Rabbit single B cell enrichment method and microfluidic screening method

Abstract

The invention discloses a rabbit single B cell enrichment method and a microfluidic screening method, and relates to the technical field of biology. The enrichment method comprises the following steps: mixing a biotinylated specific antibody and a rabbit single-cell suspension to be separated, so that the specific antibody is combined with a cell to be removed; adding magnetic beads coated by chained amycin into the cell suspension, and removing cells combined with a specific antibody by utilizing a magnetic separation principle, so as to obtain enriched rabbit single B cells; the specific antibodies comprise a T lymphocyte antibody, a CD4 monoclonal antibody, a CD8 monoclonal antibody, a CD11b monoclonal antibody, an IgM monoclonal antibody, a CD11c monoclonal antibody and a CD123 monoclonal antibody. The enrichment method does not influence the number and quality of B cells in the system, achieves the effect of quickly enriching high-purity rabbit B cells from rabbit single cells, and improves the proportion of secretory plasma B cells. The microfluidic screening method provided by the invention can be used for high-throughput rapid separation of target micro-droplets, and has the advantages of high efficiency and accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a rabbit single B cell enrichment method and a microfluidic screening method. Background Art

[0002] Rabbit monoclonal antibodies have more natural advantages than mouse monoclonal antibodies, such as a larger spleen that can obtain more B cells; rabbit antibodies can recognize antigen epitopes that mice cannot recognize, increasing antibody diversity; rabbit antibodies have higher stability and affinity than mouse antibodies; and a simpler antibody structure makes antibody optimization easier.

[0003] Due to the lack of commercial rabbit B cell marker antibodies, the main method currently used to separate rabbit B cells is to perform IgG and antigen marker detection on cell samples based on traditional flow cytometry to sort memory B cells, but the probability of obtaining effective antibody sequences is relatively low due to flux limitations and varying degrees of loss caused by the operation of B cells. CN201910125091.4 discloses a method for enriching rabbit antigen-specific B lymphocytes, a method for removing cells combined with biotinylated anti-rabbit CD4 / CD8 / CD14 by streptavidin magnetic beads, and then screening non-IgM antigen-specific cells by flow sorting, but this method has the problem that the purity of separated cells is not high enough, and only memory B cells can be screened, and flow cytometer sorting will cause cell damage.

[0004] There are currently three major technical routes for the development of rabbit monoclonal antibodies. The first is the traditional hybridoma technology platform, which screens antibody-secreting plasma B cells. Due to the instability of rabbit myeloma cell lines, rabbit hybridomas are also unstable and have relatively low fusion efficiency compared to traditional mouse hybridomas. The IgG secretion that decreases over time is more serious than that of mouse homologous hybridomas. The second is phage display technology, which directly sequences and builds libraries for B cells. The source of antibody sequences can be plasma cells or memory cells (Memory B cells), but the random combination of antibody variable region genes that occurs during library construction leads to the loss of natural homologous heavy and light chain pairing that evolved and selected in vivo during the immune response, resulting in a decrease in antibody affinity. Due to this random pairing, antibodies from the initial antibody library usually need to mature in vitro to give the antibodies higher affinity and stability; the third is single B cell direct sequencing or B cell in vitro culture screening and sequencing technology, the source of antibody sequences in this technology can be plasma cells or memory cells. The existing methods of culturing B cells and activating memory B cells for plasma B cell screening have poor stability, high cost, and will lose antibody diversity to a certain extent during the culture process. CN115639363 A discloses a method for detecting and sorting antigen-specific secretory cells, using droplet microfluidics to sort CHO cells expressing rabbit antibodies, but it has not involved the sorting of primary rabbit plasma B cells. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a rabbit single B cell enrichment method and a microfluidic screening method based on the deficiencies and defects mentioned in the background technology.

[0006] In order to solve the above problems, the present invention proposes the following technical solutions:

[0007] In one aspect, the present invention provides a method for enriching rabbit single B cells, comprising the following steps:

[0008] S1. Mixing a biotinylated specific antibody and a rabbit single cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension;

[0009] S2, adding streptavidin-coated magnetic beads to the cell suspension obtained in S1, and using the principle of magnetic separation to remove cells bound to the specific antibody, thereby obtaining enriched rabbit single B cells;

[0010] The specific antibody is a combination of one or more antibodies selected from the group consisting of T lymphocyte antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD11b monoclonal antibody, IgM monoclonal antibody, CD11c monoclonal antibody and CD123 monoclonal antibody.

[0011] A further technical solution is that in the cell suspension, the concentrations of specific antibodies are: T lymphocyte antibody 1 μg / mL-30 μg / mL, CD4 monoclonal antibody 1 μg / mL-30 μg / mL, CD8 monoclonal antibody 1 μg / mL-30 μg / mL, CD11b monoclonal antibody 1 μg / mL-50 μg / mL, IgM monoclonal antibody 1 μg / mL-40 μg / mL, CD11c monoclonal antibody 1 μg / mL-30 μg / mL and CD123 monoclonal antibody 1 μg / mL-30 μg / mL.

[0012] A further technical solution is that in the cell suspension, the concentrations of specific antibodies are: T lymphocyte antibody 5μg / mL-30μg / mL, CD4 monoclonal antibody 5μg / mL-30μg / mL, CD8 monoclonal antibody 5μg / mL-30μg / mL, CD11b monoclonal antibody 10μg / mL-50μg / mL, IgM monoclonal antibody 10μg / mL-40μg / mL, CD11c monoclonal antibody 5μg / mL-30μg / mL and CD123 monoclonal antibody 5μg / mL-30μg / mL.

[0013] A further technical solution is that the tissue sources of the rabbit single cells include rabbit spleen and rabbit peripheral blood.

[0014] Its further technical solution is that the CD4 monoclonal antibody is a rabbit anti-CD4 monoclonal antibody, the CD8 monoclonal antibody is a rabbit anti-CD8 monoclonal antibody, the CD11b monoclonal antibody is a human anti-CD11b monoclonal antibody, the IgM monoclonal antibody is a rabbit anti-IgM monoclonal antibody, the CD11c is a mouse anti-human CD11c monoclonal antibody, and the CD123 monoclonal antibody is a mouse anti-human CD123 monoclonal antibody.

[0015] A further technical solution is that the specific antibody is a combination of at least four antibodies selected from the group consisting of T lymphocyte antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD11b monoclonal antibody, IgM monoclonal antibody, CD11c monoclonal antibody and CD123 monoclonal antibody.

[0016] A further technical solution is that the specific antibody is a combination of at least five antibodies selected from the group consisting of T lymphocyte antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD11b monoclonal antibody, IgM monoclonal antibody, CD11c monoclonal antibody and CD123 monoclonal antibody.

[0017] The present invention also provides a microfluidic rabbit single B cell screening method, comprising the following steps:

[0018] According to the rabbit single B cell enrichment method described in the above scheme, an enriched rabbit single B cell suspension is obtained;

[0019] The target antigen is coated on the surface of microbeads to obtain microbead antigens;

[0020] Prepare a buffer containing a detection antibody, resuspend the rabbit single B cell suspension and the microbead antigen in the buffer, respectively, mix the resuspended rabbit single B cell suspension and the microbead antigen, and prepare a plurality of nanoliter or picoliter droplets;

[0021] incubating the droplets under appropriate conditions to allow the rabbit single B cells to secrete the target antibody, thereby obtaining incubated droplets;

[0022] The incubated droplets are passed through a droplet sorting chip one by one for fluorescence signal detection, and the target droplets are sorted and enriched according to the fluorescence signal values ​​of the droplets to obtain the target rabbit single B cells;

[0023] The detection antibody is labeled with a first fluorescent marker.

[0024] A further technical solution is that the first fluorescent label includes any one fluorescent group of Alexa Fluor 488, Alexa Fluor 647, PE or BV421.

[0025] A further technical solution is that the buffer solution further includes a dead cell fluorescent dye, and the fluorescent signal generated by the first fluorescent marker is different from the fluorescent signal generated by the dead cell fluorescent dye.

[0026] A further technical solution is that the dead cell fluorescent dye is a dye such as NucGreen, NucRed or EvaGreen that specifically binds to dead cells and has excitation and emission spectrum information. It is understandable that the dead cell fluorescent dye does not non-specifically bind to living cells, microbeads or proteins.

[0027] A further technical solution is that in the buffer, the final concentration of the detection antibody can be any concentration between 2nM and 400nM, preferably 2-200nM, more preferably 10-80nM. The final concentration of the dead cell fluorescent dye can be any concentration between 5nM and 5μM, preferably 50-1μM.

[0028] A further technical solution is to also perform reverse transcription and PCR amplification on the screened target rabbit single B cells, and obtain antibodies expressed by the corresponding rabbit B cells through sequencing and expression verification.

[0029] Compared with the prior art, the technical effects achieved by the present invention include:

[0030] The rabbit single B cell enrichment method provided by the present invention utilizes a combination of multiple specific antibodies to capture non-B cells in the system, does not affect the quantity and quality of B cells in the system, achieves the effect of rapidly enriching high-purity rabbit B cells from rabbit single cells, and greatly improves the proportion of secretory plasma B cells. The enrichment process of the present invention does not cause damage to B cells, improves the purity of rabbit plasma B cells, and the enriched rabbit plasma B cells can be directly used for downstream microfluidic rabbit plasma B antibody screening, effectively improving the downstream screening efficiency.

[0031] The microfluidic rabbit single B cell screening method provided by the present invention utilizes the aforementioned enriched rabbit single B cells for screening, and can perform high-throughput rapid sorting of target microdroplets, has the advantages of high accuracy and no damage to cells, and can shorten the work of traditional methods such as hybridoma screening methods that take several months to complete within 1 day. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0033] Figure 1 A schematic diagram of the process of the rabbit single B cell enrichment method provided by the present invention;

[0034] Figure 2 This is the B cell enrichment result of the PBMC group of the present invention;

[0035] Figure 3 This is the B cell enrichment result of the spleen cell group of the present invention;

[0036] Figure 4 ELISPOT diagram of B cell enrichment of spleen cell group of the present invention;

[0037] Figure 5 This is the B cell percentage result of the spleen cell group of the present invention;

[0038] Figure 6 Schematic diagram of the components of droplets formed in the microfluidic rabbit single B cell screening method in an embodiment of the present invention;

[0039] Figure 7 This is an agarose gel image of BCR of the product obtained by microfluidic screening in an embodiment of the present invention;

[0040] Figure 8 The experimental results of randomly selecting 20 products obtained from microfluidic screening for purified antibody concentration gradient binding. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0043] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0044] The embodiment of the present invention provides a method for enriching rabbit single B cells, comprising the following steps:

[0045] S1. Mixing a biotinylated specific antibody and a rabbit single cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension;

[0046] S2, adding streptavidin-coated magnetic beads to the cell suspension obtained in S1, and using the principle of magnetic separation to remove cells bound to the specific antibody, thereby obtaining enriched rabbit single B cells;

[0047] The specific antibody is a combination of T lymphocyte antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD11b monoclonal antibody and IgM monoclonal antibody.

[0048] In some embodiments, the concentrations of specific antibodies in the cell suspension are: T lymphocyte antibody 1 μg / mL-30 μg / mL, CD4 monoclonal antibody 1 μg / mL-30 μg / mL, CD8 monoclonal antibody 1 μg / mL-30 μg / mL, CD11b monoclonal antibody 1 μg / mL-50 μg / mL, IgM monoclonal antibody 1 μg / mL-40 μg / mL, CD11c monoclonal antibody 1 μg / mL-30 μg / mL and CD123 monoclonal antibody 1 μg / mL-30 μg / mL.

[0049] In a preferred embodiment, the concentration of specific antibodies in the cell suspension is: T lymphocyte antibody 5 μg / mL-30 μg / mL, CD4 monoclonal antibody 5 μg / mL-30 μg / mL, CD8 monoclonal antibody 5 μg / mL-30 μg / mL, CD11b monoclonal antibody 10 μg / mL-50 μg / mL, IgM monoclonal antibody 10 μg / mL-40 μg / mL, CD11c monoclonal antibody 5 μg / mL-30 μg / mL and CD123 monoclonal antibody 5 μg / mL-30 μg / mL.

[0050] In a more preferred embodiment, the concentration of specific antibodies in the cell suspension is: T lymphocyte antibody 10 μg / mL-30 μg / mL, CD4 monoclonal antibody 10 μg / mL-30 μg / mL, CD8 monoclonal antibody 10 μg / mL-30 μg / mL, CD11b monoclonal antibody 10 μg / mL-50 μg / mL, IgM monoclonal antibody 10 μg / mL-40 μg / mL, CD11c monoclonal antibody 10 μg / mL-30 μg / mL and CD123 monoclonal antibody 10 μg / mL-30 μg / mL.

[0051] The rabbit single B cell enrichment method provided by the present invention utilizes a combination of multiple specific antibodies to capture non-B cells in the system, does not affect the quantity and quality of B cells in the system, achieves the effect of rapidly enriching high-purity rabbit B cells from rabbit single cells, greatly improves the proportion of secretory plasma B cells, and the enrichment process of the present invention does not cause damage to B cells, thereby improving the purity of rabbit plasma B cells.

[0052] The present invention provides a method for enriching rabbit single B cells, wherein the tissue sources of the rabbit single cells include rabbit spleen and rabbit peripheral blood, and the single cells may be memory B cells and / or plasma B cells.

[0053] The rabbit mono B cell enrichment method provided by the present invention, the CD4 monoclonal antibody is a rabbit anti-CD4 monoclonal antibody, the CD8 monoclonal antibody is a rabbit anti-CD8 monoclonal antibody, the CD11b monoclonal antibody is a human anti-CD11b monoclonal antibody, the IgM monoclonal antibody is a rabbit anti-IgM monoclonal antibody, the CD11c is a mouse anti-human CD11c monoclonal antibody, and the CD123 monoclonal antibody is a mouse anti-human CD123 monoclonal antibody. The above specific antibodies can bind to rabbit monocytes, T lymphocytes, CD4+T lymphocytes and CD8+T lymphocytes, B cells expressing IgM and dendritic DC cells to achieve the effect of capturing non-B cells in the system.

[0054] Some embodiments of the present invention also provide a microfluidic rabbit single B cell screening method, comprising the following steps:

[0055] According to the rabbit single B cell enrichment method described in the above example, an enriched rabbit single B cell suspension is obtained;

[0056] The target antigen is coated on the surface of microbeads to obtain microbead antigens;

[0057] Prepare a buffer containing a detection antibody, resuspend the rabbit single B cell suspension and the microbead antigen in the buffer, respectively, mix the resuspended rabbit single B cell suspension and the microbead antigen, and prepare a plurality of nanoliter or picoliter droplets;

[0058] incubating the droplets under appropriate conditions to allow the rabbit single B cells to secrete the target antibody, thereby obtaining incubated droplets;

[0059] The incubated droplets are passed through a droplet sorting chip one by one for fluorescence signal detection, and the target droplets are sorted and enriched according to the fluorescence signal values ​​of the droplets to obtain target rabbit single B cells; the detection antibody carries a first fluorescent marker.

[0060] In some embodiments, the first fluorescent label comprises any one of AlexaFluor488, AlexaFluor 647, PE or BV421 fluorescent groups.

[0061] In some embodiments, the buffer further includes a dead cell fluorescent dye, and the fluorescent signal generated by the first fluorescent marker is different from the fluorescent signal generated by the dead cell fluorescent dye.

[0062] In some embodiments, the dead cell fluorescent dye is a dye that specifically binds only to dead cells and has excitation and emission spectrum information, such as NucGreen, NucRed or EvaGreen. It is understandable that the dead cell fluorescent dye does not non-specifically bind to living cells, microbeads or proteins.

[0063] In some embodiments, the final concentration of the detection antibody in the buffer may be any concentration between 2 nM and 400 nM, preferably 2-200 nM, more preferably 10-80 nM. The final concentration of the dead cell fluorescent dye may be any concentration between 5 nM and 5 μM, preferably 50-1 μM.

[0064] In some embodiments, the method further comprises reverse transcription and PCR amplification of the obtained target rabbit single B cells, and obtaining B cells expressing rabbit antibodies through sequencing and expression verification.

[0065] It can be understood that the present invention utilizes microfluidic sorting technology to screen antigen-specific plasma B cells from enriched rabbit single B cells, and can complete the screening and amplification pairing of BCR within 2 days, and has the advantages of high throughput, high sensitivity, and high screening accuracy.

[0066] The rabbit single B cell antibody screening method provided in the embodiment of the present invention can complete the enrichment, screening and subsequent BCR amplification of rabbit B cells within 2 days, greatly shortening the timeliness of antibody discovery and the success rate of obtaining antibody sequences. It can be applied in the research and development of antibody drugs and diagnostic antibodies to obtain high-affinity antibodies for subsequent clinical applications.

[0067] The following example is a method for enriching rabbit single B cells provided by the present invention. The B cell ratio and IgG secreting plasma cells in the rabbit B cells are detected by sequencing and enzyme-linked immunosorbent spot (ELISpot) technology before and after enrichment. The results are shown in Figure 2 , Figure 3 , Figure 4 It can be seen that by using the method provided by the present invention, the proportion of B cells and plasma cells after enrichment increased by 5-10 times (depending on the difference in the immune status of the rabbit). Obviously, the method of the present invention can effectively increase the proportion of rabbit plasma B cells that secrete IgG, and the cell viability is maintained at more than 90%.

[0068] PBMC Group Example 1 (Isolation-1)

[0069] This embodiment provides a method for enriching rabbit single B cells, comprising the following steps:

[0070] S1, mixing a biotinylated specific antibody and a rabbit PBMC cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension;

[0071] S2. Add streptavidin-coated magnetic beads to the cell suspension obtained in S1, and use the principle of magnetic separation to remove cells bound to the specific antibody to obtain enriched rabbit single B cells.

[0072] The rabbit single B cells enriched in this example were stained with fluorescent live and dead dyes, and the live and dead cells were counted using a fluorescent cell counter, and statistics showed that the proportion of live cells exceeded 90%.

[0073] The specific antibodies and their concentrations in this example are: T lymphocyte antibody 8 μg / mL, CD4 monoclonal antibody 6 μg / mL, CD8 monoclonal antibody 6 μg / mL, CD11b monoclonal antibody 12 μg / mL, and IgM monoclonal antibody 10 μg / mL. Figure 2 The results show that the proportion of B cells after enrichment increased by 4.7 times compared with the cells before enrichment.

[0074] PBMC Group Example 2 (Isolation-2)

[0075] This embodiment provides a method for enriching rabbit single B cells, comprising the following steps:

[0076] S1, mixing a biotinylated specific antibody and a rabbit PBMC cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension;

[0077] S2. Add streptavidin-coated magnetic beads to the cell suspension obtained in S1, and use the principle of magnetic separation to remove cells bound to the specific antibody to obtain enriched rabbit single B cells.

[0078] The rabbit single B cells enriched in this example were stained with fluorescent live and dead dyes, and the live and dead cells were counted using a fluorescent cell counter, and statistics showed that the proportion of live cells exceeded 90%.

[0079] The specific antibodies and their concentrations in this example are: T lymphocyte antibody 8 μg / mL, CD4 monoclonal antibody 10 μg / mL, CD8 monoclonal antibody 10 μg / mL, CD11b monoclonal antibody 30 μg / mL, and IgM monoclonal antibody 10 μg / mL. Figure 2 The results show that the proportion of B cells after enrichment increased by 4.6 times compared with the cells before enrichment.

[0080] Spleen cell group implementation case 1 (Isolation-1)

[0081] This embodiment provides a method for enriching rabbit single B cells, comprising the following steps:

[0082] S1, mixing a biotinylated specific antibody and a rabbit spleen cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension;

[0083] S2. Add streptavidin-coated magnetic beads to the cell suspension obtained in S1, and use the principle of magnetic separation to remove cells bound to the specific antibody to obtain enriched rabbit single B cells.

[0084] The specific antibodies and their concentrations in this embodiment are: T lymphocyte antibody 10 μg / mL, CD4 monoclonal antibody 5 μg / mL, CD8 monoclonal antibody 5 μg / mL, CD11b monoclonal antibody 10 μg / mL and IgM monoclonal antibody 10 μg / mL. Figure 3 The results show that compared with the cells before enrichment, the proportion of B cells after enrichment increased by 1.94 times, and the proportion of T cells decreased by 55%.

[0085] Spleen cell group implementation case 2 (Isolation-2)

[0086] This embodiment provides a method for enriching rabbit single B cells, comprising the following steps:

[0087] S1, mixing a biotinylated specific antibody and a rabbit spleen cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension;

[0088] S2. Add streptavidin-coated magnetic beads to the cell suspension obtained in S1, and use the principle of magnetic separation to remove cells bound to the specific antibody to obtain enriched rabbit single B cells.

[0089] The specific antibodies and their concentrations in this embodiment are: T lymphocyte antibody 20 μg / mL, CD4 monoclonal antibody 10 μg / mL, CD8 monoclonal antibody 10 μg / mL, CD11b monoclonal antibody 10 μg / mL and IgM monoclonal antibody 10 μg / mL. Figure 3 The results show that compared with the cells before enrichment, the proportion of B cells after enrichment increased by 2.7 times, and the proportion of T cells decreased by 90%.

[0090] Spleen cell group implementation case 3 (Isolation-3)

[0091] This embodiment provides a method for enriching rabbit single B cells, comprising the following steps:

[0092] S1, mixing a biotinylated specific antibody and a rabbit spleen cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension;

[0093] S2. Add streptavidin-coated magnetic beads to the cell suspension obtained in S1, and use the principle of magnetic separation to remove cells bound to the specific antibody to obtain enriched rabbit single B cells.

[0094] The specific antibodies and their concentrations in this embodiment are: T lymphocyte antibody 20 μg / mL, CD4 monoclonal antibody 10 μg / mL, CD8 monoclonal antibody 10 μg / mL, CD11b monoclonal antibody 10 μg / mL, IgM monoclonal antibody 10 μg / mL, CD123 monoclonal antibody 5 μg / mL. Figure 3 The results show that compared with the cells before enrichment, the proportion of B cells after enrichment increased by 2.55 times, the proportion of T cells decreased by 85%, and the proportion of DC decreased by 30%.

[0095] Spleen cell group implementation case 4 (Isolation-4)

[0096] This embodiment provides a method for enriching rabbit single B cells, comprising the following steps:

[0097] S1, mixing a biotinylated specific antibody and a rabbit spleen cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension;

[0098] S2. Add streptavidin-coated magnetic beads to the cell suspension obtained in S1, and use the principle of magnetic separation to remove cells bound to the specific antibody to obtain enriched rabbit single B cells.

[0099] The specific antibodies and their concentrations in this embodiment are: T lymphocyte antibody 20 μg / mL, CD4 monoclonal antibody 10 μg / mL, CD8 monoclonal antibody 10 μg / mL, CD11b monoclonal antibody 10 μg / mL, IgM monoclonal antibody 10 μg / mL, CD11c monoclonal antibody 5 μg / mL. Figure 3 The results show that compared with the cells before enrichment, the proportion of B cells after enrichment increased by 2.8 times, the proportion of T cells decreased by 85%, and the proportion of DC decreased by 50%.

[0100] Spleen cell group implementation case 5 (Isolation-5)

[0101] This embodiment provides a method for enriching rabbit single B cells, comprising the following steps:

[0102] S1, mixing a biotinylated specific antibody and a rabbit spleen cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension;

[0103] S2. Add streptavidin-coated magnetic beads to the cell suspension obtained in S1, and use the principle of magnetic separation to remove cells bound to the specific antibody to obtain enriched rabbit single B cells.

[0104] The specific antibodies and their concentrations in this embodiment are: T lymphocyte antibody 20 μg / mL, CD4 monoclonal antibody 15 μg / mL, CD8 monoclonal antibody 15 μg / mL, CD11b monoclonal antibody 10 μg / mL, IgM monoclonal antibody 10 μg / mL, CD11c monoclonal antibody 10 μg / mL, CD123 monoclonal antibody 10 μg / mL. Figure 3 The results show that compared with the cells before enrichment, the proportion of B cells after enrichment increased by 3 times, the proportion of T cells decreased by 91%, and the proportion of DC decreased by 98%.

[0105] Furthermore, the present invention performs ELISpot detection on the B cells enriched in the spleen cell group Examples 1-5, and uses the number of B cells secreting IgG to represent the enriched plasma B cells. At the same time, a rabbit spleen cell suspension that does not use the enrichment method of the present invention is used as a control. Compare the proportion of B cells in each example.

[0106] The ELISpot assay is as follows:

[0107] 1 μg / mL goat anti-rabbit IgG (H+L) polyclonal antibody was spread on a 96-well PVDF membrane plate pretreated with 35% alcohol, 100 μL per well, at 4°C overnight;

[0108] The next day, the plate was washed five times with 200 μL PBS, and the wells were blocked with 100 μL of 1640 medium containing 10% fetal bovine serum per well and incubated at 37°C for 1 h;

[0109] The plate was washed five times with 200 μL PBS, 5000 cells were seeded in each well of the first column and 1000 cells were seeded in each well of the second column, 100 μL was added to each well, and the cells were incubated in a 37°C cell culture incubator overnight;

[0110] The next day, the plate was washed five times with 200 μL PBS, 100 μL 1 μg / mL biotinylated goat anti-rabbit IgG Fc antibody was added, and incubated at room temperature for 2 h;

[0111] The plate was washed five times with 200 μL PBS, and 100 μL of 1:1000 diluted Streptavidin-ALP was added to each well and incubated at room temperature for 1 h;

[0112] The plate was washed five times with 200 μL PBS, 100 μL BCIP / NBT-plus chromogenic substrate was added, and incubated at room temperature for 10 min;

[0113] The plate was washed five times with 200 μL PBS to terminate the reaction, and the PVDF membrane was dried in an oven before taking photos and analyzing them using an upright microscope.

[0114] Results Figure 4 and Figure 5 Obviously, after the rabbit spleen cell suspension is enriched by the method of the present invention, the proportion of B cells is significantly increased. It can also be seen from the results of each embodiment that the type and concentration of specific antibodies have a great relationship with the proportion of plasma B cells finally enriched.

[0115] See also Figure 6 In this example, the B cells enriched in the spleen cell group Example 5 were subjected to microfluidic screening, and the specific operation was as follows:

[0116] Droplet generation cell phase: Take 1 million B cells enriched in Example 5, centrifuge at 500 g, gently discard the supernatant, and resuspend the cells in 83 μL cell culture medium (DMEM culture medium supplemented with 10% FBS, 16% cell density reagent Optiprep, and 0.1% cell protectant F-68);

[0117] Droplet generation reagent phase: 66 μL antigen-coated streptavidin magnetic beads were magnetically separated and the supernatant was discarded, followed by the addition of 70 μL cell culture medium (DMEM medium supplemented with 10% FBS, 16% cell density reagent Optiprep, 0.1% cell protectant F-68), 10 μL dead cell dye NucGreen dead, and 1 / 200 diluted goat anti-rabbit IgG coupled to Alexa fluro647 fluorescence detection secondary antibody. The cell phase and reagent phase were generated using the Dapu Bio droplet generation chip to generate 50 μm droplets, which were collected in a 1.5 mL centrifuge tube and placed in a 37 ° C cell culture incubator for 1 hour. The droplet was analyzed and screened by fluorescence signals using the droplet microfluidics high-throughput screening platform. Droplets with no NucGreen dead dead cell dye fluorescence and a fluorescence detection secondary antibody signal value of goat anti-rabbit IgG coupled to Alexa fluro647 that was more than 2 times the background fluorescence value were screened as positive droplets and printed into a 96-well PCR plate.

[0118] The screened cells were subjected to BCR amplification, and the amplified BCR was subjected to agarose gel to observe whether the fragment size was in line with expectations.

[0119] Results Figure 7 , it can be seen that 83% of the cells can amplify light and heavy chains, and fragments of corresponding sizes appear on the agarose gel: 450 bp for the heavy chain and 500 bp for the light chain.

[0120] Randomly select 20 paired products for expression and purification, and perform Elisa detection on the purified antibodies:

[0121] Elisa test protocol is as follows:

[0122] 1 μg / mL OVA antigen protein, 100 μL / well, was incubated with PBS buffer at 4°C overnight, then the solution in the wells was discarded, and the plate was washed three times with PBS + 0.1% Tween-20, and dried on absorbent paper;

[0123] Add 200 μL / well PBS + 1% BSA, incubate at 37°C for 1 h, wash the plate three times with PBS + 0.1% Tween-20, and dry;

[0124] Add gradient diluted purified antibodies, starting from 100 nM, and dilute 3-fold to 10 points. Incubate at 37°C for about 45 min, wash the plate 3 times, and dry it.

[0125] Add 100 μL / well of the diluted rabbit anti-human HRP secondary antibody working solution, incubate at 37°C for about 45 min, wash the plate at least 5 times, and dry it;

[0126] Add 100 μL / well of TMB substrate solution and react at 37°C in the dark for 5-10 min until a clear blue gradient appears in the wells of the serially diluted standard.

[0127] Add 50 μL / well of stop solution to terminate the enzymatic reaction, and the color changes from blue to yellow;

[0128] Within 15 minutes, measure the OD value of each well on the microplate reader at 450 nm after adjusting the blank control well to zero.

[0129] Results Figure 8 , verified by Elisa, 17 out of 20 randomly selected positive droplets were able to specifically bind to the antigen, with a positive rate of up to 85%.

[0130] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0131] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for enriching rabbit single B cells, characterized in that: The following steps are involved: S1. Mixing a biotinylated specific antibody and a rabbit single cell suspension to be separated, so that the specific antibody binds to the cells to be removed, and obtaining a cell suspension; S2, adding streptavidin-coated magnetic beads to the cell suspension obtained in S1, and using the principle of magnetic separation to remove cells bound to the specific antibody, thereby obtaining enriched rabbit single B cells; The specific antibody is a combination of one or more antibodies selected from the group consisting of T lymphocyte antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD11b monoclonal antibody, IgM monoclonal antibody, CD11c monoclonal antibody and CD123 monoclonal antibody.

2. The rabbit single B cell enrichment method according to claim 1, characterized in that: In the cell suspension, the concentrations of specific antibodies are: T lymphocyte antibody 1 μg / mL-30 μg / mL, CD4 monoclonal antibody 1 μg / mL-30 μg / mL, CD8 monoclonal antibody 1 μg / mL-30 μg / mL, CD11b monoclonal antibody 1 μg / mL-50 μg / mL, IgM monoclonal antibody 1 μg / mL-40 μg / mL, CD11c monoclonal antibody 1 μg / mL-30 μg / mL and CD123 monoclonal antibody 1 μg / mL-30 μg / mL.

3. The rabbit single B cell enrichment method according to claim 2, characterized in that: In the cell suspension, the concentrations of specific antibodies are: T lymphocyte antibody 5 μg / mL-30 μg / mL, CD4 monoclonal antibody 5 μg / mL-30 μg / mL, CD8 monoclonal antibody 5 μg / mL-30 μg / mL, CD11b monoclonal antibody 10 μg / mL-50 μg / mL, IgM monoclonal antibody 10 μg / mL-40 μg / mL, CD11c monoclonal antibody 5 μg / mL-30 μg / mL and CD123 monoclonal antibody 5 μg / mL-30 μg / mL.

4. The rabbit single B cell enrichment method according to claim 1, characterized in that: The CD4 monoclonal antibody is a rabbit anti-CD4 monoclonal antibody, the CD8 monoclonal antibody is a rabbit anti-CD8 monoclonal antibody, the CD11b monoclonal antibody is a human anti-CD11b monoclonal antibody, the IgM monoclonal antibody is a rabbit anti-IgM monoclonal antibody, the CD11c is a mouse anti-human CD11c monoclonal antibody, and the CD123 monoclonal antibody is a mouse anti-human CD123 monoclonal antibody.

5. The rabbit single B cell enrichment method according to any one of claims 1 to 4, characterized in that: The specific antibodies are a combination of at least four antibodies selected from the group consisting of T lymphocyte antibodies, CD4 monoclonal antibodies, CD8 monoclonal antibodies, CD11b monoclonal antibodies, IgM monoclonal antibodies, CD11c monoclonal antibodies, and CD123 monoclonal antibodies.

6. The rabbit single B cell enrichment method according to claim 1, characterized in that: The tissue sources of the rabbit single cells include rabbit spleen and rabbit peripheral blood.

7. A microfluidic rabbit single B cell screening method, characterized in that: The following steps are involved: Obtaining an enriched rabbit single B cell suspension according to the rabbit single B cell enrichment method according to any one of claims 1 to 6; The target antigen is coated on the surface of microbeads to obtain microbead antigens; Prepare a buffer containing a detection antibody, resuspend the rabbit single B cell suspension and the microbead antigen in the buffer, respectively, mix the resuspended rabbit single B cell suspension and the microbead antigen, and prepare a plurality of nanoliter or picoliter droplets; incubating the droplets under appropriate conditions to allow the rabbit single B cells to secrete the target antibody, thereby obtaining incubated droplets; The incubated droplets are passed through a droplet sorting chip one by one for multi-channel fluorescence signal detection, and the target droplets are sorted and enriched according to the fluorescence signal values ​​of the droplets to obtain the target rabbit single B cells; The detection antibody is labeled with a first fluorescent marker.

8. The microfluidic rabbit single B cell screening method according to claim 7, characterized in that: The buffer solution also includes dead cell fluorescent dye, and the fluorescent signal generated by the first fluorescent marker is different from the fluorescent signal generated by the dead cell fluorescent dye.

9. The microfluidic rabbit single B cell screening method according to claim 7, characterized in that: The method also includes reverse transcription and PCR amplification of the target rabbit single B cells obtained by screening, and obtaining antibodies expressed by the corresponding rabbit B cells through sequencing and expression verification.

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