEP1 enzymatic reaction, where the C-terminal 'NGL' peptide sequence is recognized, cleaved and linked to the 'GVG-K(N3)-RG' peptide (the orange dashed line shows the enzyme's cleavage site). The N3-coupled nanobodies are linked to DBCO-modified DNA oligonucleotides (DNAoligo) through a click reaction. The final product (Nb-DNAoligo) is then ready for barcoding. B. MaMBA workflow. Each antibody is incubated with a Nb-DNAoligo with a unique DNA barcode. After filtration purification, the Nb-DNAoligo binds to the Fc region of the antibody to form a DNA-barcoded complex (Ab-Nb 1 to Ab-Nb 3). The Ab-Nb-DNA oligo can then be pooled and used for assays (IHC, immunoassays, etc.).

[0043] Figure 2: MaMBA applied to multiple in situ protein imaging of misHCR. A. Schematic diagram of misHCR based on HCR reaction treated with single-stranded DNA (HCR initiating strand) and a pair of DNA hairpins (HCR amplifying strand). One-step staining is achieved by Nb-HCR initiating strand-bound antibody (Ab-HCR initiating strand). Arrows indicate the 5' to 3' direction. The HCR amplifying strand is modified with a fluorophore. B to D. Fluorescent images of mouse brain sections with or without HCR amplification. The sections were stained with anti-NeuN antibodies with Cy5 dye-conjugated Nb (Nb-Cy5) or misHCR (misHCR-Cy5) using Cy5 dye-conjugated HCR amplifying strand, and stained with anti-Th antibodies with Nb-Cy3 or misHCR-Cy3. The mean fluorescence intensity of NeuN (C) and Th (D) was measured, respectively (n = 3 brain sections per group, mean ± sem. ****P < 0.0001, ***P < 0.001; two-sided t-test). Scale bar, 100 μm. E. Multiplex misHCR imaging workflow. Antibodies barcoded with unique HCR initiating chains (Ab-i1 to Ab-in) were pooled and simultaneously applied to detection antigens (Ag.1 to Ag.n), followed by consecutive rounds of imaging via hybridization and dehybridization of orthogonal HCR amplification chains (Amp.). F. Fluorescence images of human psoriatic skin sections stained with DAPI and six other epitopes using three rounds of HCR imaging. Scale bar, 300 μm. The host species of the antibodies are shown in parentheses. Rb, rabbit; Mus, mouse; Th, tyrosine hydroxylase; PDFGRα, platelet-derived growth factor receptor A; KRT14, keratin 14; DCT, dopachrome tautomerase; αSMA, α-smooth muscle actin.

[0044] Figure 3 : Cleavable MaMBA can be used with misHCR n Increase the multiplexity of in situ protein imaging. A. Overview of the production of cleavable DNA barcoding modules. DBCO-modified DNA oligo with disulfide bonds is connected to N3-coupled Nb via a click reaction. The product (Nb-SS-DNA oligo) is ready for barcoding and the DNA oligo can be removed by reductive cleavage (TCEP treatment, etc.). TCEP, tris(2-carboxyethyl)phosphine. B. misHCR n Staining and Imaging Overview. In the first round of staining (first round misHCR), antibodies barcoded with unique and cleavable HCR initiating chains (Ab-SS-i1 to Ab-SS-in) were pooled and applied simultaneously, followed by consecutive rounds of HCR imaging (image rounds), as shown. Figure 2E. After completing the imaging round, the HCR initiation chain is removed by breaking the disulfide bond by TCEP, and then a second round of staining is performed (second round of misHCR). The antibody used in the second round of misHCR can be equipped with the same HCR initiation chain as in the first round of misHCR (Ab-SS-i1' to Ab-SS-in'), so that the same HCR amplification chain is used in the imaging round. Then after removing the HCR initiation chain, another round of misHCR can be performed, and so on. C. Fluorescent images of DAPI and 12 other epitopes stained in the dorsal raphe nucleus (DRN) of mouse brain sections by two rounds of misHCR, a total of seven imaging rounds. The imaging position is marked in the box area (red) of the mouse brain atlas shown in the lower left. The host species of the antibody are shown in brackets. Scale bar, 100 μm. nNOS, neuronal nitric oxide synthase; GFAP, glial fibrillary acidic protein; Th, tyrosine hydroxylase; NF-H, neurofilament-H; Tph2, tryptophan hydroxylase 2; GABA, gamma-aminobutyric acid; TMEM119, transmembrane protein 119; DDC, dopa decarboxylase; 5-HT, 5-hydroxytryptamine; Iba1, ionized calcium receptor protein. Magnified view of the dotted box area marked in DC. Arrows indicate three types of cells (1, DDC + Th + Tph2 - 5-HT - Cell; 2, DDC + Th - Tph2 + 5-HT + Cell; 3, DDC + Th - Tph2 - 5-HT - Cells). Scale bar, 50 μm.

[0045] Figure 4 : Cleavable MaMBA suitable for multiplex quantitative protein detection by BLISA. A. Direct antigen detection workflow of BLISA. The biomolecules of the sample are directly immobilized on the surface of a polystyrene microplate, and the target antigens (Ag.1 to Ag.3) are detected by a pool of unique and cleavable DNA-barcoded antibodies (Ab-SS-DNA barcodes). The DNA barcodes are extracted by breaking the disulfide bonds by TCEP. The released DNA barcodes are quantified by qPCR to reveal the antigen levels. B. Schematic diagram of multiplex detection of GFP, mCherry, and α-tubulin by BLISA. Purified GFP and mCherry are loaded with each other with 1 μg / mL of cell lysate (containing α-tubulin). C. Figure 4qPCR results of BLISA for the three target levels in the samples described in B. Negative control (NC) represents a sample containing only cell lysate. ΔCt = Ct(NC) - Ct(target) + 1. D. Figure 4 Dose-effect curves of GFP (left) and mCherry (right) analyzed in C. n = 3 experiments, mean ± sem. GFP, R 2 =0.9893; mCherry, R 2 =0.9972. E. Schematic diagram of the detection of endogenous proteins in HEK293T cells by BLISA and Western blotting. HEK293T cells were transiently co-transfected to express GFP and mCherry, which are controlled by drug-inducible promoters. The expression of GFP was controlled by the Tet-ON system and induced by the tetracycline derivative ATc, which binds to the rtTA transcription factor, enabling it to bind DNA at the TRE promoter to trigger gene expression. The expression of mCherry was controlled by the 3×CRE promoter and induced by FSK in the presence of 0.3 mM IBMX. ATc and FSK were titrated against each other to generate a range of culture conditions. F, G. Figure 4 E. Representative results of BLISAqPCR and western blot bands for the samples described. Three targets (GFP, mCherry, and α-tubulin) were detected by BLISA and western blot. H. Correlation of relative expression levels between BLISA and western blot for GFP (left) and mCherry (right), which were normalized to α-tubulin. Pearson correlation analysis was performed. r = 0.9718, P < 0.0001 (left); r = 0.9520, P < 0.0001 (right). ATc, anhydrotetracycline hydrochloride; TRE, tetracycline response element; CRE, cAMP response element; FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine.

[0046] Figure 5 : Sequencing-coupled BLISA for detection of anti-SARS-CoV-2 Spike RBD IgG in human serum. A. Sandwich antibody detection workflow of BLISA. Purified antigens are immobilized on microplates to capture target antibodies during sample loading. After washing away other unbound biomolecules, DNA-barcoded secondary antibodies (2 ndAb-SS-DNA barcode) to detect target antibodies. Before generating amplicons for sequencing, DNA barcodes are extracted from the plate in a solution containing TCEP and a known amount of DNA barcodes for normalization (norm bc). B. Schematic diagram of ELISA and BLISA detection of human IgG. Human IgG is immobilized on a microplate and detected with an anti-human IgG antibody (from the Rb host species) that is DNA barcoded for qPCR, or with a horseradish peroxidase (HRP)-conjugated anti-Rb IgG antibody that uses a 3,3',5,5'-tetramethylbenzidine (TMB) substrate for ELISA colorimetric readout. C. Sensitivity assessment of BLISA versus ELISA by detecting human IgG. The dose-response curve shows that BLISA has higher sensitivity and shows the dynamic range (dashed line). The dynamic range described in this article is from LOQ to the lower limit of the 95% confidence interval (dashed line). n=3 experiments per sample, mean±sem. ELISA, R 2 =0.9978, dynamic range ~3.45logs, LOD = 6.1801ng / mL; BLISA, R 2 =0.9940, dynamic range ~3.60logs, LOD=0.1407ng / mL. LOQ, limit of quantification; LOD, limit of detection. D. DNA barcode design and amplicon generation for BLISA sequencing. The DNA barcode contains an Ab barcode (Ab bc) and a unique molecular identifier (UMI). The released DNA barcode is further barcoded by a three-step PCR scheme for well barcode (well bc) addition, phased spacer sequence (PS) addition, and luminescent sequencing adapter labeling. E. Annotated amplicon sequence of the final product for luminescent sequencing. F. Schematic diagram of BLISA for human anti-SARS-CoV-2 Spike RBD IgG detection. G. BLISA sequencing results for large-scale anti-SARS-CoV-2 Spike RBD IgG detection in human serum samples. Scatter plot showing the average anti-SARS-CoV-2 Spike RBD IgG abundance normalized by normalization barcode for 505 human serum samples (unknown), listed by plate subgroup (S1 to S12). Each plate includes a blank control and a negative control (neg.ctrl). n=2 replicates. Negative control represents serum from individuals who were neither infected with SARS-CoV-2 nor vaccinated. H. Frequency distribution of IgG abundance. Relative frequencies (percentages) are shown and Gaussian distribution was performed. R 2=0.7369. I. The coefficient of variation (CV) decreased after the normalization step. The histogram (left) shows the relative distribution (percentage) of the CV for each replicate in the original (grey) or normalized (red) data. Box plots (right) of the CV distribution between the original and normalized groups were analyzed using a two-sided paired t-test ****. P < 0.0001. JL. Validation of BLISA sequencing results by ELISA. A portion of the samples (n = 13) were placed in the same well (replicate wells G9, H9) of each plate (except plate S12). Sample s1 was in plate S1, sample s2 was in plate S2, and so on; samples s12 (wells E1, F1) and s13 (wells E2, F2) were in plate S12. The results of sequencing combined with BLISA (I) and ELISA (K) are given. The correlation between sequencing combined with BLISA and ELISA was analyzed (L) (Pearson correlation. r = 0.9743, P < 0.0001). n = 2 replicates per sample, mean ± sem.

[0047] Figure 6 :2-plex sequencing combined with BLISA for detection of hepatitis B virus (HBV) antigen in human serum. A. Sandwich antigen detection workflow of BLISA. Capture antibody (Ab Cap ) is fixed on the microplate. Samples and DNA barcoded detection antibodies (Abs) are loaded simultaneously. Det -SS-DNA barcode). Then, the target antigen is captured on Ab Cap and Ab Detbetween. After the generation of amplicons for sequencing, DNA barcodes were extracted from the plate in a solution containing TCEP and known amounts of standardized barcodes. B. Schematic diagram of dual BLISA for HBsAg and HBeAg detection. CE. Detection of 2-plex antigens in control serum samples by BLISA. qPCR results of 2-plex BLISA (C) show the levels of HBsAg (blue) and HBeAg (red) in double-negative and double-positive serum samples (neg.1 to neg.3, pos.1 to pos.4) defined by commercial ELISA kits (D, E). n = 2 replicates per sample, mean ± sem. F. Summary of the average antigen levels detected in the HBsAg and HBeAg negative and positive groups. Mean ± sem. ****P < 0.0001, **P < 0.01, two-sided t-test. g. 2-plex BLISA sequencing results for HBsAg and HBeAg detection in human serum. Scatter plots showing the normalized mean abundance of HBsAg and HBeAg for 498 human serum samples listed by plate subgroup (H1 to H12). n = 2 replicates. H. Frequency distribution of HBsAg (blue) and HBeAg (red) antigen abundance. I. Correlation of normalized abundance between replicates for HBsAg (left) and HBeAg (right). Pearson correlation analysis was performed. r = 0.9973, P < 0.0001 (left); r = 0.9770, P < 0.0001 (right). J. Validation of sequencing data by ELISA. Samples with antigen abundance exceeding the overall mean in HBsAg or HBeAg validated by ELISA (n = 84). The grey lines on the X-axis represent the cut-off values ​​defined by the commercial ELISA kits, while the blue (HBsAg) and red (HBeAg) lines represent values ​​below the ELISA 'positive' point, the so-called 'cut-off' values ​​for BLISA. The dashed line represents the mean + 3 s.d. of the negative control group, which is a double negative sample as defined by ELISA. The double positive sample (#23) is marked.

[0048] Figure 7 : Multiplex detection of phosphoproteins by magnetic bead (MB)-based BLISA. A. Schematic diagram of BLISA for multiplex detection of MBs. Antigens are detected by a sandwich-type assay, in which capture antibodies are attached to MBs and identified by different DNA barcodes (bc.1 to bc.n). B. MB-based BLISA workflow. The capture antibody-bound MBs are mixed and aliquoted into sample wells of a deep well plate. Samples are loaded and incubated with MBs. After a wash step to remove unbound biomolecules using magnetic separation, the captured antigens are detected by DNA barcode detection antibodies (Ab Det -SS-DNA barcode) detection. Then, the excess Ab was washed away Det-SS-DNA barcode, DNA barcode extracted by TCEP. CE. Multiple validation of MB-based BLISA. Seven purified phosphoproteins (p-p38α, p-ERK1 / 2, p-JNK, p-AMPKα1, p-CREB, p-Src and p-Akt) were loaded into 7 wells with different concentration gradients (C). The qPCR results of BLISA for each antigen protein are described (D). n = 3 experiments, mean ± sem. The R of the dose-effect curve of each antigen protein is listed in E. 2 F. 7-plex BLISA and corresponding ELISA for detecting phosphoproteins in cell lysates of U87 and U937 cell lines. U87 and U937 cells were subcultured for 24 hours and serum starved overnight under different culture conditions (NT, 10% FBS for 10 minutes, 100 nM Aniso for 1 hour and 20 mM H2O2 for 10 minutes). The cells were then lysed and measured by 7-plex BLISA (left) and ELISA (middle). The correlation between BLISA and ELISA for each phosphoprotein target was analyzed (right) (Pearson correlation. r = 0.9241, P = 0.0010; r = 0.9541, P = 0.0002; r = 9685, P < 0.0001; r = 7684, P = 0.0259; r = 0.9845, P < 0.0001; r = 0.9829, P < 0.0001; r = 0.7636, P = 0.0275). n = 3 replicates, mean ± sem. p-p38α, phospho-p38α(T180 / Y182); p-ERK1 / 2, phospho-ERK1(T202 / Y204) / ERK2(T185 / Y187); p-JNK, phospho-JNK pan-specific; p-AMPKα1, phospho-AMPKα1(T183); p-CREB, phospho-CREB(S133); p-Src, phospho-Src(Y419); p-Akt, phospho-Akt(S473); NT, untreated; FBS, fetal bovine serum; Aniso, anisomycin.

[0049] Figure 8 : SDS-PAGE validation of Nb-DNAoligo production and unaffected spatial resolution of misHCR imaging. A. Non-reducing SDS-PAGE gel of purified nanobody-NGL-His6 (Nb-NGL-His6), OaAEP1 reaction product (Nb-NGV-N3), and Nb-DNAoligo. The DNAoligo of the same gel was first stained with nucleic acid dye, and then the protein was stained with Coomassie brilliant blue. B. The secondary antibody (2 ndFluorescence images of α-tubulin in HeLa cells stained by conventional method (left) and misHCR (right) using Alexa Fluor 647-coupled HCR amplifier chain. Magnified views are shown as markers in the boxed area. Scale bar, 10 μm. Example intensity distribution of a line drawn perpendicular to the microtubule structure shown in CB. D. Conventional method (2 nd Ab) and the spatial resolution of misHCR. nd No significant differences were observed between Ab and misHCR (for 2 nd Ab, n=26, for misHCR, n=25, mean±sem. P=0.1567, unpaired two-sided t-test). ns, not significant.

[0050] Fig. 9 : Effective signal removal of image rounds and misHCR rounds after the misHCR washing step and their reproducibility in several rounds of image round washing. AB. Fluorescence images of mouse brain slices before and after removal of HCR amplification chains or initiation chains by formamide or TCEP. Scale bar, 500 μm (A), 300 μm (B). C. Fluorescence images of NeuN, NF-H and Th in the dorsal raphe nucleus (DRN) of mouse brain slices through five rounds of HCR imaging and washing cycles. Scale bar, 100 μm. D. Signal-to-noise ratio (SNR) of each round of HCR imaging for NeuN (left) and NF-H (right). n = 3 brain slices per group, mean ± sem. One-way ANOVA test was performed. Images of each group were acquired using the same microscope settings. NF-H, neurofilament-H.

[0051] Fig.10 : Imaging of misHCR in healthy human skin sections. Fluorescence images of healthy human skin sections stained with DAPI and six other epitopes using three rounds of HCR imaging. The host species of the antibodies are indicated in parentheses. Scale bar, 200 μm. PDGFRα, platelet-derived growth factor receptor A; KRT14, keratin 14; DCT, dopachrome tautomerase; αSMA, alpha smooth muscle actin.

[0052] Fig.11: Multiple rounds of misHCR were performed on nine targets using three sets of HCR initiation and amplification chains repeatedly. A. Fluorescence images of mouse brain sections stained with DAPI and nine other targets by three rounds of misHCR, one image round each. The imaging locations are marked in the boxed area of ​​the mouse brain atlas shown in the lower right corner. Scale bar, 300 μm. Magnified view of the dotted box area marked in BA. Different target signals were not co-localized in misHCR imaging (marked by arrows). Scale bar, 25 μm. MAP2, microtubule-associated protein 2; DDC, dopa decarboxylase; nNOS, neuronal nitric oxide synthase; Iba1, ionized calcium adaptor protein 1; GFAP, glial fibrillary acidic protein; Th, tyrosine hydroxylase; NF-H, neurofilament-H; Tph2, tryptophan hydroxylase 2.

[0053] Fig.12 : Colocalization of antibody staining patterns of conventional methods and misHCR. Mouse brain sections were stained with antibodies targeting different epitopes and then, after HCR using an Alexa Fluor 647-conjugated amplifier chain (misHCR-647), incubated with an equal amount of Cy3-conjugated secondary antibody (2 nd Ab-Cy3) was incubated with Nb-HCR initiator strand. Scale bar, 100 μm.

[0054] Fig.13: Detection of endogenous proteins in HEK293T cells by BLISA and Western blot, and sequencing-coupled BLISA for multiplex detection of GFP, mCherry, and α-tubulin. A. Detection of endogenous GFP. HEK293T cells were transiently transfected to express GFP, which was controlled by the Tet-ON system and induced by ATc, a tetracycline derivative, which binds to the rtTA transcription factor, enabling it to bind DNA at the TRE promoter to trigger gene expression. B. Representative results of BLISA qPCR and Western blot bands (left panel), and their corresponding correlations with the relative expression levels of GFP (normalized to α-tubulin) (right panel). Pearson correlation analysis was performed. r=0.9724, P<0.0001. C. Detection of endogenous mCherry. HEK293T cells were transiently transfected to express mCherry, which was controlled by the 3×CRE promoter and induced by FSK in the presence of 0.3 mM IBMX. D. Representative results of BLISA qPCR and western blot bands (left panel), and their corresponding correlations with the relative expression levels of mCherry (normalized to α-tubulin) (right panel). Pearson correlation analysis was performed. r=0.9647, P<0.0001. E. DNA barcode design and amplicon generation for BLISA sequencing. The DNA barcodes contain Ab barcodes (Abbc) and unique molecular identifiers (UMIs). The released DNA barcodes were further barcoded by a two-step PCR approach for well barcode (well bc) addition and luminophore sequencing adapter labeling. F. UMI counts of the three targets (GFP, mCherry, and α-tubulin) in samples loaded with 1 μg / mL of cell lysate (α-tubulin) according to the design in the samples containing purified GFP and mCherry. G. Dose-response curves of GFP (upper panel) and mCherry (lower panel) analyzed from F. R 2 =0.9827, 0.9843. n = 3 experiments, mean ± sem. ATc, anhydrotetracycline hydrochloride; TRE, tetracycline response element; CRE, cAMP response element; FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine.

[0055] Fig.14: Reproducibility and inter-plate variability of sequencing combined with BLISA. A. Correlation of normalized abundance between replicates of IgG detection. Pearson correlation analysis was performed. r=0.9882, P<0.0001. B. Results of BLISA by qPCR for a subset of samples (n=13) localized by different plates in sequencing. n=2 replicates per sample, mean±sem. C. Consistency between the two BLISA outputs, qPCR and sequencing. The correlation between the BLISA sequencing results and the qPCR results for the 13 samples was analyzed. (Pearson correlation. r=0.9354, P<0.0001).

[0056] Fig.15 : Standard curves of purified phosphoproteins in BLISA compared to ELISA. A. Standard curves of purified phosphoproteins (p-p38α, p-ERK1 / 2, p-JNK, p-AMPKα1, p-CREB, p-Src, and p-Akt) in BLISA and ELISA. B. R of the standard curves 2 . DETAILED DESCRIPTION

[0057] Before describing the methods and compositions of the present invention, it should be understood that the present invention is not limited to the specific methods or compositions described, and thus may vary. It should also be understood that the terminology used herein is only used to describe specific embodiments and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

[0058] As modular linkers, nanobodies (single-domain antibodies derived from camel heavy-chain antibodies) are becoming increasingly popular as powerful tools in cell biology, structural biology, and therapeutics due to their outstanding features (small size, easy production, flexible operation, etc.). Using secondary nanobodies (nanobodies targeting antibodies) with monovalent properties, different antibodies even sharing the same host species can be simply pre-incubated with the secondary nanobodies and then applied to the sample together, which is not feasible for divalent secondary antibodies. In this study, we provide MaMBA, an efficient modular barcoding strategy that is generally applicable to antibodies for simple and site-specific DNA barcoding via nanobodies. First, we used this strategy to develop misHCR, a multiplexed in situ protein imaging method that combines HCR technology with immunohistochemistry using orthogonal HCR priming chains-barcoded antibodies. To achieve higher imaging throughput, we developed a cleavable MaMBA by using a disulfide linker between nanobodies and DNA, and further applied it to multiple rounds of versions of misHCR (misHCR n). Next, we extended the cleavable MaMBA strategy to the field of immunoassays and developed BLISA, a DNA barcode-driven multiplex quantitative protein assay using qPCR or high-throughput DNA sequencing readouts. To investigate the potential of BLISA for population-scale testing diagnostics, we performed sequencing-coupled BLISA for anti-SARS-CoV-2 Spike RBD IgG detection in approximately 500 human serum samples, and further performed 2-plex BLISA for HBV antigens (HBsAg and HBeAg) detection in approximately 500 human serum samples. Overall, MaMBA provides a simple and effective strategy to barcode antibodies with DNA, which has the feasibility and versatility to be applied to different immunoassays.

[0059] Fluorescent immunostaining has become a routine method in biological and clinical laboratories. However, due to the spectral overlap of fluorophores, traditional methods provide limited multiplexing targets. Although strategies such as sequential antibody staining (stripping bound antibodies and starting a new round of staining) have achieved enhanced multiplexing, it requires multiple rounds of antibody incubation (hours or even days per cycle) and takes weeks to image dozens of targets. In contrast, the DNA barcoding method enables antibodies to be simultaneously bound to orthogonal DNA barcodes, followed by sequential docking chain readings to achieve faster multiplexing imaging. However, due to the overlap of antibody species, DNA conjugation should be applied to antibodies, which makes the signal of sequential docking chain imaging weaker than traditional immunostaining because it loses the signal enhancement of using secondary antibodies. Therefore, DNA-based signal amplification methods such as rolling circle amplification (RCA), exchange reaction signal amplification (SEBAR), and HCR are applied. HCR uses a pair of HCR hairpins, which are triggered by complementary HCR initiator chains and repeatedly opened. The signal amplification of HCR occurs in situ and can be regulated by changing the concentration of HCR amplification chains. Therefore, misHCR not only achieves multiplexing that relies on the power of DNA barcodes, but also obtains amplified signal levels through HCR reactions. In addition, the potential of misHCR is not limited to fluorescence imaging. Recently, Raman microscopy-based methods have been developed for multiplexed protein imaging, which utilizes orthogonal Raman dyes, whose vibrational peaks are much narrower than fluorescence. Raman dye imaging shows the advantage of single-shot multiplexing, which can greatly speed up the experimental process.

[0060] Immunoassays based on DNA-barcoded antibodies have shown the advantage of significantly improving the sensitivity of traditional immunoassays. For example, using DNA amplification technology, immuno-PCR and its related methods have shown excellent performance in ultrasensitive detection of low-abundance proteins, usually improving the sensitivity by 10 to 10,000 times compared with similar enzyme amplification immunoassays. Therefore, in combination with sequencing technology, various immunoassay methods using DNA-barcoded antibodies have been developed and demonstrated for highly multiplexed and high-throughput protein detection (such as ID-seq), further single-cell level readouts (such as CITE-seq), and even transcriptome joint expression mapping (such as SCITO-seq). However, antibody-DNA oligo conjugates are mostly produced by a one-to-one biochemical conjugation method, which means that each combination of antibody and DNA barcode should undergo a conjugation process (activation, conjugation, purification, and detection), which reveals a labor-intensive and expensive procedure. In addition, despite the large number of readily available commercial antibodies, different suppliers usually provide different buffer formulations for antibody preservation (salt, sodium azide, glycerol, etc.), which may affect the chemical reaction and introduce inharmonious conjugate ratios for different antibodies. To avoid this, antibodies should be buffer exchanged before conjugation, which further increases the workload. Therefore, the use of a one-to-one biochemical conjugation strategy is not conducive to large-scale antibody production and hinders its widespread application. To overcome these problems, BLISA adopts the MaMBA strategy, using secondary nanobodies as modular mediators to non-covalently and site-specifically barcode antibodies. The pipeline-synthesized nanobody DNA conjugates can be directly mixed with antibodies, and then each purified DNA-barcoded antibody can be assembled in multiplexes. This simple DNA barcoding workflow is designed to be suitable for large-scale pipeline production, which enables technologies based on DNA-barcoded antibodies to be more widely used with higher efficiency and lower cost.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials cited in these publications. It should be understood that in the event of a conflict, the content of the present disclosure supersedes any disclosure of the incorporated publication.

[0062] definition

[0063] Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0064] It should be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "recombinant AAV viral particle" includes a plurality of such viral particles, reference to a "microglia" includes one or more microglia known to those skilled in the art and equivalents thereof, and so forth.

[0065] The term "hybridization chain reaction" or "HCR" as used herein is a technique based on a chain reaction of recognition and hybridization events between two sets of DNA hairpin molecules, providing an enzyme-free alternative for rapid detection of specific DNA sequences. HCR uses a pair of complementary, kinetically trapped hairpin oligomers to propagate a chain reaction of hybridization events.

[0066] The term "hybridization chain reaction initiation chain" or "HCR initiation chain" used herein refers to a nucleic acid region that can cause two metastable HCR hairpin monomer species to polymerize to form an HCR amplified polymer. The exposed HCR initiation chain is functional and initiates the polymerization of metastable HCR hairpin monomers under polymerization conditions. The chelated HCR initiation chain is inoperative and does not initiate the polymerization of metastable HCR hairpin monomers under polymerization conditions. When another molecule is combined with the chelating molecule, the chelated HCR initiation chain (therefore, initially inoperative) can be exposed (therefore, becomes functional).

[0067] The term "Nanobody" as defined herein includes, but is not limited to, VHH sequences. In some embodiments, the Nanobodies of the present disclosure specifically bind to the Fc domain of a primary antibody.

[0068] The term "unique molecular identifier" or "UMI" as used herein includes random oligonucleotide sequences that are increasingly used in high-throughput sequencing.

[0069] The following examples are intended to provide a complete disclosure and description for those of ordinary skill in the art to guide how to make and use the variant AAV capsids disclosed herein, and are not intended to limit the scope of the invention disclosed herein. In addition, the following examples do not represent that the following experiments are all or only experiments.

[0070] method

[0071] Reagents and reagent preparation.

[0072] DNA oligos were synthesized by Thermo Fisher Scientific, Genewiz, and Sangon Biotech. The detailed sequences and modifications of DNA oligos are listed in Table 1. All oligos were dissolved in nuclease-free water (Thermo Fisher Scientific, AM9932) and stored at -20°C.

[0073] Sodium tetraborate decahydrate (NaB; S9640), Triton X-100 (T8787), dextran sulfate (D8906), tris(2-carboxyethyl)phosphine (TCEP; 646547), Tween-20 (P9416), protease inhibitor cocktail (P8340), and 3,3',5,5'-tetramethylbenzidine (TMB; T0440) were purchased from Sigma-Aldrich. 20× sodium chloride citrate (SSC) buffer (AM9763), sheared salmon sperm DNA (AM9680), cell extraction buffer (FNN0011), and protein-free blocking buffer (PFBB; 37572) were purchased from Thermo Fisher Scientific.

[0074] Table 1. Detailed sequences and modifications of DNA oligos.

[0075]

[0076]

[0077]

[0078] The sequences of the antibody-specific barcodes are underlined.

[0079] Plasmid construction.

[0080] DNA fragments were amplified by PCR using primers with 17-20 bp overlap (Genewiz) for Gibson assembly, and plasmid sequences were verified using Sanger sequencing. To purify the protein, the DNA fragments were amplified by PCR using primers with 17-20 bp overlap (Genewiz) for Gibson assembly, and plasmid sequences were verified using Sanger sequencing. 1pET28a-His6-Ubiquitin-OaAEP1(C247A) was constructed, and DNA sequences encoding TP897-NGL-His6 and TP1107-NGL-His6 were synthesized (Genewiz) and cloned into the pET28a vector. EGFP and mCherry sequences were cloned into the pET28a vector and fused to the C-terminal His6 tag sequence. To evaluate the multiplicity of BLISA in endogenous protein detection, EGFP and rtTA sequences were cloned into the pLJM1 vector (Addgene, 91980) under the control of the TRE promoter and PGK promoter, respectively. 2 (by replacing the original puromycin N-acetyltransferase gene), and the sequence encoding mCherry was cloned into another pLJM1 vector with a 3×CRE promoter.

[0081] Protein expression and purification.

[0082] Recombinant OaAEP1 (C247A) was expressed and obtained. Briefly, pET28a-His6-Ubiquitin-OaAEP1 (C247A) was transformed into E. coli SHuffle, and protein expression was induced with 1 mM IPTG overnight at 18°C. The cells were collected by centrifugation, resuspended in lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole), and lysed by ultrasonication. Cell debris was removed by centrifugation at 39,000 g for 1 hour at 4°C. The supernatant was bound to Ni-NTA resin, washed with AEP wash buffer (50 mM NaH2PO4, pH 8.0, 0.3 M NaCl, 20 mM imidazole), and eluted with elution buffer (50 mgM NaH2PO4, pH 8.0, 3 M NaCl, 250 mM imidazole). The protein was further purified by a size exclusion column (Superdex 75 Increase 10 / 300GL) pre-equilibrated in PBS buffer. For autoactivation of OaAEP1, fractions containing the protein were pooled, incubated overnight at room temperature (RT), 1 mM EDTA and 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride were added to the solution, and the pH was adjusted to 4.0 by titration of glacial acetic acid. After filtration with a 0.22 μm membrane and dilution 1:8 in 50 mM acetate, pH 4.0, the mature protein was further purified by cation exchange (HiTrap SP column) and eluted with a salt gradient (0-100% storage buffer (50 mM sodium acetate, pH 4.0, 10% glycerol, 1 m NaCl); 10 column volumes. Fractions containing mature AEP were pooled and concentrated using a 10K MWCO concentrator (Sartorius). The final product was analyzed by SDS-PAGE and the protein molecular weight and extinction coefficient were determined by A 280 reading( N60, IMPLEN) for quantification and then stored in aliquots at -80°C until use.

[0083] Nanobodies (TP897 and TP1107) against IgG Fc domains were expressed and purified. In brief, pET21a-TP897-NGL-His6 and pET21a-TP1107-NGL-His6 were transformed into E.coli Shuffle. Expression was induced with 1mM IPTG at 28°C for 16-18 hours. After collection by centrifugation, the cells were resuspended in Nb lysis buffer (50mM HEPES, pH 7.5, 300mM NaCl, 5mM imidazole, 10% glycerol) and lysed with ultrasound. Cell debris was removed by centrifugation, and the supernatant was applied to Co-NTA resin. The resin was washed with Nb wash buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 10 mM imidazole, 10% glycerol) and His-tagged nanobodies were eluted with Nb elution buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 500 mM imidazole, 10% glycerol). The nanobodies were further purified and buffer exchanged in Nb exchange buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 10% glycerol) by size exclusion column (SEC). The fractions containing nanobodies were pooled and concentrated using an Amicon 3KMWCO concentrator (Millipore). As described above, the nanobodies were purified by A 280 The protein concentration was determined by reading. The nanobodies were stored in aliquots at -80°C until use. The amino acid sequences of TP897-NGL-His6 and TP1107-NGL-His6 are listed in Table 2.

[0084] pET28a-GFP and pET28a-mCherry were transformed into E. coli BL21 (DE3). Expression was induced with 0.5 mM IPTG at 16 °C for 16-18 h. GFP and mCherry were purified using Co-NTA resin as described above and further purified by SEC in PBS. Proteins were concentrated using an Amicon 10K MWCO concentrator and purified by A 280 Readings were quantitative.

[0085] Table 2. Amino acid sequences of Nanobodies.

[0086]

[0087] C-terminal labeling of nanobodies was performed via OaAEP1 reaction.

[0088] Purified NGL-tagged nanobodies were C-terminally labeled with GVG-K(N3)-RG via the OaAEP1 reaction. This was performed by gently shaking 50 μM nanobodies, 1 mM GVG-K(N3)-RG, and 750 nM OaAEP1(C247A) in reaction buffer (100 mM sodium phosphate buffer, pH 6.5, supplemented with 2 mM DTT) at room temperature overnight. The reaction mixture was then diluted 1:1 in Nb lysis buffer and bound to Ni-NTA resin to remove GL-His6 and unreacted His-tagged nanobodies. The resin was washed with Nb lysis buffer until A 280 The flowthrough and wash fractions containing the azide-labeled Nanobody were pooled and the remaining GVG-K(N3)-RG was removed using a 3,000 MWCO concentrator. 280 Readouts were quantified and analyzed by SDS-PAGE.

[0089] Nanobody-fluorophore conjugation.

[0090] 40 μM azide-labeled nanobody was mixed with 10-fold molar excess of DBCO-Cy3 (777366, Sigma-Aldrich) or DBCO-Cy5 (777374, Sigma-Adrich) and reacted at room temperature for 4 hours. Excess fluorophore was removed twice using a Zeba spin desalting column (7k MWCO). N60 measures protein concentration and degree of labeling (DOL) with dye correction. Fluorophore-conjugated nanobodies were analyzed by SDS-PAGE.

[0091] Nanobody-DNA oligonucleotide conjugation.

[0092] For Nb-SS-HCR priming strands and Nb-SS-DNA barcodes, we used DBCO-PEG3-SS-NHS (10 mM in anhydrous DMSO; CP-2089, Conju-Probe, LLC) as a linker. HCR priming strands and DNA barcodes modified with 5'NH2-C6 were reacted with DBCO-PEG3-SS-NHS (20-fold molar excess) in 0.091 M NaB (pH 8.5) at room temperature for 2 hours with gentle shaking. To remove excess linkers, oligonucleotides were precipitated by mixing with one-tenth volume of 3 M sodium acetate, pH 5.2 and 2 volumes of cold anhydrous ethanol and incubated at -20°C for 1 hour. After spinning at 20,000g for 30 minutes at 4°C, the supernatant was removed and the precipitate was carefully rinsed twice with 70% cold ethanol. The precipitate was air-dried and redissolved in TE buffer. Excess cross-linker was further removed by Zeba spin desalting columns (7K MWCO).

[0093] HCR priming strands modified with 5' DBCO moieties (2-fold molar excess) or DBCO-PEG3-SS-conjugated oligo (2-fold molar excess) were mixed with 25 μM azide-labeled Nanobodies and reacted with shaking at 4° C. for 16 h. DNA oligo-conjugated Nanobodies were analyzed by SDS-PAGE.

[0094] DNA barcoding of antibodies via the MaMBA strategy.

[0095] At room temperature, in 50 μL premix buffer (0.1% BSA, 0.05% Triton X-100, 5mM EDTA in PBS), the antibody was premixed with a 3-fold molar excess of DNA oligo-coupled nanoantibodies (Nb HCR priming chain, Nb-SS-HCR priming chain or Nb-SS-DNA barcode) and shaken for 3-5 hours. The mixture was then added to the Amicon Ultra-0.5100K centrifugal filter and washed 10 times with 400 μL PBS containing 0.05% Tween-20 and 5mM EDTA. The purified DNA-barcoded antibodies were then stored at 4°C with 0.03% NaN3 or stored at -20°C with 50% glycerol until assayed.

[0096] Mouse.

[0097] The care and use of animals were approved by the Animal Care and Use Committee of the National Institute of Life Sciences in Beijing (approval number: NIBSLuoM15C) in accordance with the Regulations on Laboratory Animals of China. C57BL / 6N mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (China), and adult (8–12 weeks old) mice of either sex were used. Mice were maintained with a 12-h light / dark photoperiod (lights on at 8 a.m.) and provided with food and water freely.

[0098] Tissue sample preparation for immunohistochemistry.

[0099] Mice were anesthetized with an overdose of pentobarbital and intracardially perfused with PBS followed by 4% paraformaldehyde (PFA; 4% (w / v) in PBS). The brain was dissected out and postfixed in 4% PFA for 4 hours at room temperature. The samples were cryoprotected in 30% sucrose until they sank to the bottom. Coronal sections (35 μm) were prepared on a cryostat (Leica CM1950).

[0100] Human skin samples were surgically obtained from the arm skin of a 28-year-old female with psoriasis and a 23-year-old healthy male. The samples were embedded in OCT compound, frozen at -80°C, and cut into 30 μm thick specimens. The skin sections were fixed in 4% PFA for 15 minutes and washed three times with PBS.

[0101] misHCR / misHCR n Immunostaining.

[0102] Table 3 lists the detailed information about the antibodies used and Nb-HCR trigger chains (or Nb-SS-HCR trigger chains). The samples were permeated with 0.3% Triton X-100 in PBS (PBST) and blocked for 1 hour with blocking buffer (2% BSA, 5mM EDTA, 0.3% Triton X-100 in PBS) at room temperature. According to the type of nanoantibodies used above, all prepared Ab-HCR trigger chains (or Ab-SS-HCR trigger chains) by the MaMBA strategy were pooled into incubation buffer (1% BSA, 0.1% Triton X-100, 5mM EDTA, 0.5mg / mL sheared salmon sperm DNA, 1% dextran sulfate, 150mM NaCl, 0.05% NaN3) supplemented with excessive Nb-NGL-His6. The sections were then incubated with a pool of Ab-HCR trigger chains (or Ab-SS-HCR trigger chains) at 4°C for 12-16 hours and then washed three times with wash buffer (2% BSA, 0.1% Triton X-100 in PBS) for 10 minutes each. The samples were washed twice with PBS for 5 minutes each and then post-fixed with 5mM BS(PEG)5 (A35396, Thermo Fisher Scientific) in PBS for 1 hour at room temperature and then quenched in Tris-buffered saline (TBS) for 10 minutes. For control experiments, the sections were incubated with antibodies diluted in blocking buffer at 4°C for 12-16 hours and then washed three times in PBST. 1) The samples were incubated with Nb-fluorophores or Nb-HCR trigger chains in incubation buffer for 2 hours at room temperature and then washed three times with PBST. 2) The samples were incubated with a mixture containing equal amounts of Cy3-conjugated anti-rabbit IgG antibody (111-165-008, Jackson ImmunoResearch) and Nb-HCR priming chain (B4 I1) in incubation buffer for 2 h at room temperature and then washed three times with PBST.

[0103] Before the next round of misHCR, tissue sections with anchored Ab-SS-HCR priming chains were incubated with 50 mM TCEP in PBS for 15 min at room temperature (to remove HCR priming chains by reductive cleavage) and then washed three times with PBST. Samples were blocked in blocking buffer for 1 h at room temperature and subjected to a new round of staining as described above.

[0104] Cultured cell samples were fixed in 4% PFA for 10 min and washed three times with PBST. After blocking with blocking buffer for 30 min at room temperature, cells were anti-stained with an antibody against α-tubulin (1:4000 dilution; T5168, Sigma-Aldrich) followed by an Alexa Fluor 647-conjugated secondary antibody (1:1,000 dilution; 715-605-151, Jackson ImmunoResearch) or stained with an equal amount of Ab-HCR priming chain (B4 I1) against α-tubulin followed by HCR using an Alexa Fluor 647-conjugated amplification chain.

[0105] HCR amplification.

[0106] The basic HCR amplification process is universal in all experiments. The samples were blocked in amplification buffer (5×SSC buffer, 0.1% (v / v) Tween-20, 10% dextran sulfate) for 1 hour at room temperature. Using a PCR instrument, heat at 95°C for 90 seconds, and each pair of fluorophore-coupled HCR amplification chains were quickly cooled in 5×SSC buffer solution, immediately cooled on ice for 5 minutes, and then stored in the dark at room temperature for 30 minutes. The amplification chains were added to the amplification buffer to a final concentration of 12.5nM for each pair of amplification chains. The samples were incubated overnight with the HCR amplification chains at room temperature and then washed three times with 5×SSCT (5×SSC buffer, 0.1% Tween-20) before signal detection to remove free amplification chains.

[0107] To perform multiple rounds of imaging, the hybridized amplified strands were first removed twice with 50% formamide in 0.1× PBS for 5 min each at 37° C. The samples were then washed three times with PBST and blocked again in amplification buffer for 1 h at room temperature before the basic HCR amplification process.

[0108] Before removing the HCR priming strand for a new round of staining, the hybridized amplified strand should be removed.

[0109] Fluorescence imaging.

[0110] Samples were mounted on microscope slides in 50% glycerol containing 1 μg / mL DAPI (D9542, Sigma-Aldrich) for image acquisition. Confocal microscopy was performed on a Zeiss LSM880 using a 20× / NA-0.8 objective or on a Leica SP8 using a 20× / NA-0.75 or 10× / NA-0.40 objective. Alexa Fluor (AF) 488 was visualized with a 488-nm laser; Cy3, AF546, and AF594 were visualized with a 552-nm laser; and Cy5 and AF647 were visualized with a 638-nm laser. Images were 1024 × 1024 pixels or 512 × 512 pixels and acquired using a two-frame averaging method. Images were processed using Zeiss ZEN, Leica LAS X, and FIJI, and colorization was performed using FIJI and Photoshop. For multiple rounds of images, basic alignment and registration were performed using FIJI based on the DAPI channel of each image using the descriptor-based registration plugin.

[0111] For the 6-color multiplex of human skin samples, the entire experiment was performed using one round of staining and three rounds of images (round 1, PDGFRα, KRT14, and DCT; round 2, CD31; round 3, αSMA and CD45). For the 9-color multiplex of mouse brain sections, three rounds of staining were performed, with one round of images per staining (1 st round misHCR, MAP2, DDC and nNOS; 2 nd round misHCR, NeuN, Iba1 and GFAP; 3 rd For 12-color multiplexing of mouse brain sections, two rounds of misHCR were performed, with 4 rounds of images (round 1, nNOS and GFAP; round 2, NeuN, Th and NF-H; round 3, Tph2 and Orexin A; round 4, GABA) and 3 rounds of images (round 1, Tmem119 and DDC; round 2, 5-HT; round 3, Iba1), respectively.

[0112] Cell culture and transfection.

[0113] HEK293T cells (ATCC CRL-3216) and HeLa cells (ATCC CCL-2) were incubated in Dulbecco's modified Eagle medium (DMEM, Gibco) supplemented with 10% (v / v) fetal bovine serum (FBS, Gibco) at 37°C and 5% CO according to standard procedures. Cells were seeded in 24-well plates and grown to a viability of 70-90%. For immunostaining, cells were cultured on coverslips pretreated with poly-L-lysine solution (P8920, Sigma-Aldrich). Two hours before transfection, the cell culture medium was replaced with fresh complete medium. For each well, 0.5 μg of plasmid and 0.5 μL of DNA transfection reagent (Neofect) were mixed in 50 μL of serum-free medium and incubated at room temperature for 15-20 minutes. The mixture was added to the cell culture medium, the cells were incubated for 18-24 hours, and then the transgene expression assay was performed. For cells transfected with TRE-EGFP-PGK-rtTA, Tet system-approved FBS (Biological Industries) was used instead.

[0114] Drug treatment.

[0115] The drugs used in this study were anhydrotetracycline hydrochloride (ATc; 541642, J&K), 3-isobutyl-1-methylxanthine (IBMX; I5879, Sigma-Aldrich), and forskolin (FSK; F3917, Sigma-Adrich).

[0116] Cells transfected with TRE-EGFP PGK-rtTA were induced with increasing doses of ATc as described. Cells transfected with 3×CRE-mCherry-hPEST were induced with 0.3 mM IBMX and increasing doses of FSK as described. Cells co-transfected with TRE-EGFP PGK-rtTA and 3×CRE-mCherry-hPEST were treated with 0.3 mM IBMX, increasing doses of ATc, and decreasing doses of FSK as described. After incubation for 6-7 hours at 37°C, 5% CO2, cells were lysed in cell extraction buffer supplemented with 1 mM PMSF and protease inhibitor cocktail according to the manufacturer's instructions, quantified using the BCA protein assay (23225, Thermo Fisher Scientific), and stored in aliquots at -80°C until use.

[0117] Serum sample collection.

[0118] Human serum samples were collected from volunteers who were laboratory staff and from routine physical examinations for health screening in hospitals, approved by the Human Research Ethics Committee of the Beijing Institute of Brain Science and Brain Inspiration (Beijing). Serum samples for anti-SARSCoV-2 Spike RBD IgG testing were collected in October 2021, and samples for HBV antigen testing were collected in June 2021.

[0119] Microplate surface BLISA assay.

[0120] Details of the antibodies used and the Nb-SS-DNA barcodes are listed in Table 2. HRP-conjugated secondary antibodies against rabbit IgG (1:30,000 dilution; 31460, Thermo Fisher Scientific) and mouse IgG (1:30,000 dilution; 31430, ThermoFisher Scientific) were used.

[0121] For samples containing purified GFP and mCherry, 50 μL of purified protein diluted in coating buffer (50 mM carbonate buffer, pH 9.6) containing 1 μg / mL wild-type HEK293t cell lysate was added to a polystyrene microplate (449824, Thermo Fisher Scientific) and incubated at 37°C for 2 hours. The wells were aspirated and washed three times with plate wash buffer (5 mM EDTA, 0.05% Tween-20 in PBS). The samples were then blocked in 200 μL of blocking buffer (0.05% Tween-20 in PFBB) for 1 hour at room temperature and washed three times with plate wash buffer. All Ab-SS-DNA barcodes prepared by the MaMBA strategy were pooled into the incubation solution (blocking buffer supplemented with 5 mM EDTA, 0.4 mg / mL sheared salmon sperm DNA, 2 μM TP1107-NGL-His6, and 2 μM TP 897-NGL-His6). The samples were then incubated with the Ab-SS-DNA barcode pool for 1 hour at 37°C, washed five times with plate wash buffer, and washed three times with PBS containing 5mM EDTA. The DNA barcodes were extracted with 50 μL of elution buffer (20mM TCEP in PBS) per well and incubated for 0.5 hours at room temperature. The extracted DNA solution was measured immediately or stored at -20°C until analysis. A volume of 1 μL was used as a template for qPCR or sequencing library preparation. For ELISA control experiments, samples were incubated with antibodies (diluted in PFBB) for 1 hour at 37°C and washed three times with plate wash buffer. The samples were then incubated with HRP-conjugated secondary antibodies diluted in PFBB for 1 hour at 37°C and washed five times with plate wash buffer. HRP-driven colorimetric readings were performed by adding 50 μL of TMB solution and incubating for 10 minutes at room temperature. Then, 50 μL of 1.8 N H2SO4 was added to stop the reaction, and the absorbance of the sample was immediately measured at 450 nm, and the value of the blank well was subtracted from the result.

[0122] For endogenously expressed GFP and mCherry samples, 1 μg / mL of cell lysate diluted in coating buffer was added to the microplate following the BLISA protocol described above.

[0123] For anti-SARS-CoV-2 Spike RBD IgG detection, a sandwich immunoassay was used to coat the microplate with SARS-CoV-2 Spike RBD-His recombinant protein (40592-V08B, Sino Biological, Beijing) (1 μg / mL, diluted in PBS) overnight at room temperature and washed with a microplate washer (Wellwash). TMVersa Microplate Washer, Thermo Fisher Scientific) was washed three times with plate wash buffer. After incubation in blocking buffer for 1 hour at room temperature after three washes, 1 μL of serum and 49 μL of PBS were added to the coated microplate and incubated at 37°C for 1 hour. After five washes, the captured anti-SARS-CoV-2 Spike RBD IgG was detected by Ab-SS-DNA barcode (antibody against human IgG) diluted in incubation buffer after washing and DNA extraction as described above.

[0124] For HBsAg and HBeAg detection, after washing and DNA extraction, 40 μL of serum, 10 μL of PBS and 50 μL of Ab-SS-DNA barcode pool (detection antibodies against HBsAg and HBeAg) were added to the microplate coated with the captured antibodies.

[0125] Table 3. Antibodies and Nb-DNA oligo (or Nb-SS-DNA oligo).

[0126]

[0127]

[0128]

[0129] BLISA assay on magnetic beads.

[0130] Table 3 lists the detailed information of the antibody pairs and DNA barcodes. In short, each vial of capture antibody was coupled to 18 mg of epoxy magnetic beads (Beijing Yunci Technology). The beads were mixed with 0.1 M sodium phosphate (pH 7.4), capture antibody and 1 M ammonium sulfate. The mixture was incubated at 37 ° C with rotation (5 rpm) for 16-24 hours. The beads were then washed three times with PBS containing 1% Triton X-100 and then blocked with PBS containing 1% BSA and 0.05% Tween-20 for 2 hours at room temperature. After magnetic separation, the beads were maintained at a concentration of 10 mg / mL in PBS containing 0.1% BSA, 0.05% Tween-20 and 0.03% NaN3 and stored at 4 ° C. For multiple capture, the antibody-coupled beads of all targets were mixed before use.

[0131] After washing twice with bead wash buffer (0.1% Triton X-100 in PBS), the beads were resuspended in wash buffer and distributed to each well of a 96-well deep-well plate (501102, NEST). The beads were then mixed with 100 μL of cell lysate sample or standard protein at room temperature for 30 minutes in a plate mixer (800 rpm). After 3× washing steps with bead wash buffer, the beads were separated using a 96-well magnetic stand. The beads captured by Ab-SS-DNA barcode detection antigen were pooled in bead incubation buffer (0.5% BSA, 0.05% Triton X-100, 0.1% dextran sulfate, 0.4 mg / mL sheared salmon sperm DNA, 5 μM TP1107-NGL-His6, 2 μM TP897-NGL-His6 in PBS) for 30 minutes at room temperature on a plate mixer (800 rpm), followed by 5× washes with bead wash buffer. The beads were rinsed twice with PBS and the DNA barcodes were extracted with 50 μL elution buffer (20 mM TCEP in PBS) for 5-10 minutes at room temperature on a plate mixer (1000 rpm). After magnetic separation, the DNA extracted solution was transferred to a new tube and measured immediately or stored at -20°C until analysis.

[0132] Design of DNA barcode sequences.

[0133] The DNA barcode sequence (62 bp) was designed to include a 6-bp antibody-specific barcode (Ab bc) and a 15-bp unique molecular identifier (UMI). For qPCR, the DNA barcodes were designed with different sequences at both ends and different primer pairs were used for specific amplification. Table 1 lists the sequences of all DNA barcodes.

[0134] Quantitative PCR (qPCR) measurements.

[0135] After thorough vortexing, qPCR (20 μL / reaction, 3 replicate wells for each sample) was performed using 1 μL of the DNA extraction solution and SYBR qPCR reaction mixture (Q712, Vazyme) in a CFX 96 instrument (Bio-rad).

[0136] Sequencing library preparation.

[0137] For the two-step PCR protocol to further barcode the DNA barcode released from each well, 25 μL PCR was performed for each sample, each containing 1 μL of released DNA barcode, 0.1 mM dNTP, 0.25 μL Q5 High Fidelity DNA Polymerase (NEB), 1×Q5 reaction buffer, 0.4 μM well-specific forward primer (6-bp well barcode) and 0.4 μM reverse primer (Table 4-Table 6). The PCR thermal cycling conditions performed were as follows (PCR Condition 1): (1) 98°C for 30 seconds; (2) 98°C for 10 seconds; (3) 50°C for 30 seconds; (4) 72°C for 10 seconds; (5) repeat steps 2-4 nine times; (6) 72°C for 2 minutes; and (7) 12°C∞. 1 μL of each well-barcoded DNA from one plate was pooled and purified using 1.8×AMPure XP beads (Beckman Coulter) according to the manufacturer's protocol. The well-barcoded DNA pool was eluted with 30 μL of nuclease-free water. 29 μL of eluted DNA, 0.2 mM dNTP, 0.5 μL Q5 polymerase, 1×

[0138] Another 50 μL PCR was prepared with Q5 reaction buffer, 0.5 μM adapter forward primer (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG-3', SEQ ID NO: 63) and 0.5 μM adapter reverse primer containing i7 index (5'-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACG-3', SEQ ID NO: 64). PCR condition 1 was performed and the product was purified with 1.8× AMPure XP beads. The final sequencing library was eluted in 30 μL nuclease-free water.

[0139] For the three-step PCR protocol with phase spacer addition, 25 μL PCR was performed per well, where each well contained 1 μL DNA barcode (released DNA and norm.bc.), 0.1 mM dNTPs, 0.25 μL Q5 high-fidelity DNA polymerase, 1× Q5 reaction buffer, 0.5 μM well-specific forward primer, and 0.5 μM reverse primer (Table 4). PCR condition 1 was performed. 0.73 μL of barcoded DNA from each well of the same plate was pooled, purified with 1.8× AMPure XP beads, and eluted in 85 μL nuclease-free water. The eluted well barcoded DNA pool (10 μL each) was then divided into 8 PCR reactions (25 μL) for phase spacer addition, which contained 0.1 mM dNTPs, 0.25 μL Q5 high-fidelity DNA polymerase, 1× Q5 reaction buffer, 0.5 μM PS forward primer, and 0.5 μM PS reverse primer (Table 4). The conditions for PCR thermal cycling were as follows: (1) 98°C for 30 seconds; (2) 98°C for 10 seconds; (3) 55°C for 30 seconds; (4) 72°C for 10 seconds; (5) repeat steps 2-4 nine times; (6) 72°C for 2 minutes; (7) 12°C∞. 8.75 μL of each phase spacer-labeled DNA product (70 μL) was pooled, purified with 1.8×AMPure XP beads, and eluted in 30 μL of nuclease-free water. A 50 μL PCR was then prepared using 5 μL of eluted DNA, 0.2 mM dNTPs, 0.5 μL Q5 polymerase, 1× Q5 reaction buffer, 0.5 μM forward primer 151AR with i5 index, and 0.5 μM reverse primer 117JvA with i7 index (plate index). The conditions for PCR thermal cycling were as follows: (1) 98°C for 30 seconds; (2) 98°C for 10 seconds; (3) 60°C for 30 seconds; (4) 72°C for 10 seconds; (5) repeat steps 2-4 nine times; (6) 72°C for 2 minutes; (7) 12°C∞. The adapter-tailed DNA library was purified with 1.8× AMPure XP beads and eluted with 20 μL of nuclease-free water. The libraries on each plate were analyzed and quantified for pooling together for sequencing.

[0140] Sequencing was performed on a HiSeq2500 system according to the manufacturer's instructions (Illumina).

[0141] Table 4. Two-step PCR (Primers for Step 1)

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] Table 5. Three-step PCR (primers for step 1)

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155] Table 6. Three-step PCR (primers for step 2)

[0156]

[0157]

[0158] Sequencing data analysis.

[0159] To extract and quantify pore barcodes (pore bcs), antibody-specific barcodes (Ab bcs), and UMIs, we designed a custom pipeline using a combination of UMI tools and unix commands. The pipeline efficiently quantified barcode and UMI combinations while accounting for potential sequencing errors.

[0160] First, a whitelist of barcode and UMI combinations was generated from positions 1-6, 21-26, 27-41 of the fastq reads using the “UMI-tools whitelist” application. The whitelist was then cleaned against a benchmark true barcode list to reduce the false positive rate for read quantification. The barcodes and UMIs were then extracted using the “UMI-tools extract” application while base pair correction was performed on barcode-UMI pairs with a 2 Hamming Distance from the whitelist barcode-UMI pairs. Finally, well barcodes, antibody-specific barcodes, and UMIs were demultiplexed using unix commands to obtain the final count table for stored barcode-UMI quantification.

[0161] Example

[0162] Example 1. Design of MaMBA.

[0163] As a linker, nanobodies targeting the fragment crystallizable (Fc) region of IgG were recombinantly expressed and fused to the Asn-Gly-Leu (NGL) tripeptide recognition motif and a 6×His tag (for protein purification) at the C-terminus. To functionalize the nanobodies, an enzymatic reaction was utilized in which the NGL motif of the nanobodies was recognized by a protein ligase, which was recombinant Oldenlandia affinis asparaginyl endopeptidase (OaAEP1). OaAEP1 cleaved the NGL motif, ligated the GV-based azide-modified peptide GVGK(N3)RG, and produced a nanobody-NGVGK(N3)RG-product that was difficult to recognize by OaAEP1, enabling its efficient production. The azide-functionalized nanobodies were then coupled to DBCO-modified (azide-reactive) DNA oligonucleotides (DNAoligo) via a click reaction ( Figure 8 A and Figure 1 A). DNA oligo-conjugated nanobodies (Nb-DNA oligo) are ready for barcoding antibodies to perform multiplexed assays.

[0164] For antibody barcoding, each antibody is mixed with antibody-specific Nb-DNA oligo (specific DNA sequence). DNA oligo is coupled to the Fc domain of the antibody through site-specific binding of the nanobody to form a DNA-barcoded complex (Ab-Nb1 to Ab-Nbn), and excess Nb-DNA oligo is removed by filtration. Then, each DNA oligo-coupled antibody can be directly pooled and applied to different assays such as immunostaining, quantitative protein detection, etc. ( Figure 1 B).

[0165] Example 2. MaMBA enables multiplexed in situ protein imaging via misHCR.

[0166] First, we applied MaMBA to immunostaining for multiplexed in situ protein imaging. Previously, we developed isHCR, a DNA-based method for immunofluorescence signal amplification that utilizes a pair of fluorophore-modified DNA hairpins (HCR amplification strands) to repeatedly open the DNA hairpins through multi-step binding triggering of an initiator DNA strand (HCR priming strand) that binds to an epitope (the antigenic target of the antibody). However, redundant schemes made anchoring the HCR priming strands to the epitope laborious and time-consuming, while secondary antibody staining strategies also limited the multiplexity. After barcoding antibodies with different orthogonal HCR priming strands (Ab-HCR priming strands), we could easily reduce the HCR priming strand anchoring process to only one step and harness the power of DNA to expand the multiplexity ( Figure 2 A).

[0167] To examine the performance of misHCR, we immunostained mouse brain sections with antibodies against NeuN or tyrosine hydroxylase (Th) conjugated with equal amounts of HCR-trigger chain-coupled nanobodies and fluorophore-coupled nanobodies. The signal-amplified HCR-trigger chain-coupled nanobodies (misHCR-Cy5 / Cy3) showed a significant signal increase compared to the fluorophore-coupled nanobodies (Nb-Cy5 / Cy3), and target specificity was confirmed by the same pattern shown in the high-contrast images of Nb-Cy5 / 2Cy3 staining ( Figure 2 B-2D). We also confirmed that misHCR does not affect the spatial resolution of diffraction-limited confocal imaging performed by conventional methods ( Figure 8 B-8D).

[0168] Next, we performed misHCR for multiplexed immunostaining and imaged through several rounds of HCR and washes (using formamide to dehybridize the HCR amplifier strands). MaMBA-based HCR priming strand-barcoded antibodies allow us to perform a one-step antibody incubation independent of the antibody host species, followed by several rounds of hybridization and dehybridization of the HCR amplifier strands to sequentially image multiple epitopes ( Figure 2 E). We compared the images before and after washing (dehybridization of the HCR amplified strand) between imaging rounds, showing that the signal was effectively removed after removal of the HCR amplified strand ( Fig. 9 A). We also evaluated the chain binding integrity of misHCR for antibodies against NeuN, NF-H, and Th by performing five repeated HCR and washing cycles. It showed highly reproducible localization patterns of the signal at each HCR, and we found that more than 80% of the signal was recovered even after four HCR amplified chain washes ( Fig. 9 C and Fig. 9 D).

[0169] Next, we applied misHCR to skin sections from healthy and psoriasis patients against six markers, including platelet-derived growth factor receptor A (PDGFRα) as a progenitor marker for mesenchymal cells, keratin 14 (KRT14) as a marker for keratinocytes in the epidermis, dopachrome tautomerase (DCT) as a marker for melanoblasts, CD31 as a marker for endothelial cells, α-smooth muscle actin (α-SMA) as a marker for myofibroblasts, and CD45 as a marker for hematopoietic cells other than erythrocytes and platelets, consisting of four rabbit (Rb) IgG antibodies and two mouse (Mus) IgG1 antibodies ( Fig.10 and Figure 2F). Each antibody showed clear imaging patterns in healthy and psoriatic skin. Psoriatic skin showed vascular dilation, leukocyte accumulation, and abnormal proliferation of keratinocytes compared to healthy skin, which is consistent with previous reports. Therefore, using MaMBA for antibody barcoding and orthogonal HCR priming chains, misHCR can achieve specific and multiplexed in situ protein imaging in a streamlined workflow.

[0170] Example 3. Cleavable MaMBA through multiple rounds of misHCR (misHCR n ) enables increased multiplicity.

[0171] Given the challenges in designing orthogonal DNA hairpins, it is unrealistic to produce large quantities of HCR amplification strands to meet the demand for higher imaging throughput. In addition, antibody stripping techniques are also challenging due to the risk of cross-reactivity and impaired epitope integrity. Therefore, directly removing the HCR initiator strand from the antibody would be a viable approach. To obtain enhanced multiplexity and increase imaging throughput, we developed cleavable MaMBAs, in which azide-modified nanobodies are conjugated to DNA oligos with disulfide linkers and DBCO. The disulfide linker can be cleaved by a reducing agent ( Figure 3 A).

[0172] After one round of misHCR imaging, the HCR initiating strands can be released by reductive cleavage and washed away. Then, the next round of misHCR can be performed and the same set of HCR initiating strands can be reused ( Figure 3 B). We compared images before and after washing between misHCR rounds, effectively removing the signal by removing the HCR priming strand ( Fig. 9 B).

[0173] We then validated multiple rounds of misHCR in 35 μm-thick mouse brain cryosections. n ) and imaged nine targets through three rounds of misHCR that repeatedly used three orthogonal HCR priming chains (B1 I1, B4 I1, and B5 I1) ( Fig.11 A). We selected antibodies, all derived from the Rb host species, against several targets with established imaging distribution patterns, such as NeuN, glial fibrillary acidic protein (GFAP), neurofilament-H (NF-H), tryptophan hydroxylase 2 (Tph2), and ionized calcium adaptor protein 1 (Iba1). We validated the specificity of the barcoded antibodies by comparing the colocalization of staining between misHCR and conventional indirect immunofluorescence ( Fig.12We also compared the staining patterns of different cell type markers (NeuN for neurons, Iba1 for microglia, and GFAP for astrocytes) as well as different neuronal markers (nNOS for nNOS-expressing neurons, Th for dopaminergic neurons, and Tph2 for serotonergic neurons), and found no cross-reactivity between barcoded antibodies within or between misHCR rounds ( Fig.11 B).

[0174] We further demonstrated imaging of 12 target proteins in the dorsal raphe nucleus (DRN) of the mouse brain by two rounds of misHCR using nine orthogonal HCR priming chains conjugated to antibodies all derived from the Rb host species ( Figure 3 C). By comparing the staining patterns, we observed three types of DDC-expressing neurons in the DRN, including dopaminergic neurons (DDC + Th + Tph2 - 5-HT - cells), serotonergic neurons (DDC + Th - Tph2 + 5-HT + cells) and so-called D neurons (DDCs + Th - Tph2 - 5-HT - cell)( Figure 3 D) Therefore, misHCR n By performing multiple rounds of misHCR to increase imaging throughput, the cleavable MaMBA was utilized to easily remove the anchored HCR initiator strands by reductive cleavage, thereby efficiently recycling the HCR initiator strands.

[0175] Example 4. Extension of cleavable MaMBA to multiplex quantitative protein detection by BLISA.

[0176] To further demonstrate the flexibility and diversity of MaMBA applications, we applied cleavable MaMBA to ELISA-like assays and developed BLISA for multiplexed quantitative protein detection. Utilizing the DNA-based barcoding properties and its site-specific conjugation to antibodies, BLISA enables the simultaneous detection and quantification of multiple target proteins. The DNA barcodes are released by reductive cleavage, which reduces interference from other DNA adhered or nonspecifically bound to the immobilized surface, and then quantified by qPCR or sequencing technology.

[0177] We first validated BLISA in cell lysate by directly probing samples immobilized on the microplate surface. DNA barcoded antibodies were then loaded to detect their targets on the plate. After washing away unbound antibodies, the DNA barcodes were extracted by a reducing agent such as TCEP and quantified by qPCR ( Figure 4 A).

[0178] Considering that BLISA is generated not only by nonspecific antibody binding but also by nonspecific binding of DNA conjugates, we used protein-free blocking buffer (PFBB) and added sheared salmon sperm DNA (ssDNA) to the antibody incubation solution to block the unnecessary binding of antibodies and DNA barcodes. To evaluate whether BLISA can distinguish different target proteins in a single well, we immobilized purified GFP and mCherry with known concentration gradients on the microplate, mixed with 1 μg / mL wild-type cell lysate (to simulate the cell matrix environment), and then detected three targets including GFP, mCherry, and α-tubulin (from cell lysate) ( Figure 4 B) GFP and mCherry detected by BLISA and their designed gradients (R 2 =0.9893 and 0.9972) have good correlation and no significant interference between them ( Figure 4 C and Figure 4 D).

[0179] We also applied BLISA to endogenously expressed proteins in cells by transiently transfecting target genes driven by drug-inducible promoters. We introduced the Tet-ON system (TRE promoter triggered by drug-bound rtTA transcription factor) for GFP expression controlled by anhydrotetracycline hydrochloride (ATc, a tetracycline derivative) and the c-AMP response element (3×CRE) for mCherry expression induced by forskolin (FSK) ( Figure 4 E. Fig.13 A and Fig.13 C). We then simultaneously detected GFP, mCherry, and α-tubulin (as a normalized loading control) by BLISA and compared with Western blots, which served as our "gold standard" to measure the relative expression levels of GFP and mCherry under a range of triggers with ATc and FSK. We expressed these two drug-triggered systems separately in HEK293T cells. The results of BLISA (α-tubulin normalized ΔCt) and Western blot (α-tubulin normalized density ratio) were highly correlated (r = 0.9724, P < 0.0001 for GFP; r = 0.9647, P < 0.0001 for mCherry) ( Fig.13B and Fig.13 D). We then combined them by co-transfection in HEK293T cells, and the results showed that the results of the 3-plex BLISA assay were still highly correlated with those of Western blotting (r = 0.9718 for GFP, P < 0.0001, r = 0.9520 for mCherry, P < 0.0001) ( Figure 4 F-4H). Thus, taking advantage of the feasibility of MaMBA, BLISA is able to perform multiplex quantitative protein detection assays.

[0180] To combine BLISA with high-throughput sequencing, DNA barcodes were designed to contain a 6-bp antibody-specific barcode (Ab-bc) and a 15-bp unique molecular identifier (UMI). The well-specific barcode (well-bc) was added to the extracted DNA barcodes through PCR amplification. Each well-specific DNA barcode was then pooled to prepare an index library for sequencing ( Fig.13 E) Each target protein in each well is identified by its antibody barcode and well barcode and quantified by UMI counts. We evaluated Figure 4 The BLISA sequencing reads of the samples designed in C were used to verify the dose-effect dependence curves of GFP and mCherry (R 2 =0.9827 and 0.9843)( Fig.13 F and Fig.13 G), demonstrating the feasibility of sequencing combined with BLISA.

[0181] Example 5. Sequencing-combined BLISA for detection of anti-SARS-CoV-2 Spike RBD IgG in human serum.

[0182] To investigate whether BLISA combined with high-throughput sequencing can accurately detect protein levels in human samples, we first performed BLISA for the detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike receptor binding domain (RBD) IgG in human serum. SARS-CoV-2 uses the Spike RBD to bind to the cellular receptor ACE2, triggering the virus to infect cells. Therefore, high-throughput serological testing for antibodies against the SARS-CoV-2 Spike RBD is an important component of evaluating the efficacy of SARS-CoV-2 vaccines, especially in susceptible and high-risk population subgroups such as health care workers.

[0183] To achieve higher sensitivity, we applied a sandwich-type immunoassay in which the analyte (anti-SARS-CoV-2 Spike RBD IgG in serum) was captured by an immobilized antigen (purified SARS-CoV-2 Spike RBD protein) and by a DNA-barcoded di(2 nd ) anti-human IgG antibody. After preparing the sequencing library, the DNA barcodes ( Figure 5 A and Figure 5 F).

[0184] To evaluate the sensitivity of BLISA, we compared the human IgG detection profiles between BLISA and ELISA. For BLISA, known amounts of human IgG were immobilized on microplates and detected by DNA-barcoded anti-human IgG antibodies after qPCR readout. For ELISA, immobilized human IgG was detected by anti-human IgG antibodies (from the Rb host species), which were detected by horseradish peroxidase (HRP)-conjugated anti-Rb IgG antibodies, followed by colorimetric readout with 3,3',5,5'-tetramethylbenzidine (TMB) substrate ( Figure 5 B). The results showed that compared with ELISA, BLISA was about 44 times more sensitive and had a dynamic range of about 0.15 logarithmic times wider ( Figure 5 C).

[0185] As library preparation for sequencing, the extracted DNA barcodes and normalized barcodes were further barcoded by three-step PCR amplification, which sequentially added well bc, phase spacer, and sequencing adapter ( Figure 5 D and Figure 5 E). At the phase of spacer addition, the well-barcoded DNA was pooled and split into 8 PCR reactions to add different numbers of bases as spacers at both ends, for a total of 7 bases (0 bases and 7 bases, 1 base and 6 bases, 2 bases and 5 bases, etc.), which provided higher input for amplicon sequencing by enriching base diversity. We performed sequencing-combined BLISA for anti-SARS-CoV-2 SpikeRBD IgG, and repeated sequencing of approximately 500 human serum samples was performed in 12 microplates (S1 to S12) with a throughput of more than 1000 wells ( Figure 5 G). The results of sequencing data were fitted with Gaussian distribution (R 2 =0.7369)( Figure 5 H).

[0186] To show IgG abundance, the data were normalized by dividing the UMI count of IgG by the UMI count of the normalized barcode. We found that the variability of the signal was significantly reduced after normalization, with 98.4% of the CVs below 0.2, and the correlation between repeated sequencing was high (r = 0.9882, P < 0.0001), demonstrating the reproducibility of sequencing combined with BLISA with the participation of normalized barcodes ( Figure 5 I and Fig.14 A). At the same time, we randomly selected a portion of sera (n = 13; s1 to s13) and placed them on different plates for sequencing. The results of the two BLISA readouts (qPCR and sequencing) were consistent (r = 0.9354, P < 0.0001), and the IgG abundance shown by sequencing was highly correlated with the ELISA results (r = 0.9743, P < 0.00001), indicating that BLISA has high accuracy and low inter-plate variability ( Figure 5 JL, Fig.14 B and Fig.14 C).

[0187] Example 6.2 - Resequencing - Combined BLISA for detection of hepatitis B virus (HBV) antigens in human serum.

[0188] To further examine whether sequencing-coupled BLISA can simultaneously detect multiple targets in large-scale human samples, we performed 2-plex BLISA to detect hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in human serum, which are essential antigenic markers for the diagnosis of hepatitis B. While the anti-SARS-CoV-2 Spike RBD IgG assay used a sandwich strategy for IgG detection, the HBsAg and HBeAg assays used a sandwich strategy for antigen detection, using a pair of antibodies, namely, capture antibodies (Abs Cap ) and detection antibody (Ab Det )( Figure 6 A and Figure 6 B).

[0189] Analyte (HBsAg or HBeAg) is immobilized Ab Cap Capture and barcode Ab by DNA Det Detection, Ab Det We first validated the 2-plex BLISA by qPCR in double-negative and double-positive human serum samples defined by commercial ELISA kits ( Figure 6 C-6E). In the HBsAg and HBeAg tests, there were significant differences between negative and positive samples ( Figure 6F). Next, we performed 2-plex sequencing combined with BLISA for HBsAg and HBeAg, sequencing approximately 500 human serum samples in duplicate in 12 microplates (H1 to H12) with a throughput of more than 1000 wells ( Figure 6 G and Figure 6 H). The results of repeated sequencing of HBsAg and HBeAg groups were highly correlated (r=0.9973 for HBsAg, P<0.0001; r=0.9770 for HBeAg, P<0.0001) ( Figure 6 I). In cases where the abundance of HBsAg or HBeAg groups exceeded the overall mean, we validated 84 samples by ELISA and found 22 HBsAg + HBeAg - Sample and 1 HBsAg + HBeAg + Sample (#23) correlated with BLISA results (HBsAg, r = 0.9517, P < 0.0001; HBeAg, r = 0.7390, P < 0.0011) ( Figure 6 J).

[0190] Example 7. Multiple phosphorylated proteins in cell lysates by magnetic bead-based BLISA.

[0191] To enhance the multiplexability of BLISA, we switched to using magnetic beads (MBs) as the assay binding surface. We performed a sandwich immunoassay in which the capture antibody was conjugated to the epoxy-modified MBs and the antigen was identified by a unique DNA barcode extracted from the detection antibody ( Figure 7 A) First, MBs conjugated to capture antibodies for each target protein are mixed and aliquoted into each well of a deep-well plate. Samples are loaded, target proteins are captured, and then a wash step is performed to remove unbound biomolecules. Det -SS-DNA barcode mixture to detect target protein. After washing away excess Ab Det After -SS-DNA barcoding, DNA barcodes were extracted and quantified by TCEP ( Figure 7 B). We validated the standard curves of seven phosphoproteins in BLISA, which showed that BLISA has higher sensitivity compared with ELISA ( Fig.15 To verify the multiplexity of MB-based BLISA, we simultaneously detected seven purified phosphoproteins (p-p38α, p-ERK1 / 2, p-JNK, p-AMPKα1, p-CREB, p-Src, and p-Akt) by loading them into seven sample wells of different concentration gradient frames ( Figure 7C). All phosphoproteins detected by MB-based BLISA were highly correlated with their designed gradient framework (R 2 = 0.9258, 0.9499, 0.9190, 0.9935, 0.9882, 0.9892 and 0.8979), with no significant interference between them ( Figure 7 D and Figure 7 E). Next, we performed a 7-plex BLISA assay for phosphoproteins in cell lysates of U87 (adherent) and U937 (suspension) cell lines cultured under different conditions and measured their seven target levels, and also performed the corresponding ELISA ( Figure 7 F). The results shown by 7-plex BLISA were highly correlated with those of ELISA (r=0.9241, P=0.0010; r=0.9541, P=0.0002; r=9685, P<0.0001; r=7684, P=0.0259; r=0.9845, P<0.0001; r=0.9829, P<0.0001; r=0.7636, P=0.0275), indicating the feasibility of MB-based BLISA for multiplex detection in cell lysates.

Claims

1. A composition comprising an antibody linked to an oligonucleotide via a disulfide bond, wherein the oligonucleotide is specific for the antibody.

2. The composition according to claim 1, wherein the disulfide bond can be cleaved by a reducing agent, preferably tris(2-carboxyethyl)phosphine, dithiothreitol or β-mercaptoethanol, thereby releasing the oligonucleotide.

3. The composition of claim 1, wherein the oligonucleotide comprises a hybridization chain reaction (HCR) initiator chain.

4. The composition of claim 1, wherein the oligonucleotide comprises a first barcode sequence specific for the antibody to which it is attached.

5. The composition of claim 4, wherein the oligonucleotide further comprises a unique molecular identifier (UMI) sequence, preferably, the UMI sequence comprises a length of 10 to 20 nucleotides, more preferably, a length of 12 to 18 nucleotides. The composition according to claim 4 , wherein the first barcode sequence comprises a length of 5 to 15 nucleotides, preferably a length of 5 to 10 nucleotides.

7. The composition of claim 4, wherein the oligonucleotide further comprises a second barcode sequence that is specific to information associated with the sample to be detected.

8. The composition according to claim 1, wherein the antibody is a secondary antibody selected from anti-IgE antibody, anti-IgM antibody, anti-IgG antibody, anti-IgA antibody and anti-IgD antibody, Preferably, the oligonucleotide-associated antibody is a nanobody.

9. The composition of claim 8, wherein the oligonucleotide-associated antibody specifically binds to the Fc region of the anti-IgE, anti-IgM, anti-IgG, anti-IgA or anti-IgD antibody. 10 . A kit for detecting multiple targets, comprising the composition according to any one of claims 1 to 9.

11. The kit according to claim 10, wherein the kit comprises a plurality of oligonucleotide-associated antibodies, each comprising an antibody associated with a specific HCR triggering chain, and one or more amplification chains, Preferably, each amplifier strand is coupled to a detectable label selected from chemical groups such as biotin, digoxigenin, acrylamide, amine, succinimidyl ester, thiol, azide, TCO, tetrazine, alkyne and DBCO; and / or fluorophores such as FITC, cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluor dyes.

12. A method for detecting one or more targets in a sample, comprising the steps of: a) incubating a first primary antibody with the composition as defined in any one of claims 1 to 9 to form a complex with the first primary antibody associated with the composition.

13. The method according to claim 12, wherein the antibody of the composition is a secondary antibody, and the complex is formed by the interaction between the first primary antibody and the secondary antibody.

14. The method according to claim 12 or 13, further comprising the steps of: b) contacting the sample with the complex; c) performing an amplification reaction to produce a detectable amplification product; as well as d) Optionally, adding a reducing agent and incubating to cleave the disulfide bonds, and repeating steps a) to c).

15. The method according to claim 14, wherein the oligonucleotide comprises an HCR priming strand and the amplification reaction in step c) is a hybridization chain reaction.

16. The method according to claim 12 or 13, further comprising the steps of: b) contacting the sample with the complex; c) adding a reducing agent and incubating to cleave the disulfide bonds; d) collecting the specific oligonucleotides and performing an amplification reaction to detect the one or more targets; and e) optionally, repeating steps a) to d), Wherein the oligonucleotide comprises a first barcode sequence.

17. The method according to any one of claims 12 to 16, wherein the method comprises, before step b), a step of incubating the sample with a carrier associated with a target-specific binding substrate to capture the target in the sample, preferably, the target-specific binding substrate is a second primary antibody or an antigen.

18. The method of claim 17, wherein the carrier is selected from the group consisting of a microplate and magnetic beads.

19. The method according to any one of claims 12 to 18, wherein: In step a), one or more first primary antibodies are incubated with one or more compositions, each of which is linked to an oligonucleotide specific for the antibody to which it is linked.

20. The method according to any one of claims 12 to 19, wherein the method is used for high throughput sequencing.

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