Method for obtaining single B cell and screening antibody

Abstract

The invention discloses a method for obtaining a single B cell and screening an antibody, and relates to the technical field of monoclonal antibody development. According to the invention, B cells are enriched through a magnetic bead anion selection method, and target B cells are screened by identifying a specific fluorescence signal mode. The method provided by the invention is helpful for improving the enrichment efficiency of the target B cells, reducing the enrichment cost, increasing the proportion of the positive cells after enrichment, also helpful for screening high-affinity antibodies, and improving the efficiency of antibody discovery.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody development technology, and more specifically, to a method for obtaining single B cells and screening antibodies. Background Technology

[0002] Monoclonal antibodies (mAbs), as highly specific therapeutic and diagnostic tools, are typically produced through screening from B cells of a specific host. However, the development of monoclonal antibodies faces a major bottleneck: the lack of commercially available antibodies targeting B cell surface markers limits effective enrichment strategies. Currently, the main method for isolating B cells is based on traditional flow cytometry to detect IgG and antigen labels in cell samples, thereby sorting them into memory B cells. However, due to throughput limitations and varying degrees of loss caused by handling B cells, the probability of obtaining effective antibody sequences is relatively low. CN201910125091.4 discloses a method for enriching antigen-specific rabbit B lymphocytes. This method involves using streptavidin magnetic beads to bind biotinylated anti-CD4 / CD8 / CD14 antibodies to remove cells expressing CD4 / CD8 / CD14. Then, flow cytometry is used to screen for non-IgM antigen-specific cells. However, in this method, the biotinylated antibodies, due to steric hindrance, reduce the binding efficiency between the antibody and the target antigen, resulting in insufficient cell separation efficiency and cell damage during flow cytometry sorting.

[0003] Currently, single B-cell screening mainly relies on three technologies. The first is a combination of photoconductive systems and microfluidic chip technology: precise single-cell sorting is achieved through photoelectric positioning and microfluidic design, but this technology does not pre-enrich B cells. Due to the vast number of spleen or peripheral blood cells (500 million to 1 billion cells after removing red blood cells), and the fact that a single chip can only screen 7,000 to 20,000 cells, the screening efficiency is extremely low and the cost of consumables is high, making it difficult to widely apply in scientific research. The second is droplet microfluidic technology: single spleen or peripheral blood cells are encapsulated in epithelial droplets, and target cells are screened based on signal intensity. However, this method also lacks a B-cell enrichment step, resulting in insufficient screening throughput and high cost. The third is magnetic negative selection combined with droplet microfluidics: spleen or peripheral blood cells are pre-enriched through magnetic sorting, and then specific B cells are screened through a high-throughput droplet system. Although this method increases the proportion of target cells, the number of positive cells after enrichment is still limited, and antibody affinity cannot be guaranteed.

[0004] In summary, existing technologies suffer from low efficiency, high cost, or unstable antibody quality, necessitating the development of more efficient single B cell enrichment and screening strategies to fully realize the potential of monoclonal antibodies.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for obtaining single B cells and screening antibodies (ASC-CC).

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a method for obtaining single B cells, comprising the following steps: S1: Isolate spleen cells and / or peripheral blood cells from animals immunized with antigens; S2: Negative selection of B cells using the first antibody combination that binds to non-target cells, the antibody combination against the first antibody, and the first solid phase against the above-mentioned spleen cells and / or peripheral blood cells; Preferably, the first solid phase is selected from microspheres; the first antibody combination includes at least one of the following antibodies: T lymphocyte antibody, CD11b monoclonal antibody and IgM monoclonal antibody; the antibody combination against the first antibody is from a Stemcell kit; More preferably, the first solid phase is selected from magnetic microspheres; the first antibody combination includes T lymphocyte antibody, CD11b monoclonal antibody and IgM monoclonal antibody; the final concentrations of T lymphocyte antibody, CD11b monoclonal antibody and IgM monoclonal antibody in the first antibody combination are 0.3 μg / mL-0.6 μg / mL, 0.3 μg / mL-0.6 μg / mL and 0.3 μg / mL-0.6 μg / mL, respectively; preferably 0.5 μg / mL, 0.5 μg / mL and 0.5 μg / mL, respectively.

[0008] S3: Negatively selected spleen cells and / or peripheral blood cells are incubated with a second solid phase coated with the target antigen, and then incubated with a fluorescently labeled second antibody. The fluorescence signals are detected at the initial stage of incubation and after the cells produce antibodies. Preferably, the second solid phase is selected from microspheres, plates, or membranes; More preferably, the second solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, nylon membranes and / or nitrocellulose membranes; S4: Based on the fluorescence signal screening rules, target single B cells are selected using a Cell celector instrument; The fluorescence signal screening rules include: selecting cells with no fluorescence signal in the initial stage of incubation, and cells with a fluorescence signal centered on the cell and spreading to the surrounding area after the cells produce antibodies, as target single B cells.

[0009] Preferably, the initial incubation stage refers to 0-0.5 hours of incubation after adding the fluorescently labeled second antibody, and the antibody production stage refers to 4-24 hours of incubation after adding the fluorescently labeled second antibody.

[0010] Secondly, the present invention provides a method for screening antibodies, comprising the following steps: S1: Obtain single B cells using the method described above; S2: The cDNA obtained from the single B cells was amplified by PCR to obtain PCR product DNA. After sequencing and analysis, repetitive sequences were removed to obtain antibody nucleic acid sequence pairs. S3: Recombinantly express the antibody nucleic acid sequence pair to obtain antibody protein; S4: Determine the binding capacity and / or affinity of the obtained antibody proteins, and select antibodies that meet the requirements. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 The image shows the positive signal results of the specific target site (Human-LPA) of the target protein pore under bright field (BF) and fluorescent field (TRITC) conditions. Figure 2 The results of the signal of the irrelevant target (IL3) in the control well under bright field (BF) and fluorescence field (TRITC) are shown. Figure 3 The image shows the ELISA results of 54 Human-LPA antibody-containing cell culture supernatants. Figure 4 The image shows the ELISA results of 46 Human-IFN-gamma antibody-containing cell culture supernatants. Figure 5 The image shows the ELISA results of the culture supernatant of 33 Human-IL 22 antibody-containing cells. Figure 6 A scatter plot of the output metrics for antibodies screened from 32 targets; Figure 7 A comparison of the efficiency of monoclonal antibody production for eight targets, including human-CD19, between the ASC-CC method and the MemBe-sFACS method; Figure 8 This is a scatter plot showing the various indicators after screening for specific antibodies binding to seven recombinant antigens lacking fluorescent labels using the ASC-CC method. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0014] In a first aspect, the present invention provides a method for obtaining single B cells (ASC-CC), comprising the following steps: S1: Immunize animals with antigens and isolate spleen cells and / or peripheral blood cells from the animals immunized with antigens; S2: Negative selection of B cells was performed using the first antibody combination that binds to non-target cells, the antibody combination against the first antibody, and the first solid antibody against the above-mentioned spleen cells and / or peripheral blood cells; the antibody combination against the first antibody was obtained from the Stemcell kit. S3: Negatively selected spleen cells and / or peripheral blood cells were incubated with the second solid phase coated with antigen, and then incubated with a fluorescently labeled second antibody. The fluorescence signals were detected at the initial stage of incubation and after the cells produced antibodies. S4: Based on the fluorescence signal screening rules, target single B cells are selected using a Cell celector instrument; The fluorescence signal screening rules include: selecting cells with no fluorescence signal in the initial stage of incubation, and cells with a fluorescence signal centered on the cell and spreading to the surrounding area after the cells produce antibodies, as target single B cells.

[0015] To achieve efficient enrichment of B cells in spleen or peripheral blood cells, this invention provides a negative selection (or negative screening) method with better enrichment effect. An enrichment reagent containing a combination of first antibodies targeting non-target cells and an antibody combination against the first antibody is used to bind (or label) non-target cells (such as T lymphocytes, monocytes / macrophages, granulocytes). These labeled non-target cells are then removed using a first solid-state binder to obtain an enriched cell suspension.

[0016] After separating and removing non-target cells, this invention utilizes the principle of immunofluorescence. Specifically, the specific antibody secreted by the target cells binds to the target antigen coated on a second solid phase. A fluorescently labeled second antibody binds to the specific antibody secreted by the cells, forming a specific fluorescent signal. Rabbit monoclonal B cells secreting the target antibody are then selected using a cell detector according to specific fluorescence signal screening rules. In one embodiment, to improve the efficiency of positive cell wells obtained through screening, the state of the cells after plating is observed at the initial stage of incubation. Cells in poor condition and those exhibiting fluorescence are excluded, thus initially reducing the number of cell wells required for subsequent screening and reducing workload. After a certain incubation period, cell morphology is observed, and cell wells with abnormal cell division or death are excluded. The remaining cell wells are then screened according to a preset fluorescence signal screening rule. After a period of incubation, the target monoclonal B cells express the target antibody, and the expressed antibody binds to the antigen coated on the plate. A fluorescently labeled second antibody is then added for incubation. The fluorescently labeled second antibody binds to the antibody expressed by the cells, and fluorescence is displayed upon excitation. Cells expressing the target antibody exhibit fluorescence signals centered on the cell and spreading outwards under a fluorescence field, indicating that the cell is a target mono-B cell.

[0017] In a preferred embodiment of the present invention, the first solid phase is selected from microspheres; The second solid phase is selected from microspheres, plates, or membranes.

[0018] In a preferred embodiment of the present invention, the first solid phase is selected from magnetic microspheres; The second solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, nylon membranes and / or nitrocellulose membranes.

[0019] In a preferred embodiment of the present invention, the latex microspheres are selected from monodisperse triblock polymer latex microspheres, monodisperse polystyrene latex microspheres, and monodisperse polymethyl methacrylate latex microspheres. In a preferred embodiment of the present invention, the first antibody combination includes at least one of the following antibodies: T lymphocyte antibody, CD11b monoclonal antibody, and IgM monoclonal antibody. In a preferred embodiment of the present invention, the first antibody combination includes: T lymphocyte antibody, CD11b monoclonal antibody, and IgM monoclonal antibody. Through the combination of these three antibodies, non-target cells can be specifically labeled, and the binding of non-target cells can be achieved using the first solid phase, thereby separating the non-target cells from B cells to obtain purer B cells. In a preferred embodiment of the present invention, during the negative selection of B cells, the first antibody combination binding to non-target cells and the antibody combination against the first antibody are first incubated with spleen cells and / or peripheral blood cells, and then incubated with the first solid phase; in the incubation system containing cell suspension, the final concentrations of the T lymphocyte antibody, CD11b monoclonal antibody, and IgM monoclonal antibody in the first antibody are 0.3 μg / mL-0.6 μg / mL, 0.3 μg / mL-0.6 μg / mL, and 0.3 μg / mL-0.6 μg / mL, respectively.

[0020] For example, the final concentrations of T lymphocyte antibodies, CD11b monoclonal antibodies, and IgM monoclonal antibodies were 0.3 μg / mL, 0.3 μg / mL, and 0.3 μg / mL; 0.3 μg / mL, 0.3 μg / mL, and 0.4 μg / mL; 0.3 μg / mL, 0.4 μg / mL, and 0.3 μg / mL; 0.4 μg / mL, 0.3 μg / mL, and 0.4 μg / mL, respectively. g / mL; 0.4μg / mL, 0.4μg / mL and 0.3μg / mL; 0.4μg / mL, 0.4μg / mL and 0.4μg / mL; 0.4μg / mL, 0.4μg / mL and 0.5μg / mL; 0.4μg / mL, 0.5μg / mL and 0.5μg / mL; 0.5μg / mL, 0.5μg / mL and 0.5μg / mL; 0.5μg / mL, 0.5μg / mL and 0.6μg / mL.

[0021] In a preferred embodiment of the present invention, in an incubation system containing cell suspension, the final concentrations of the T lymphocyte antibody, CD11b monoclonal antibody, and IgM monoclonal antibody in the first antibody are 0.5 μg / mL, 0.5 μg / mL, and 0.5 μg / mL, respectively.

[0022] In a preferred embodiment of the present invention, the initial incubation stage refers to 0-0.5 hours of incubation after adding the fluorescently labeled second antibody. Cell antibody production refers to incubation after adding the fluorescently labeled second antibody for 4-24 hours, for example, 4-10 hours, 5-15 hours, 5-20 hours, 5-24 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

[0023] Secondly, the present invention provides a method for screening target antibodies, comprising the following steps: S1: Obtain single B cells using the method described above; S2: The nucleic acids obtained from the single B cells are amplified by PCR, sequenced, and analyzed to obtain antibody nucleic acid sequence pairs; S3: Recombinantly express the antibody nucleic acid sequence pair to obtain cell culture supernatant containing antibody protein and antibody protein; S4: Detect the binding capacity and / or affinity of the antibody proteins in the supernatant, and select the antibody that meets the requirements as the target antibody.

[0024] Step S2 includes: performing PCR amplification on the cDNA of the screened single B cells to obtain PCR product DNA, sequencing and analyzing it, removing repetitive sequences, and obtaining antibody nucleic acid sequence pairs.

[0025] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0026] The single B-cell antibody screening method (ASC-CC) provided in this invention can complete the enrichment, screening, and subsequent amplification of B cells within 2 days, greatly shortening the development cycle of new antibodies and improving the success rate of obtaining antibody sequences. It is suitable for the development and application of antibody drugs and diagnostic antibodies. The method for obtaining single B cells provided by this invention is highly efficient and versatile.

[0027] Example 1 In this embodiment, the rabbit monoclonal B cell antibody screening method (ASC-CC) was used to screen for target rabbit monoclonal B cells and antibodies.

[0028] 1. Obtaining spleen cells from rabbits after immunization: Spleens from New Zealand white rabbits immunized with Human-LPA-His recombinant protein (ACRObiosystems, LPA-H52H3) and with acceptable titers were harvested, ground, and resuspended to obtain spleen cell suspension. Cell counting was then performed using a cell counter (Newton Optics), and the cell count was determined to be 1.3 × 10⁻⁶. 9 indivual.

[0029] 2. Red blood cell lysis: Take the spleen cells from step 1 and lyse them for 5 minutes at room temperature using 15 ml of red blood cell lysis buffer (Tianjin Haoyang, NH4CL2009). Collect 5 × 102 lysed spleen cells. 7 After centrifugation, the cells were resuspended in 450 μL Easy Buffer (Stemcell) to prepare a spleen cell resuspension.

[0030] 3. Preparation of enrichment reagents: Add 1.5ug Mouse anti-Rabbit T Lymphocytes (BIO-RAD), 1.5ug Mouse anti-Rabbit CD11b (BIO-RAD), and 1.5ug BD Pharmingen TM Biotin Mouse antiRabbit IgM (BD) was incubated with 30 μL of component A and 30 μL of component B from the Stemcell kit ("Do it yourself Positive Selection Kit II") at 37°C for 5 hours to prepare an antibody mixture, which was then brought to a final volume of 300 μL with PBS.

[0031] 4. Enrichment of B cells: Take 50 μL of the enrichment reagent prepared in step 3, add it to the spleen cell resuspension in step 2, mix well, and incubate at room temperature for 15 min. Then add 25 μL of Stemcell magnetic beads (from "Do it yourself Positive Selection Kit II"), mix well, and incubate at room temperature for 10 min. Make up the volume to 2.5 ml with Easy Buffer, mix well, and incubate in a magnetic field at room temperature for 10 min. Pour off the incubated cell suspension; the cells poured off are the enriched target cells. Non-target cells bound to the magnetic beads are retained. Count the enriched cells; the cell count is 2.0 × 10⁻⁶. 6 The enrichment efficiency is 25 times.

[0032] 5. Plating: Take 1×10⁻⁶ cells from the enriched cells described in step 4. 5 Nuclei were evenly seeded into 6-well nanopores (Nanowell U40-40 1M Sartorius plates) coated with Human-LPA-His recombinant protein and non-LPA protein without His tag modification, respectively. 15 μg of a fluorescently labeled secondary antibody (549 anti-RabbitIgG Fc Recombinant Ab, Thermo Fisher (CAT: A78956)) was added to each 6-well nanopore and incubated at 37°C. The nanopores coated with Human-LPA-His recombinant protein were used as target wells; the nanopores coated with the irrelevant target protein IL-3 (ACRObiosystems, IL3-H5115) without His tag modification were used as control wells to detect fluorescence signals caused by non-immune effects.

[0033] 6. Signal identification and selection: Using a Cell Celector instrument (Sartorius, CC1314), bright-field and fluorescence field images were scanned on cell wells with fluorescently labeled secondary antibodies after 0 hours and 20 hours of incubation, respectively. Wells with single cells were selected for signal pattern screening.

[0034] There are three types of reference hole signal modes: (1) No fluorescence signal was observed in the 0h fluorescence field and no fluorescence signal was observed in the 20h fluorescence field; (2) No fluorescence signal appears in the 0h fluorescence field, and an irregularly bright fluorescence signal appears in the 20h fluorescence field; (3) Fluorescent signals appeared in the fluorescence field at 0h, and irregularly bright fluorescent signals appeared in the fluorescence field at 20h; There are four target hole signal modes: (1) No fluorescence signal was observed in the 0h fluorescence field and no fluorescence signal was observed in the 20h fluorescence field; (2) No fluorescence signal appeared in the 0h fluorescence field, and an irregular, extremely bright fluorescence signal appeared in the 20h fluorescence field, consistent with the signal pattern (2) of the control well. (3) A fluorescence signal appeared in the fluorescence field at 0h, and an irregular, extremely bright fluorescence signal appeared in the fluorescence field at 20h, consistent with the signal pattern (3) of the control well. (4) No fluorescence signal was observed in the 0h fluorescence field, and the 20h fluorescence field showed a fluorescence signal that was significantly different from the control well signal and diffused outward from the cell center. For the selected target holes (see) Figure 1 ) to count. Figure 2 This is a diagram showing the bright field and fluorescence field signals of cells in the control wells. Figure 2 As shown, the fluorescence signal of cells in the control wells became blurred, discontinuous, and granular after 20 hours of incubation. Therefore, target cells can be screened by comparing fluorescence signals.

[0035] 0h-BF refers to a bright field image of cells after 0h incubation; 0h-TRITC refers to a fluorescence field image of cells after 0h incubation; 20h-BF refers to a bright field image of cells after 20h incubation with a fluorescently labeled secondary antibody; and 20h-TRITC refers to a fluorescence field image of cells after 20h incubation with a fluorescently labeled secondary antibody.

[0036] In this embodiment, the state of cells after plating was first observed at 0h, and cells in poor condition and fluorescent cells were removed from the target wells. Then, after 20h of incubation, the morphology of the cells was observed, and cells with abnormal division and death were removed. Cells that met the preset fluorescence signal pattern were selected, and single cells from the selected target protein wells were picked into cell lysis buffer using the Cell Celector robotic arm. A total of 96 cells were picked.

[0037] 7. SMART (Switching Mechanism at 5' end of RNA Template) technology was used to synthesize and amplify cDNA from 96 selected cells. After obtaining purified PCR product DNA via antibody-specific PCR, first-generation sequencing was performed. The sequencing results were analyzed and screened using SnapGene software. The antibody DNA sequence analysis and screening involved the following three steps: (1) Use SnapGene to confirm the expression profile of the obtained antibody DNA sequence and delete the sequence with the stop codon.

[0038] (2) Match the light and heavy chain DNA sequences of each remaining clone one by one.

[0039] (3) Remove the CDR3 repeating DNA sequence and record the number of repeating sequences.

[0040] Finally, 74 pairs of sequences that met the requirements were obtained, and 54 unique sequences were obtained after deduplication (unique CDR3s). 8. Recombinant Expression and Detection: The 54 different sequences obtained after sequencing screening were cloned into the vector pcDNA3.1 (Thermo Fisher, V79020) to construct recombinant eukaryotic expression vector plasmids containing the above 54 antibodies. These plasmids were transfected into HEK293 cells for expression. After 72 hours of culture, ELISA was performed on the 54 cell culture supernatants. The results showed that all 54 antibodies were monoclonal antibodies specifically binding to the Human-LPA recombinant protein. See [link to results]. Figure 3 Furthermore, according to BLI testing, 20 of the antibodies showed an affinity of 10. -12 At levels above M, the affinity of 23 antibodies reached 10. -10 At the M level, the affinity of the other antibodies was all above 10. -9 M level, see Table 1.

[0041] Table 1. Affinity assay results of supernatants from 54 Human-LPA antibody expression samples.

[0042] Figure 3The meanings of each column in the table shown are as follows: In the leftmost column, Sample ID represents the sample number; In the second column from the left, OD450 represents the reading shown by coating the target protein (Human-LPA-His) + cell culture supernatant + fluorescently labeled secondary antibody; In the left third column, OD450-Blank0 represents the OD450 value in the left second column minus the reading of the target protein (Human-LPA-His) coated only with fluorescent secondary antibody when there is no sample to be tested (the purpose is to remove the background value of the coated plate). In the left fourth column, OD450 represents the reading of coated control protein (Human-VCAM-1-His) + cell culture supernatant to be tested + fluorescently labeled secondary antibody; In the left 5 columns, OD450-Blank0 represents the OD450 value in the left 4 columns minus the reading of the control protein (Human-VCAM-1-His) coated only with fluorescent secondary antibody in the absence of test sample (the purpose is to remove the background value of the coated plate). In the left 6 columns, blanks indicate uncoated proteins, and the values ​​are those containing the test sample plus a fluorescently labeled secondary antibody.

[0043] Example 2 This embodiment screens for anti-Human-IFN-gamma monoclonal antibodies.

[0044] Referring to steps 1-7 of the method in Example 1, screening of Human-IFN-gamma antibody single B cells and antibody sequence pairs was performed. 89 pairs of complete rabbit antibody bands were amplified from the 96 selected cells, and 46 different antibody sequence pairs were obtained after sequencing analysis.

[0045] Referring to step 8 of method 1 in Example 1, cell culture supernatant containing anti-Human-IFN-gamma monoclonal antibodies was obtained. ELISA detection showed that all 46 antibodies were monoclonal antibodies specifically binding to the recombinant Human-IFN-gamma protein. The detection results are shown in [link to relevant documentation]. Figure 4 Furthermore, BLI analysis showed that 12 of the antibodies had an affinity of 10. -12 At the above levels, the affinity (KD value) of 8 antibodies reached 10. -10 The affinity of the remaining antibodies is mostly around 10. -9 The affinity statistics for each antibody are shown in Table 2.

[0046] Table 2. Affinity assay results of supernatants from the expression of 46 Human-IFN-gamma antibodies.

[0047] ELISA results of 46 Human-IFN-gamma antibody cell culture supernatants were obtained as follows: Figure 4 As shown.

[0048] Example 3 This embodiment screens for anti-Human-IL-22 monoclonal antibodies. Following steps 1-7 of the method in Example 1, screening of Human-IL-22 antibody single B cells and antibody sequence pairing were performed. 62 pairs of complete rabbit anti-bands were amplified from the 96 selected cells, and 33 different antibodies were obtained after sequencing.

[0049] Following step 8 of the method in Example 1, cell culture supernatant containing anti-Human-IL-22 monoclonal antibody was obtained. ELISA detection revealed that 33 of the 38 cell culture supernatants contained monoclonal antibodies specifically binding to the recombinant Human-IL-22 protein. The detection results are shown below. Figure 5 Furthermore, BLI analysis showed that all 33 antibodies exhibited high affinity levels; specific data are shown in Table 3. BLI analysis also revealed that four of these antibodies had an affinity (KD value) of 10. -10 At the level, the affinity (KD value) of the three antibodies reached 10. -10 The affinity of the remaining antibodies is mostly around 10. -9 level.

[0050] Table 3. Affinity test results of supernatants from 33 Human-IL 22 antibody expression samples.

[0051] ELISA results of 33 Human-IL 22 antibody-containing cell culture supernatants were obtained as follows: Figure 5 As shown.

[0052] Example 4: Antibody production efficiency test using the ASC-CC method Referring to the method in Example 1, this example performed antibody affinity testing on 29 targets (see Table 4) other than Human-LPA, Human-IFN-gamma, and Human-IL-22.

[0053] The number of logarithmic pairs of the light and heavy chain V region genes amplified from selected cells is denoted as "H+L pairs". Analysis of the IMGT database shows that antibodies differing by only one amino acid in the CDR3 region of either the light or heavy chain from the amplified and sequenced light and heavy chain V region genes, denoted as "unique CDR3s". Antibodies obtained by recombinant expression of the light and heavy chain genes from "unique CDR3s" clones, with an affinity KD value <10 nM, are denoted as "nM clones". All index values ​​are proportionally corrected to the actual number of cells obtained, with index values ​​obtained per 96 cells. The data is displayed as a scatter plot, representing the median and the first quartile, showing the median value. The results are as follows: Figure 6 As shown, the ASC-CC method can meet the needs of antibody development.

[0054] Table 4: Figure 4 The specific selection of the 32 projects and the output indicators.

[0055] Example 5 This embodiment tests the enrichment effect of the finished B cell sorting kit (Dap, RRF0011) and the rabbit B cell enrichment step in the ASC-CC method provided in Example 1.

[0056] ASC-CC cell enrichment procedure: Following steps 1-2 in Example 1, obtain rabbit spleen cells and lyse erythrocytes after immunization. Specifically, immunize New Zealand white rabbits with Human-IL21 recombinant protein, then perform erythrocyte lysis. Following steps 3-4 in Example 1, collect 5 × 10⁵ spleen cells after erythrocyte lysis. 7 Enrichment was performed on individual cells, yielding 2.0 × 10⁶ enriched spleen cells. 6 The enrichment efficiency is 25 times.

[0057] Finished B-cell sorting kit: Following steps 1-2 in Example 1, immunize New Zealand white rabbits with Human-IL21 recombinant protein, then perform erythropoiesis on spleen cells. Collect 2 × 103 spleen cells after erythropoiesis. 7 Rabbit B cells were enriched using the Rabbit PanB Isolation Kit (RRF0011) from Dapoxetine: The isolation and enrichment procedure was performed according to the manufacturer's instructions, yielding 4.0 × 10⁶ cells. 6 The enrichment efficiency is 5 times.

[0058] Referring to step 5 in Example 1, 5.0 × 10⁵ cells were taken from each of the cell products obtained by the two methods described above. 4 Cells were plated, wells were sealed, fluorescently labeled secondary antibodies were added, and the cells were incubated.

[0059] In step 6 of the reference embodiment, signal identification and selection are performed to screen out wells that do not show fluorescence at 0 hours but show fluorescence signals centered on the cell and spreading outwards at 20 hours for counting.

[0060] The number of positive signal wells after plating cells obtained using the ASC-CC method was 550; while the number of positive signal wells after plating cells obtained using the Dap Biotech Rabbit PanB Isolation Kit was 197. Therefore, the method provided by this invention can increase the proportion of positive cells among the enriched spleen cells.

[0061] Example 6 This embodiment compares the efficiency of the ASC-CC method and MemB-sFASCS in screening specific antibodies.

[0062] ASC-CC method: Following the steps shown in Example 1, monoclonal antibodies targeting eight targets, including human-CD19, were screened.

[0063] The steps for MemB-sFACS are as follows: 1. Immunize different New Zealand white rabbits from the same batch using the same antigen from the ASC-CC method, according to the MemB-sFACS method.

[0064] 2. The recombinant protein of the target to be screened was labeled with fluorescent molecules. The spleen of the immunized rabbit was taken and subjected to density gradient centrifugation to obtain a single cell suspension of the rabbit spleen. 3. Antigen-specific memory B cell samples were stained and sorted using anti-Rabbit IgM antibody (Abcam Cas: AB150095) labeled with fluorescent signal A, anti-Rabbit IgG antibody (Southern Biotech Cas: 4090-09) labeled with fluorescent signal B, and recombinant proteins of various targets labeled with fluorescent signal C.

[0065] 4. Using a flow cytometer (Sony SH800 fully automated flow cytometer), single lymphocyte populations were obtained by gating with FSC-A / BSC-A; adhesion was removed by FSC-A / FSC-H to exclude cell duplexes or multiples; B cell populations with surface antibody subtype IgG were obtained by gating with anti-Rabbit IgM fluorescence signal A (Abcam Cas: AB150095) and anti-Rabbit IgG fluorescence signal B (Southern Biotech Cas: 4090-09); and single antigen-specific B cells were obtained by gating with target recombinant protein labeled with fluorescence signal C.

[0066] 5. Referring to step 7 in Example 1, the obtained cells were subjected to cDNA synthesis and amplification to obtain the deduplicated sequence (unique CDR3s), which was then constructed and expressed. 6. Referring to step 8 in Example 1, the obtained unique CDR3s were recombinantly expressed, and the affinity data of each antibody were obtained by BLI assay.

[0067] The experimental design and statistical analysis methods are the same as in Example 4, and the experimental results are as follows: Figure 7 As shown in Table 5, the results indicate that the method provided by this invention produces specific antibodies with comparable efficiency to that produced by the MemB-sFACS method, while incurring lower time costs.

[0068] Table 5: Efficiency data of SC-CC and MemB-sFASCS for screening specific antibodies

[0069] Example 7 This embodiment uses the ASC-CC method to screen for specific antibodies that bind to recombinant antigens lacking fluorescent labels. Specific antibodies that bind to recombinant antigens lacking fluorescent labels are not suitable for traditional flow cytometry screening.

[0070] Seven recombinant antigens lacking fluorescent labeling were selected, and specific antibodies were screened using the ASC-CC method, following the steps in Example 1. The experimental protocol and statistical analysis methods are as shown in Example 5, and the results are as follows: Figure 8 As shown, the ASC-CC technology yielded nM-level affinity antibodies suitable for downstream development in all seven experiments involving recombinant antigens lacking fluorescent labels.

[0071] Table 6: Indicator parameters for screening recombinant antigen-specific antibodies lacking fluorescent labeling by the ASC-CC method

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for obtaining single B cells, characterized in that, Includes the following steps: S1: Isolate spleen cells and / or peripheral blood cells from animals immunized with antigens; S2: Negative selection of B cells using a first antibody combination that binds to non-target cells, an antibody combination against the first antibody, and a first solid phase against the spleen cells and / or peripheral blood cells; S3: Incubate the negatively selected spleen cells and / or peripheral blood cells with the second solid phase coated with the antigen, add the fluorescently labeled second antibody for incubation, and detect the fluorescence signal at the initial stage of incubation and after the cells produce antibodies, respectively; S4: Based on the fluorescence signal screening rules, target single B cells are selected using a Cell celector instrument; The fluorescence signal screening rules include: cells with no fluorescence signal in the initial stage of incubation, and cells with a fluorescence signal centered on the cell and spreading outwards after the cells produce antibodies, are selected as target single B cells.

2. The method according to claim 1, characterized in that, The first solid phase is selected from microspheres; The second solid phase is selected from microspheres, plates, or membranes.

3. The method according to claim 2, characterized in that, The first solid phase is selected from magnetic microspheres; The second solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, nylon membranes and / or nitrocellulose membranes.

4. The method according to claim 1, characterized in that, The first antibody combination includes at least one of the following antibodies: T lymphocyte antibody, CD11b monoclonal antibody, and IgM monoclonal antibody.

5. The method according to claim 4, characterized in that, The first antibody combination includes: T lymphocyte antibody, CD11b monoclonal antibody and IgM monoclonal antibody.

6. The method according to claim 5, characterized in that, During the negative selection of B cells, the first antibody combination binding to non-target cells and the antibody combination against the first antibody are first incubated with the spleen cells and / or peripheral blood cells, and then incubated with the first solid phase; the final concentrations of T lymphocyte antibody, CD11b monoclonal antibody and IgM monoclonal antibody in the first antibody combination are 0.3 μg / mL-0.6 μg / mL, 0.3 μg / mL-0.6 μg / mL and 0.3 μg / mL-0.6 μg / mL, respectively.

7. The method according to claim 6, characterized in that, The final concentrations of the T lymphocyte antibody, CD11b monoclonal antibody, and IgM monoclonal antibody in the first antibody combination are 0.5 μg / mL, 0.5 μg / mL, and 0.5 μg / mL, respectively.

8. The method according to claim 1, characterized in that, The initial incubation phase refers to 0-0.5 hours of incubation with a fluorescently labeled second antibody, and the antibody production in cells refers to 4-24 hours of incubation with a fluorescently labeled second antibody.

9. A method for screening antibodies, characterized in that, Includes the following steps: S1: Obtaining single B cells according to any one of claims 1-8; S2: The nucleic acids obtained from the single B cells are amplified by PCR, sequenced, and analyzed to obtain antibody nucleic acid sequence pairs; S3: Recombinantly express the antibody nucleic acid sequence pair to obtain antibody protein; S4: Determine the binding capacity and / or affinity of the obtained antibody proteins, and select antibodies that meet the requirements.

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