Method for enabling plasma cell to capture antibody secreted by plasma cell

Abstract

The present invention relates to the technical field of biology, and provides a method for enabling a plasma cell to capture an antibody secreted by the plasma cell. Specifically, the method comprises: labeling the plasma cell with a capture reagent; and performing single-cell encapsulation treatment on the labeled plasma cell to capture an antibody, wherein the capture reagent has the activity of binding the antibody secreted by the plasma cell, and the plasma cell has the activity of secreting the antibody. By means of the method, the antibody secreted by the plasma cell can be recaptured onto the membrane surface of the plasma cell to form a structure similar to a BCR of a memory B cell, enabling a corresponding antigen protein to specifically recognize and bind to the BCR on the membrane surface of the plasma cell, thereby enabling screening and sequencing of an antigen-specific plasma cell.

Description

Methods for plasma cells to capture antibodies secreted by themselves Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for plasma cells to capture antibodies secreted by themselves. Background Technology

[0002] Monoclonal antibodies (mAbs) are among the most important types of biopharmaceuticals on the pharmaceutical market. Currently, over 100 monoclonal antibodies have been approved by the US FDA for various diseases, including cancer, infectious diseases, autoimmune diseases, and neurological disorders. Their safety profile and high target specificity make them one of the most promising classes of biopharmaceuticals. Furthermore, the reactivity of antibodies determines the effectiveness and durability of immune protection after viral infection or vaccination, playing a crucial role in various autoimmune diseases and cancers. Therefore, the efficient development of fully human antibodies for antibody drug development remains an important topic, especially during viral pandemics, when there is an urgent need for highly effective virus-neutralizing antibodies to treat diseases caused by viral infections.

[0003] The variable region of the B cell antigen receptor (BCR) determines the antigen specificity of B cells, thus exhibiting great diversity. This diversity is mainly achieved through mechanisms such as recombination, linker diversity, and somatic mutations in the gene encoding the antibody variable region V(D)J. Therefore, developing a high-throughput, high-efficiency method for screening antigen-specific B cells is of great importance. In mammals, antibodies are classified into five main isotypes or classes based on their heavy chains: IgG, IgA, IgD, IgE, and IgM. Each type of antibody has different structures, location distributions, and functional characteristics. For example, IgE is mainly found in the skin, lungs, and mucous membranes; upon binding to mast cells, it activates histamine release, causing allergic reactions. IgA is mainly found in saliva, tears, mucus, breast milk, and intestinal fluid, and can prevent the ingestion and inhalation of pathogens. IgM is mainly found in the blood and lymphatic system, serving as the first line of defense against infection and also playing an important role in immune regulation. IgG is the most common antibody, accounting for approximately 70% to 75% of all immunoglobulins in the body; it is mainly found in blood and tissue fluid, protecting the body from viral and bacterial infections. Therefore, high-throughput acquisition of complete BCR sequence information is crucial for a more comprehensive understanding of antibody response characteristics.

[0004] Antigen-specific B cells include terminally differentiated plasma cells and memory B cells. Plasma cells secrete large amounts of antibodies, enabling antibody-mediated humoral immunity, and are therefore also known as antibody-secreting cells (ASCs). Based on their lifespan, they can be divided into short-lived plasma cells and long-lived plasma cells, with long-lived plasma cells determining long-term antibody immunity levels in the body. ASC responses play a crucial protective role in pathogen infection and vaccine immunization; however, the secretion of antibodies against self-antigens by ASCs has become a detrimental factor in many autoimmune diseases. Despite the importance of ASCs, research on their differentiation, phenotypic characteristics, heterogeneity, and mechanisms for maintaining their long lifespan is very limited. Currently, most high-throughput methods for sorting antigen-specific B cells focus primarily on memory B cells, neglecting ASCs, thus limiting a more comprehensive and accurate analysis of antibody immune responses.

[0005] Therefore, it is important to develop a platform that can recapture antibodies secreted by plasma cells onto the surface of plasma cell membranes and study them. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0007] Therefore, in a first aspect, the present invention provides a method for plasma cells to capture their own secreted antibodies. According to an embodiment of the present invention, the method includes: labeling the plasma cells with a capture reagent; encapsulating the labeled plasma cells into single cells to achieve antibody capture; wherein the capture reagent has the activity of binding antibodies secreted by the plasma cells, and the plasma cells have the activity of secreting antibodies. In order to simultaneously obtain information on antibodies secreted by plasma cells during the analysis or sequencing of antigen-specific B cells, the inventors have creatively proposed labeling the surface of plasma cells with an assembled capture reagent, enabling the secreted antibodies to be recaptured back to the cell surface, forming a structure similar to the BCR of memory B cells. This allows the corresponding antigen protein to specifically recognize and bind to the BCR on the surface of the plasma cell membrane, thereby screening and sequencing antigen-specific plasma cells. During subsequent single-cell library construction and sequencing, simultaneous analysis of plasma cells and memory B cells can be achieved. Furthermore, this method is convenient, efficient, and low-cost, making it suitable for adoption by different laboratories.

[0008] According to embodiments of the present invention, the method for plasma cells to capture their own secreted antibodies may further include at least one of the following additional technical features:

[0009] According to an embodiment of the present invention, the capture reagent comprises rabbit anti-mouse CD138 antibody and bispecific nanobody (VHH-anti-rabbit-IgG & VHH-anti-mouse-kappa).

[0010] According to an embodiment of the present invention, the single-cell encapsulation process is performed in a droplet generation device.

[0011] According to an embodiment of the present invention, the single-cell encapsulation treatment further includes: incubating the single-cell encapsulation treatment product; and demulsifying the incubated product to obtain plasma cells containing autosecretory antibodies.

[0012] According to an embodiment of the present invention, the labeling treatment of the rabbit anti-mouse CD138 antibody is carried out at 1-5°C for 20-40 min.

[0013] According to an embodiment of the present invention, the labeling treatment of the bispecific nanobody (VHH-anti-rabbit-IgG & VHH-anti-mouse-kappa) is carried out at 1-5°C for 20-40 min.

[0014] According to an embodiment of the present invention, the incubation treatment is carried out at a temperature of 30°C to 40°C for 1 to 2 hours.

[0015] According to an embodiment of the present invention, the demulsification treatment is carried out in the presence of a demulsifier.

[0016] According to an embodiment of the present invention, the demulsification treatment is performed at 20–30°C for 3–10 min.

[0017] In a second aspect, the present invention provides a plasma cell. According to an embodiment of the invention, the plasma cell membrane surface contains auto-secreted antibodies, which are captured by the method described in the first aspect. By recapturing the auto-secreted antibodies of the plasma cell onto the plasma cell membrane surface using the method described in the present invention, the plasma cell acquires a structure similar to the BCR of a memory B cell, which is beneficial for subsequent screening and sequencing of the plasma cell.

[0018] In a third aspect, the present invention provides a method for screening antigen-specific plasma cells. According to an embodiment of the present invention, the method includes: antigen-labeling the plasma cells described in the second aspect with a protein antigen labeled with an oligo-barcode; sorting the antigen-labeling product to obtain the antigen-specific plasma cells; wherein the protein antigen has the activity of binding to antibodies secreted by the plasma cell membrane. Through the method described in this invention, the corresponding protein antigen can specifically recognize and bind to the BCR on the surface of the plasma cell membrane, thereby screening for antigen-specific plasma cells.

[0019] According to embodiments of the present invention, the method for screening antigen-specific plasma cells may further include at least one of the following additional technical features:

[0020] According to an embodiment of the present invention, the antigen labeling treatment is performed at 1°C to 5°C for 20 min to 40 min.

[0021] According to an embodiment of the present invention, the sorting process is performed by flow cytometry.

[0022] According to an embodiment of the present invention, the sorting process is performed by: binding the antigen-labeled treatment product with an anti-His tag dye; staining the binding product with DAPI; and obtaining antigen-specific plasma cells based on the fluorescence signal of the staining product.

[0023] In a fourth aspect of the invention, the invention proposes the use of antigen-specific plasma cells obtained by screening using the method described in the third aspect in the construction of single-cell sequencing libraries and single-cell sequencing. Beneficial effects:

[0024] 1) Low cost, high throughput and easy operation: This technical solution does not require the development of new instruments and equipment. With the droplet generation method and equipment independently developed by BGI Life Sciences, it is very convenient, low cost and high throughput to modify millions of plasma cells, so that the antibodies secreted by the cells themselves can be recaptured on the cell membrane surface.

[0025] 2) By labeling the surface of plasma cells with capture reagents that can capture secreted antibodies, the antibodies secreted by plasma cells can be recaptured onto the cell membrane surface, forming a structure similar to the BCR of memory B cells. This allows for the simultaneous analysis of sequencing antigen-specific plasma cells and memory B cells.

[0026] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0028] Figure 1 is an experimental flowchart according to an embodiment of the present invention;

[0029] Figure 2 is a droplet generation device according to an embodiment of the present invention;

[0030] Figure 3 is a flow cytometry detection diagram of CD45R and CD138 expression on the surface of mouse hybridoma cells according to an embodiment of the present invention.

[0031] Figure 4 is a graph showing the ability of flow cytometry to capture antibodies after hybridoma-labeled catch reagent is used according to an embodiment of the present invention.

[0032] Figure 5 shows the single-cell encapsulation efficiency according to an embodiment of the present invention;

[0033] Figure 6 shows the culture viability of a single hybridoma cell encapsulated by a droplet according to an embodiment of the present invention;

[0034] Figure 7 illustrates the validation of the antigen protein marker oligo according to an embodiment of the present invention;

[0035] Figure 8 illustrates the flow cytometry detection of the binding of secreted antibodies and fluorescent antigens to a single hybridoma cell, according to an embodiment of the present invention.

[0036] Figure 9 is a schematic diagram of single-cell library construction and sequencing of plasma cell transcriptomes, BCR, and corresponding antigen oligo library construction according to an embodiment of the present invention. Detailed Implementation

[0037] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0038] The present invention will be further described below through specific embodiments. It should be noted that the embodiments described below are only for explaining the present invention and are not intended to limit the present invention.

[0039] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] In this document, unless otherwise stated, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0041] In this document, unless otherwise stated, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may or may not occur as described below, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0042] In this document, unless otherwise stated, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0043] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] In this document, unless otherwise stated, whether it is "cell barcode" or "oligo-barcode," it refers to a nucleic acid molecule, which can be either RNA or DNA. The different expressions are used only to indicate the different functions of these nucleic acid sequences. Those skilled in the art can understand the function of the sequence represented by each expression based on the context.

[0045] The existing technologies have the following drawbacks: 1) Low throughput, long processing time, and large workload: such as hybridoma technology; 2) Limited detection parameters: such as hybridoma technology and phage display technology, which can only obtain antigen-specific antibody variable region sequence information; flow cytometry can detect information on multiple cell surface proteins, but cannot bind BCR sequence information to the same cell; newly developed Beacon technology, due to the limited detection channels of the instrument itself, can often only detect 2 to 4 parameters; 3) Types of B cells to be detected: most technologies can only detect antibody-secreting cells or only memory B cells, making it difficult to efficiently detect both; 4) Lack of convenience and applicability: newly developed Beacon technology has high instrument costs and limited detection parameters, which is not conducive to widespread application. Therefore, this invention uses a capture reagent labeled on the surface of plasma cells to capture secreted antibodies. A single plasma cell is then encapsulated in a droplet and incubated, allowing the antibodies secreted by the individual plasma cell to be recaptured onto the cell membrane surface, forming a structure similar to the BCR of memory B cells. Antigen-specific B cells are then identified and screened using oligo and fluorescently labeled antigen proteins. Based on these antigen-specific B cells, subsequent single-cell library construction and sequencing can be performed, integrating the full-length BCR sequence (including antibody variable and constant regions), transcriptome expression, and antigen-specific information within the same single cell. This invention combines the advantages of flow cytometry and droplet microfluidics, antigen protein fluorescent labeling and oligo-barcode dual labeling, single-cell sequencing, and single-molecule sequencing analysis technologies. It fully utilizes the advantages of BGI's previously developed convenient negative-pressure droplet generation technology and sequencing platform, enabling the new method to be applied to antibody mining with high efficiency at a lower cost, and further facilitating a comprehensive analysis of antibody-mediated immune response mechanisms.

[0046] Specifically, this invention proposes a method for plasma cells to capture their own secreted antibodies. By capturing the antibodies secreted by the plasma cells onto their cell membrane surface, antigen-specific plasma cells can be screened and obtained. Based on these antigen-specific plasma cells, high-throughput analysis, sorting, and sequencing are performed. These methods will be described in detail below.

[0047] Methods for plasma cells to capture antibodies secreted by themselves

[0048] This invention proposes a method for plasma cells to capture their own secreted antibodies. According to an embodiment of the invention, the method includes: labeling the plasma cells with a capture reagent; and encapsulating the labeled plasma cells into single cells to capture the antibodies. The capture reagent has the activity of binding to the antibodies secreted by the plasma cells, and the plasma cells have the activity of secreting antibodies. Normally, after plasma cells generate antibodies, they release them into the surrounding tissue fluid and do not attach to the surface of the plasma cells. In order to simultaneously obtain information about the antibodies secreted by plasma cells during subsequent analysis and sequencing of antigen-specific B cells, the inventors creatively propose labeling the surface of plasma cells with an assembled capture reagent, enabling the secreted antibodies to be recaptured back to the cell membrane surface, forming a structure similar to the BCR of memory B cells, thus achieving the goal of simultaneously analyzing and sequencing plasma cells and memory B cells. Furthermore, this method is convenient, efficient, and low-cost, making it suitable for adoption by different laboratories.

[0049] According to some embodiments of the present invention, the capture reagent comprises a rabbit anti-mouse CD138 antibody and a bispecific nanobody (VHH-anti-rabbit-IgG & VHH-anti-mouse-kappa). The rabbit anti-mouse CD138 antibody specifically binds to mouse plasma cells, and the bispecific nanobody (VHH-anti-rabbit-IgG & VHH-anti-mouse-kappa) binds to the rabbit anti-mouse CD138 antibody while simultaneously capturing antibodies secreted by plasma cells, ultimately achieving the effect of capturing the secreted antibodies back to the cell membrane surface. This capture reagent can specifically recognize plasma cells, and the combination is flexible and variable, allowing for alterations and modifications to adapt to different research needs.

[0050] To ensure that each plasma cell captures only its own secreted antibodies, the labeled plasma cells are encapsulated in single cells. This encapsulation process utilizes droplet microfluidics, which encapsulates individual cells within tiny droplets, with each droplet containing a single cell, thus guaranteeing that each plasma cell captures only its own secreted antibodies.

[0051] According to some embodiments of the present invention, the single-cell encapsulation process is performed in a droplet generation device. The droplet generation device used in this invention is a convenient negative pressure-based droplet generation device previously developed by BGI Genomics, and the droplet generation device uses the DNBelab C4 device developed by BGI Genomics.

[0052] To obtain B cells, the single-cell encapsulation treatment further includes: incubating the single-cell encapsulation product; and demulsifying the incubated product to obtain plasma cells containing their own secreted antibodies. The incubation treatment is to enable the plasma cells to secrete antibodies, thus better capturing their own secreted antibodies; the demulsification treatment is to break up the droplets generated by the encapsulation treatment and recover the single cells within the droplets. At this point, the single cells within the droplets are plasma cells that have captured their own secreted antibodies, meaning that the cell membrane surface of the plasma cells carries their own secreted antibodies (in this invention, plasma cells carrying their own secreted antibodies are referred to as B cells).

[0053] According to some embodiments of the present invention, rabbit anti-mouse CD138 antibody labeling is performed at 1–5°C for 20–40 min, for example, at 1°C, 2°C, 3°C, 4°C, 5°C for 20 min, 25 min, 30 min, 35 min, 40 min, etc. Therefore, the capture reagent can be better labeled on the surface of the plasma cell membrane, thus laying the foundation for better capture of antibodies secreted by the plasma cells themselves.

[0054] According to some embodiments of the present invention, the bispecific nanobody (VHH-anti-rabbit-IgG & VHH-anti-mouse-kappa) labeling is performed at 1–5°C for 20–40 min, for example, at 1°C, 2°C, 3°C, 4°C, 5°C, etc., for 20 min, 25 min, 30 min, 35 min, 40 min, etc. Therefore, the capture reagent can be better labeled on the surface of the plasma cell membrane, thus laying the foundation for better capture of antibodies secreted by the plasma cells themselves.

[0055] According to some embodiments of the present invention, the incubation treatment is carried out at a temperature of 30 to 40°C for 1 to 2 hours, for example, at 30°C, 32°C, 35°C, 37°C, 40°C, etc. for 1 hour, 1.5 hours, 2 hours, etc.

[0056] According to some embodiments of the present invention, the demulsification treatment is carried out in the presence of a demulsifier.

[0057] According to some embodiments of the present invention, the demulsification treatment is carried out at 20-30°C for 3-10 minutes, for example, at 20°C, 22°C, 25°C, 27°C, 30°C, etc., for 3 minutes, 5 minutes, 7 minutes, 10 minutes, etc. Therefore, the droplets can be completely ruptured, releasing the single cells within the droplets.

[0058] plasma cells

[0059] This invention proposes a plasma cell. According to an embodiment of the invention, the plasma cell membrane surface contains self-secreted antibodies, which are captured by the method described in the first aspect. By recapturing the self-secreted antibodies of the plasma cell onto the plasma cell membrane surface using the method of this invention, the plasma cell acquires a structure similar to the BCR of a memory B cell, which is beneficial for subsequent screening and sequencing of the plasma cell.

[0060] Methods for screening antigen-specific plasma cells

[0061] This invention proposes a method for screening antigen-specific plasma cells. According to an embodiment of the invention, the method includes: treating the plasma cells with a protein antigen labeled with an oligo-barcode; sorting the antigen-labeling product to obtain the antigen-specific plasma cells; wherein the protein antigen has the activity of binding to antibodies secreted by the plasma cell membrane. Through the method described in this invention, the corresponding protein antigen can specifically recognize and bind to the BCR on the surface of the plasma cell membrane, thereby screening for antigen-specific plasma cells.

[0062] Antigen labeling involves labeling antibodies on the cell membrane of plasma cells with antigens to analyze antibody capture and screen for antigen-specific plasma cells. Sorting is used to obtain antigen-specific plasma cells capable of recognizing and binding to antigen molecules. These two steps enable the rapid screening of antigen-specific plasma cells, laying the foundation for subsequent library construction and sequencing.

[0063] According to some embodiments of the present invention, the antigen labeling treatment is carried out at 1°C to 5°C for 20 to 40 minutes, for example, at 1°C, 2°C, 3°C, 4°C, 5°C for 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, etc. Therefore, it can fully bind to antibodies on the cell membrane surface.

[0064] According to some embodiments of the present invention, the sorting process is performed by flow cytometry.

[0065] To obtain antigen-specific plasma cells, the sorting process is performed as follows: the antigen-labeled treatment product is bound to an anti-His-tagged dye; the bound product is stained with DAPI; and antigen-specific plasma cells are obtained based on the fluorescence signal of the stained product. The anti-His-tagged dye binds to the antigen on the surface of the plasma cell membrane in the antigen-labeled treatment product, causing the plasma cells to fluoresce. Through further staining and analysis, plasma cells carrying the antigen can be screened out, and these plasma cells possess antigen specificity.

[0066] use

[0067] This invention proposes the use of antigen-specific plasma cells obtained by screening antigen-specific plasma cells in the construction of single-cell sequencing libraries and single-cell sequencing.

[0068] According to some embodiments of the present invention, the RNA of the antigen-specific plasma cells is subjected to reverse transcription; and the reverse transcription product is then subjected to next-generation sequencing to form a transcriptome library. This method lays the foundation for subsequent sequencing of the antigen-specific plasma cells and for obtaining their transcriptome information and target antigen protein information.

[0069] According to some embodiments of the present invention, the RNA of the antigen-specific plasma cells is reverse transcribed; and the reverse transcription product is enriched with BCR, wherein the BCR enrichment is performed using a DNA probe targeting the constant region of the BCR; the BCR-enriched product is then subjected to single-molecule library construction and third-generation single-molecule long-length sequencing. Using the method described in this invention, BCR can be enriched and used as a template for library construction, laying the foundation for subsequent sequencing of the BCR to obtain its sequence information.

[0070] It should be noted that the "BCR" mentioned in this invention refers to antibodies bound to the surface of plasma cells.

[0071] According to some embodiments of the present invention, the DNA probe is modified with biotin.

[0072] According to some embodiments of the present invention, the DNA probe is complementary to at least a portion of the cDNA sequence in the BCR constant region.

[0073] According to some embodiments of the present invention, the enrichment process is performed using magnetic beads.

[0074] According to some embodiments of the present invention, the enrichment process specifically involves hybridization of a DNA probe targeting the BCR constant region with cDNA from the BCR constant region, followed by enrichment of the BCR using magnetic beads. The magnetic beads are capable of capturing biotin-labeled DNA probes, thereby enriching the BCR.

[0075] The flowchart of the method of the present invention is shown in Figure 1, which is mainly divided into 5 parts: (1) preparation of plasma cell single cell suspension; (2) cell staining and labeling of capture reagents; (3) droplet encapsulation of single cells; (4) oligo-barcode labeling of antigens; (5) flow cytometry analysis and sorting of antigen-specific plasma cells; (6) single cell library construction and sequencing: including transcriptome and BCR library construction and sequencing.

[0076] Figure 9 shows a schematic diagram of single-cell sequencing of plasma cell transcriptome, BCR, and corresponding antigen oligo library construction. During the reverse transcription of plasma cells to generate cDNA, the cell's RNA and antigen oligo both carry cell tag sequences. Part of the cDNA is used for transcriptome and Antigen-barcode sequencing, and the other part of the cDNA is used for BCR enrichment. Specifically, after hybridization using biotin-modified DNA probes specific to the BCR constant region, the cDNA is enriched with streptavidin magnetic beads and then amplified by PCR. After single-molecule PCR full-length sequencing, bioinformatics integration analysis is performed on the transcriptome, full-length BCR, and corresponding antigen information of a single B cell.

[0077] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0078] Example 1

[0079] 1. Testing and optimization of labeling for cell membrane surface capture reagents:

[0080] Cells were labeled using rabbit anti-mouse CD138 antibody and bispecific nanobodies (VHH-anti-rabbit-IgG & VHH-anti-mouse-kappa). The specific steps are as follows:

[0081] (1) Take 2×10 6 Antibody-secreting cells (hybridoma or immunized mouse plasma cells) were resuspended in 200 μl of cell staining solution and divided into two tubes (100 μl each). One tube (experimental group) was treated with 5 μl of Abflo 488 Rabbit anti-mouse CD138 (Abclonal, Cat: A24153), stained at 4°C for 30 minutes, then 1 ml of cell staining solution was added, centrifuged at 300 g for 5 minutes, and washed twice. The other tube (control group) was placed on ice without any further action.

[0082] (3) After washing, the cells in the experimental group were resuspended in 100 μl of cell staining solution containing different concentrations (0 ng / μl, 400 ng / μl, 4000 ng / μl) of bispecific nanobodies (VHH-anti-rabbit-IgG & VHH-anti-mouse-kappa) (reference: Tino Pleiner, et al. 2017). The cells were incubated at 4℃ for 45 minutes, washed with 1 ml of cell staining buffer (centrifuged at 300 g for 5 min), and 100 μl of cell staining solution containing 5 μl of FITC mouse anti-human CD31 (Biolegend, catalog number: 303104) was added. The capture efficiency of the capture reagent for mouse IgG antibodies was tested. The results are shown in Figure 4. As the concentration of the added bispecific antibody increased, the capture ability of the capture reagent for mouse antibodies also increased.

[0083] (4) Discard the supernatant and resuspend the cells in 500 μl of working buffer (composed of RPMI 1640 + 20% fetal bovine serum FBS + 1% penicillin antibody P / S + 100 μM non-essential amino acids + 100 μM β-thioethanol + 0.1% F68) for both the experimental and control groups. The cell concentration was 1000 cells / μl.

[0084] 2. Droplet generation, single-cell encapsulation, and incubation:

[0085] In this step, the droplet generation device will be assembled. The droplet generation device and process are shown in Figure 2. Droplet generation buffer, cell suspension, and droplet generation oil will be added to the droplet generation chip to generate droplets. The specific operating steps are as follows:

[0086] (1) Assemble the droplet generation device. Add 200 μl of cell suspension, 200 μl of working buffer and 800 μl of droplet generation oil (BIO-RAD#cat:1864006) to the sample inlet of the droplet generation chip. Pull the syringe from 12 ml to 20 ml and generate droplets by generating negative pressure.

[0087] (2) Take out the generated droplets and transfer them to a 15ml centrifuge tube, and incubate them in an incubator at 37℃ for 1-2 hours.

[0088] 3. Detection and optimization of cell viability within droplets:

[0089] (1) Take 0.5 × 10 6Antibody-secreting cells (hybridoma or plasma cells from immunized mice) were added to the cell suspension at a ratio of 1:5000 with the cell viability dye calcein-AM (cat:750001885). After staining at 37°C for 20 minutes, 1 ml of cell staining solution (PBS + 1% FBS) was added, and the cells were centrifuged at 300g for 5 minutes and washed twice.

[0090] (2) After washing, the cells were resuspended in 500 μl of working buffer (composed of RPMI 1640 + 20% fetal bovine serum FBS + 1% penicillin antibody P / S + 100 μM non-essential amino acids + 100 μM β-thioethanol + 0.1% F68) to form droplets with a cell concentration of 1000 cells / μl.

[0091] (3) Gently transfer the droplet to a 15ml centrifuge tube and incubate it in a 37°C cell culture incubator. At 1h, 2h and 4h after incubation, take a small amount of droplet and take a fluorescence image on a cell counting plate. At the same time, take 100μl of droplet to break the emulsion and recover B cells. Flow cytometry is used to detect the fluorescence intensity of the cell viability dye calcein am, as shown in Figure 6. The fluorescence intensity of the viability dye displayed in the cells in the droplet in the fluorescence image and the flow cytometry results show that the cells maintained a good viability state within 4 hours of culture.

[0092] 4. Oligo-barcode labeling of antigens:

[0093] The steps for oligo-barcode antigen labeling are as follows:

[0094] (1) Activation: First, mix 100 μl of antigen protein with 1 μl of DBCO-PEG5-NHS (molar ratio of 1:25) and incubate at room temperature for 30 min. After incubation, add 10 μl of 1M Tris (pH 7.4) to terminate the reaction and incubate at room temperature for 10 min.

[0095] (2) Purification: Add all the activated product to an ultrafiltration tube and centrifuge at 13000g for 10 min at 4°C. Collect the filtrate into a 1.5 ml centrifuge tube, then add 500 μl of PBS to the ultrafiltration tube and centrifuge at 13000g for 5 min at 4°C. Collect the filtrate into a 1.5 ml centrifuge tube (repeat the operation once). Finally, invert the ultrafiltration column and centrifuge off the protein-DBCO retained on the ultrafiltration membrane. (Ensure that the remaining liquid is at least 100 μL)

[0096] (3) Oligo conjugation: Take out oligo powder and centrifuge at 10,000 rpm for 10 min. Add the collected protein-DBCO liquid to the oligo powder and incubate at 37℃ for 24 h to obtain antigen-oligo.

[0097] (4) SDS-PAGE verification of coupling effect: Pour the loading buffer into the electrophoresis tank, take 5 μl of marker for loading, take 8 μl of coupling product and mix with 2 μl of SDS protein loading buffer for loading.

[0098] 5. Flow cytometry analysis for sorting antigen-specific B cells:

[0099] After incubation, the droplets are removed and the B cells are recovered via demulsification. A portion of the cells can then be used for flow cytometry analysis to sort out antigen-specific B cells. The specific operating steps are as follows:

[0100] (1) Demulsification: Take out 200 μl of liquid droplet, add 20 μl of demulsifier (1H,1H,2H,2H-perfluoro-1-octanol; 370533), place at room temperature for 4-5 min, after demulsification, add culture medium and gently blow up the upper B cells, and collect the B cells;

[0101] (2) Staining: Centrifuge the collected B cells at 300g for 5 min, discard the supernatant, then resuspend the cells in 100 μl of staining buffer, add 1 μl of antigen-oligo from step 4, incubate at 4℃ for 30 min, after incubation, add 2 ml of PBS (containing 0.1% F68), centrifuge at 300g for 5 min, wash twice, then resuspend the cells in 100 μl of staining buffer, add 1 μl of anti-histag-AF647, incubate at 4℃ for 30 min, after incubation, add 2 ml of PBS (containing 0.1% F68), centrifuge at 300g for 5 min, wash twice;

[0102] (3) Flow cytometry detection: Collected B cells were centrifuged at 300g for 5min, the supernatant was discarded, and the cells were resuspended in 200μl of PBS. Then, 0.2μl of DAPI (cat:564907) was added, and the cells were detected by flow cytometry and sorted based on the fluorescence signal.

[0103] 6. Single-cell transcriptome, antigen oligo, and BCR library construction and sequencing analysis:

[0104] This step involves cell preparation, followed by single-cell transcriptomics, single-molecule library construction, sequencing, and analysis. The specific steps are as follows:

[0105] (1) B cells sorted by flow cytometry are used for library construction and sequencing analysis according to the standard procedure of single-cell transcriptomics. The sequencing results are compared with known oligo information. Since the transcriptomics information and antigen protein oligo information of the same B cell have the same cell-barcode, bioinformatics integration analysis can be performed to realize the integration of antigen protein and corresponding B cell transcriptomics and BCR information.

[0106] (2) A portion of cDNA was extracted, and BCR was enriched using RNA probes targeting the constant region of the antibody. Finally, the full-length sequence of BCR was obtained through single-molecule sequencing analysis.

[0107] Example 2

[0108] The specific process for analyzing COVID-19-specific hybridoma cells is as follows:

[0109] 1. COVID-19-specific hybridoma cell marker capture reagent:

[0110] (1) First, the expression of CD138 on the cell surface was tested: Plasma cells were the main cell type studied in this method. The representative membrane protein of plasma cells is CD138. The antibody capture reagent used this protein as the anchoring target to label the cells. Therefore, the expression of CD138 protein on the mesangial surface of hybridoma cells was first verified. By staining with a fluorescent antibody against mouse CD138, as shown in Figure 3, 8D3 hybridoma cells highly expressed CD138 compared with the control group Jurkat cells.

[0111] (2) Then, the hybridoma cell line was labeled with the capture reagent. The specific process was as follows: Take 1×10 6 Hybridoma cells (8D3 or 2H2 cell lines) were resuspended in 100 μl of cell staining solution (PBS + 1% FBS), and 1 μl of rabbit anti-CD138-streptavidin (sinobiological, 50641-R004) was added. After staining at 4°C for 30 minutes, 2 ml of cell staining solution (PBS + 1% FBS) was added, and the cells were centrifuged at 300 g for 5 minutes and washed twice. After washing, the cells were then stained with 100 μl of... After staining with 400 ng / μl of bispecific nanobodies (VHH-anti-rabbit-IgG & VHH-anti-mouse-kappa) at 4℃ for 30 minutes, add 2 ml of cell staining solution, centrifuge at 300g for 5 min, and wash twice; discard the supernatant, and resuspend the cells in 1000 μl of working buffer (composed of RPMI 1640 + 20% fetal bovine serum FBS + 1% penicillin antibody P / S + 100 μM non-essential amino acids + 100 μM β-thioethanol + 0.1% F68) to a cell concentration of 1000 cells / μl.

[0112] 2. Droplet generation, single-cell encapsulation, and incubation:

[0113] (1) Assemble the droplet generation device as shown in Figure 2. Add 200 μl of cell suspension, 200 μl of working buffer and 800 μl of droplet generation oil (BIO-RAD#cat:1864006) to the sample inlet of the droplet generation chip. Quickly pull the syringe from 12 ml to 20 ml to generate droplets by generating negative pressure.

[0114] (2) The device can encapsulate hundreds of thousands of B cells in 10 minutes. As shown in Figure 5, more than 80% of the cells are in a single-cell encapsulation state, and nearly 20% of the cells are in a multi-cell encapsulation state. Of course, due to the Poisson distribution problem, nearly 75% of the generated droplets are empty droplets without encapsulated cells.

[0115] (3) Take out the generated droplets and transfer them to a 15ml centrifuge tube, and incubate them in an incubator at 37℃ for 1-2 hours.

[0116] 3. Oligo-barcode labeling of the receptor-binding region (RBD) of the SARS-CoV-2 spike protein:

[0117] (1) Activation: First, mix 100 μl of RBD protein with 1 μl of DBCO-PEG5-NHS (molar ratio of 1:25) and incubate at room temperature for 30 min. After incubation, add 10 μl of 1M Tris solution (pH 7.4) to terminate the reaction and incubate at room temperature for 10 min.

[0118] (2) Purification: Add all the activated product to the ultrafiltration tube and centrifuge at 13000g for 10 min at 4℃. Collect the filtrate into a 1.5 mL centrifuge tube, then add 500 μl of PBS to the ultrafiltration tube and centrifuge at 13000g for 5 min at 4℃. Collect the filtrate into a 1.5 mL centrifuge tube (repeat the operation once). Finally, invert the ultrafiltration column and centrifuge off the protein-DBCO retained on the ultrafiltration membrane (ensure that the remaining liquid is 100 μl).

[0119] (3) Oligo coupling: Take out oligo powder and centrifuge at 10,000 rpm for 10 min. Add the collected protein-DBCO liquid to the oligo powder and incubate at 37℃ for 24 h to obtain RBD-oligo.

[0120] oligo sequence: TTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGACGTCCTTTCTGCGTGACGTCCTTCCTTCCNNNNNNNNNNBAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 1)

[0121] SDS-PAGE verification of coupling effect: Pour loading buffer into the electrophoresis tank, load 5 μl of marker, and mix 8 μl of the coupling product with 2 μl of SDS protein loading buffer before loading. The results of RBD protein coupling are shown in Figure 7, which shows that RBD protein can be successfully coupled with oligo.

[0122] 4. Flow cytometry analysis for sorting SARS-CoV-2-specific hybridoma cells:

[0123] (1) Demulsification: Take out 200 μl of the droplet after incubation in step 2, add 20 μl of demulsifier (1H,1H,2H,2H-perfluoro-1-octanol; 370533), place at room temperature for 4-5 min, add culture medium and gently blow up the upper layer of cells to collect hybridoma cells;

[0124] (2) Staining: Centrifuge the collected hybridoma cells at 300g for 5min, discard the supernatant, then resuspend the cells in 100μl of staining buffer, add 1μl of RBD-oligo prepared in step 3, incubate at 4℃ for 30min, after incubation, add 2ml of cell staining solution, centrifuge at 300g for 5min, wash twice, then resuspend the cells in 100μl of staining buffer, add 1μl of anti-histag fluorescent antibody anti-histag-AF647 (biolegend, 362611), incubate at 4℃ for 30min, after incubation, add 2ml of cell staining solution, centrifuge at 300g for 5min, wash twice;

[0125] (3) Flow cytometry detection: The collected hybridoma cells were centrifuged at 300g for 5min, the supernatant was discarded, and the cells were resuspended in 200μl of PBS. Then, 0.2μl of DAPI (cat:564907) was added, and the cells were detected by flow cytometry and sorted based on the fluorescence signal.

[0126] The 8D3 experimental group had capture antibodies labeled on the cell membrane surface, while the NC group served as a control and was not labeled with capture antibodies. After incubating both groups of cells in droplets, the droplets were broken to recover the cells. The antigen was then fluorescently labeled with APC to obtain RBD-APC, which bound to the cells. Flow cytometry was used to detect the intensity of antigen binding on the cell surface. The experimental results are shown in Figure 8, demonstrating that the capture reagent can efficiently capture secreted antibodies.

[0127] 5. Single-cell transcriptome, RBD-oligo and hybridoma cell antigen receptor library construction and sequencing analysis:

[0128] (1) Hybridoma cells sorted by flow cytometry were used for library construction and sequencing analysis according to the standard procedure of single-cell transcriptomics to obtain transcriptomics information and antigen-specific information;

[0129] (2) In the single-cell library construction process shown in Figure 9, after all mRNAs were reverse transcribed into cDNA and tagged with cell tags, a portion of the cDNA was taken out and enriched with the hybridoma cell antigen receptor using RNA probes targeting the constant region of the antibody. Finally, the full-length sequence of the hybridoma cell antigen receptor was obtained by single-molecule sequencing analysis (using Oxford Nanopore or PacBio instruments). The antigen receptor sequence information of 2H2 used in this experiment has been submitted to GenBank, and the query codes are 2H2-VH (MW271803) and 2H2-VL (MW271804). The antigen receptor sequence information of 8D3 and its structural information on binding with the corresponding antigen protein have been submitted to the Protein database, and the query codes are 8D3- (7W9F).

[0130] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0131] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method of capturing antibodies secreted by plasma cells, characterized in that, The method comprises: labeling the plasma cells with a capture reagent; performing single-cell encapsulation on the labeled plasma cells to capture the antibodies; wherein the capture reagent has an activity of binding to the antibodies secreted by the plasma cells, and the plasma cells have an activity of secreting the antibodies.

2. The method of claim 1, wherein, The capture reagent comprises a rabbit anti-mouse CD138 antibody and a bispecific nanobody.

3. The method of claim 1, wherein, The single-cell encapsulation is performed in a droplet generation device.

4. The method of claim 3, wherein, The single-cell encapsulation is further performed by: incubating the single-cell encapsulation product; and demulsifying the incubation product to obtain the plasma cells containing the self-secreted antibodies.

5. The method of claim 2, wherein, The labeling of the rabbit anti-mouse CD138 antibody is performed at 1-5°C for 20-40 min. The labeling of the bispecific nanobody is performed at 1-5°C for 20-40 min.

6. The method of claim 4, wherein, The incubation is performed at a temperature of 30-40°C for 1-2 h.

7. The method of claim 4, wherein, The demulsification is performed in the presence of a demulsifier. Optionally, the demulsification is performed at 20-30°C for 3-10 min.

8. A plasma cell, characterized in that, The plasma cells have a membrane surface containing self-secreted antibodies, which are obtained by the method of any one of claims 1-7.

9. A method of screening for antigen-specific plasma cells, characterized in that, The method comprises: performing antigen labeling on the plasma cells of claim 8 with protein antigens labeled with oligo-barcode; performing sorting on the antigen labeling product to obtain the antigen-specific plasma cells; wherein the protein antigens have an activity of binding to the antibodies secreted by the plasma cells on the membrane surface.

10. The method of claim 9, wherein, The antigen labeling is performed at 1-5°C for 20-40 min. Optionally, the sorting is performed by flow cytometry sorting.

11. The method of claim 10, wherein, The sorting is performed by: performing binding on the antigen labeling product with an anti-His tag dye; performing DAPI staining on the binding product; and obtaining the antigen-specific plasma cells based on the fluorescence signal of the staining product.

12. Use of the antigen-specific plasma cells obtained by the method of any one of claims 9-11 in constructing a single-cell sequencing library and single-cell sequencing.

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