Method for screening single b cells

Abstract

This application discloses a method for screening single B cells. The method provided in this application uses magnetic beads coated with IL-6 antigen, combined with fluorescently labeled secondary antibody, and employs droplet microfluidic technology to rapidly sort B cells. The obtained IL-6 positive B cells are then directly cultured, and the generated antibodies are subjected to ELISA and IL-6 antigen-antibody affinity detection to obtain the target cell line. The entire process is greatly simplified and the workload is significantly reduced.

Description

Technical Field

[0001] This application relates to the field of cell biology, and in particular to a method for screening single B cells. Background Technology

[0002] Single-cell B-cell antibody preparation technology boasts advantages such as short cycle time, high efficiency, and mature technology, enabling the direct acquisition of fully human antibodies with natural structures, which is beneficial for subsequent clinical translation. Based on cell sorting and sequencing, this technology requires only a small number of B cells to obtain antibody genes, and rapidly generates large quantities of antigen-specific monoclonal antibodies through in vitro expression. Currently, this technology has been successfully applied in anti-tumor and antiviral fields (such as Zika virus and influenza virus), becoming an important platform for the development of diagnostic and therapeutic antibodies.

[0003] In existing B-cell antibody preparation technologies, key steps rely on phycoerythrin (PE)-labeled secondary antibodies or antigens. Taking patent CN112098640A as an example, its PE-antibody conjugation method is cumbersome, involving multiple centrifugations, ultrafiltration, liquid changes, and multiple chemical reactions. Its main drawbacks are: complex operation and high cost: the process uses many types of reagents, is time-consuming and labor-intensive; and the PE-antibody molar ratio of 5:1, due to the high price of PE protein, significantly increases the cost of the final product. Harsh reaction conditions, easily affecting protein activity: the entire process requires multiple steps at 25℃, lasting approximately 6 hours. Under these conditions, both PE and antibody may degrade or become inactive, thus reducing the performance of the fluorescent antibody. The B-cell screening process is cumbersome and difficult to standardize: existing methods mostly rely on fluorescently labeled antigens, but antigen molecular characteristics vary greatly, requiring individual optimization of labeling methods, making standardization impossible and increasing the initial workload. Subsequent steps include flow cytometry sorting of positive B cells, and extensive sequencing, cloning, and expression, making the overall process complex and labor-intensive. In summary, there is a need in this field to develop new antigen labeling methods that are unified, highly active, simple, and lower cost for the preparation of single B cell antibodies.

[0004] In recent years, novel B-cell screening technologies such as droplet microfluidics and hydrogel-based micro / nanopore arrays are replacing traditional techniques, simplifying processes, increasing throughput, and reducing costs. For example, a Harvard University team developed a droplet microfluidics-based process that encapsulates B cells, magnetic beads coated with capture antibodies, and fluorescently labeled antigens within microdroplets. If the antibodies secreted by the cells bind to the antigens, the fluorescent signal is retained on the magnetic beads and detected by the instrument, thus achieving single-cell screening based on antibody function. However, this method uses fluorescently labeled antigens, and each antigen sequence varies greatly, requiring individual exploration of fluorescent labeling methods for each antigen. This significantly increases the experimental difficulty, and the specificity and binding affinity of the antibodies secreted by the resulting B cells need further improvement. Summary of the Invention

[0005] The purpose of this application is to provide a method for screening single B cells.

[0006] Another objective of this application is to provide a method for preparing an antibody.

[0007] To address the aforementioned technical problems, a first aspect of this application provides a method for screening single B cells, the method comprising the steps of: Step 1: Biotinylate the IL-6 antigen to obtain the biotinylated IL-6 antigen. Step 2: Coat the biotinylated IL-6 antigen with immunomagnetic beads to obtain immunomagnetic beads coated with IL-6 antigen. Step 3: Polyorubin is conjugated with secondary antibody to obtain PE-secondary antibody conjugation product; Step 4: Mix the treated IL-6 hybridoma cells with the PE-secondary antibody conjugation product, then add the biotinylated IL-6 antigen-coated immunomagnetic beads, and wait for droplet formation; Step 5: Incubate the droplets and perform cell sorting.

[0008] In some preferred embodiments, in step 1, the biotin may be a PEG-based biotin, such as Biotin-PEG4-NHS (CAS No.: 459426-22-3).

[0009] In some preferred embodiments, step 1, the biotinylation of the IL-6 antigen, includes the steps of: mixing the IL-6 antigen solution with a solution containing dissolved Biotin-PEG4-NHS and then incubating.

[0010] In some preferred embodiments, step 1, the biotinylation of the IL-6 antigen, includes the step of having a biotin to IL-6 antigen molar ratio of approximately 40:1.

[0011] In some preferred embodiments, step 1, the biotinylation of the IL-6 antigen, includes the steps of adding a biotin solution to the IL-6 antigen solution to be labeled and then incubating it.

[0012] In some preferred embodiments, in step 1, after adding biotin solution to the IL-6 antigen solution to be labeled, the incubation temperature is approximately 25°C, and the incubation time is approximately 40-60 minutes. Preferably, the incubation is carried out at 25°C in the dark for 40-60 minutes to obtain the biotinylated IL-6 antigen.

[0013] In some preferred embodiments, in step 1, the solution containing dissolved Biotin-PEG4-NHS is mixed with the IL-6 antigen solution at a biotin to IL-6 antigen molar ratio of approximately 40:1, and then incubated at 25°C in the dark for 40-60 min to obtain biotinylated IL-6 antigen.

[0014] In some preferred embodiments, step 2, which involves coating the biotinylated IL-6 antigen onto the immunomagnetic beads, includes the following steps: taking magnetic beads containing streptavidin, resuspending them, adding a solution of the biotinylated IL-6 antigen, and incubating them to obtain the biotinylated IL-6 antigen-coated immunomagnetic beads.

[0015] In some preferred embodiments, step 2 includes incubation at room temperature for 60-90 minutes, followed by removal of the supernatant and incubation at room temperature for 30 minutes with the addition of 100 μL of Blocking buffer; then removal of the supernatant and incubation at room temperature for 30-60 minutes with the addition of 200 μL of Cell buffer.

[0016] In some preferred embodiments, step 2 includes incubation at room temperature for 60-90 minutes, followed by removal of the supernatant, addition of 100 μL of Blocking buffer, and incubation at room temperature for 30 minutes; removal of the supernatant, addition of 200 μL of Cell buffer, and incubation at 4°C overnight.

[0017] In some preferred embodiments, step 3, which involves conjugating phycoerythrin (PE) with a secondary antibody, includes the following steps: PE is coupled with a crosslinking agent to obtain PE-crosslinking agent; Then the secondary antibody and reducing agent are mixed and reacted to obtain the reduced secondary antibody; Then, the reduced secondary antibody is coupled with a PE crosslinking agent, and a blocking agent is added for blocking, thus obtaining the desired result.

[0018] In some preferred embodiments, in step 3, the crosslinking agent is LC-SMCC (succinimide-[4-(N-maleimidemethyl)]-cyclohexane-1-carboxylic acid-(6-aminohexanoate), CAS No.: 125559-00-4).

[0019] In some preferred embodiments, in step 3, when PE is coupled with the crosslinking agent, the coupling is carried out at a feeding ratio of 2:1 for PE to crosslinking agent by mass.

[0020] In some preferred embodiments, in step 3, the PE crosslinking agent is PE-SMCC.

[0021] In some preferred embodiments, in step 3, the secondary antibody is a goat anti-mouse secondary antibody.

[0022] In some preferred embodiments, in step 3, the sealing agent is ethyl maleimide.

[0023] In some preferred embodiments, in step 3, the reducing agent is a thiol reducing agent, such as TCEP (tris(2-carboxyethyl)phosphine).

[0024] In some preferred embodiments, step 3, the mixing reaction of the secondary antibody and the reducing agent, includes the following steps: adjusting the concentration of the secondary antibody to 1~2 mg / ml, the concentration of TCEP to 5~10 mM / L, and incubating at 25°C with shaking at 600 rpm / min for 10~20 min to obtain the final product.

[0025] Preferably, after the secondary antibody and reducing agent are mixed and reacted, the step further includes treating the reduction product with ammonium sulfate solution. The step of treating the reduction product with ammonium sulfate solution includes: adding 5 times the volume of 65% ammonium sulfate solution to the reduction product, incubating in an ice bath for 10 min, centrifuging at 13000 rpm and 4°C for 10 min, discarding the supernatant, and reconstituteing the precipitate with PBS at an 80% recovery rate.

[0026] In some preferred embodiments, step 3, the coupling of the reduced secondary antibody with the PE crosslinking agent, includes the step of coupling the secondary antibody with the PE crosslinking agent at a mass ratio of approximately 1:1. Preferably, the reduced secondary antibody and the PE crosslinking agent are reacted at 20-25°C for 50-80 min or at 18-20°C for 80-100 min to obtain the desired result.

[0027] In some preferred embodiments, in step 3, the reduced secondary antibody and PE-crosslinking agent are incubated at 20°C with shaking at 600 rpm / min for 90~120 min to obtain the desired result.

[0028] In some preferred embodiments, after obtaining the PE-secondary antibody conjugate product, a purification step using molecular sieves is also included.

[0029] In some preferred embodiments, after step 3, the immunofluorescence assay can be used to determine whether the obtained IL-6-coated magnetic beads can be used for single B cell screening.

[0030] In some preferred embodiments, in step 4, the treated IL-6 hybridoma cells are obtained by the following steps: taking negative and positive IL-6 hybridoma cells with a viability greater than 99%, mixing them at a negative cell:positive cell ratio of 3:1, and resuspending them in PBS and CSB-14 in sequence.

[0031] In some preferred embodiments, step 4, the mixing of the treated IL-6 hybridoma cells with the PE-secondary antibody conjugate product, includes the following steps: mixing the PE-secondary antibody conjugate product with immunomagnetic beads coated with IL-6 antigen to form a reagent phase; then mixing the treated IL-6 hybridoma cells with the PE-secondary antibody conjugate product to form a cell phase; and injecting the reagent phase and the cell phase into a microfluidic chip system (e.g., a DG300 microfluidic chip system) to generate droplets.

[0032] In some preferred embodiments, in step 4, the desired package rate λ = 0.3.

[0033] In some preferred embodiments, the generated droplets are collected in an EP tube containing a special oil and immediately stored at a controlled temperature of 4°C.

[0034] In some preferred embodiments, step 5, incubating the droplet includes the following steps: placing the droplet at 37°C for about 1 hour, so that the secreted antibody binds to the antigen on the magnetic bead within the droplet, and is subsequently labeled by PE-secondary antibody to form a fluorescent signal.

[0035] In some preferred embodiments, in step 5, the droplets are observed using a microscope and sorted.

[0036] In some preferred embodiments, in step 5, droplets that can emit specific fluorescent signals are identified and selected, and sorted to obtain individual target B cells.

[0037] In some preferred embodiments, the target cell line is screened by culturing the individual target B cells and performing ELISA and IL-6 antigen-antibody affinity assays on the generated antibodies.

[0038] In a second aspect, the present invention provides a method for preparing an antibody, comprising the step of: culturing mono-B cells obtained by the method as described in any one of claims 1-10 to induce them to secrete antibodies.

[0039] In some preferred embodiments, individual target B cells obtained in the first aspect of the present invention are cultured to secrete antibodies; Antibodies obtained by ELISA and IL-6 antigen-antibody affinity assays were used to screen target B cell lines for culture, thereby inducing them to secrete large amounts of antibodies.

[0040] Based on the prior art, the present invention has at least the following advantages: The method provided by this invention utilizes biotin-activated magnetic beads, then coats streptavidin-activated magnetic beads, and finally uses fluorescently labeled secondary antibodies, effectively solving the problem of inconsistent methods for fluorescently labeled antigens.

[0041] The method provided by this invention obtains positive B cells through droplet microfluidic technology, and the cells are cultured. The antibodies produced are then detected using ELISA and antigen-antibody affinity, which can rapidly obtain the target cell line, greatly simplifying the screening process and significantly reducing the workload.

[0042] The method provided by this invention reduces the molar ratio of fluorescent protein to secondary antibody, significantly reduces the amount of fluorescent protein (PE) used by 3-4 times, and obtains an effective fluorescent antibody, thus greatly saving costs.

[0043] The method provided by this invention can significantly reduce the reaction time required for fluorescent proteins and secondary antibodies. Compared with the existing technology, which has a total reaction time of 6 hours at 25°C, this invention only requires a reaction time of up to 2 hours at a comparable or lower temperature. The reaction conditions are milder and the reaction time is shorter, effectively avoiding the degradation and inactivation of PE and CD16.

[0044] It should be understood that, within the scope of this application, the above-described technical features of this application and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0045] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0046] Figure 1 This is a graph showing the coupling rate results of PE-labeled goat anti-mouse secondary antibodies according to an embodiment of the present invention; Figure 2 This is a fluorescence spectral detection result diagram according to an embodiment of the present invention; Figure 3 This is the OD560 emission spectrum of IL-6 antigen-coated magnetic beads according to an embodiment of the present invention; Figure 4 This is the OD560 emission spectrum peak diagram of IL-6 antigen-coated magnetic beads according to an embodiment of the present invention; Figure 5 This is a diagram showing the cell droplet generation results according to an embodiment of the present invention (left, bright field; right, fluorescence). Figure 6 This is a diagram showing the cell sorting results according to an embodiment of the present invention; Figure 7 This is a diagram showing the cell sorting results according to an embodiment of the present invention. Detailed Implementation

[0047] Existing technologies mostly involve fluorescently conjugating IL-6 antigen, labeling B cells immunized with the corresponding antigen, screening using flow cytometry, and finally performing extensive sequencing and molecular cloning for expression, followed by further screening for IL-6 target antibodies. The entire process is extremely complex, especially sequencing, cloning, and antibody expression, which are incredibly labor-intensive. Through extensive and in-depth research, the inventors have developed a method using IL-6 antigen-coated magnetic beads combined with fluorescently labeled secondary antibodies. This method utilizes droplet microfluidic technology to rapidly sort B cells. The resulting IL-6-positive B cells can then be directly cultured, and the generated antibodies can be analyzed using ELISA and IL-6 antigen-antibody affinity testing to obtain the target cell line. This significantly simplifies the entire process and greatly reduces the workload.

[0048] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the present application is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0049] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0050] Example 1: IL-6 antigen-coated magnetic beads 1.1 Antigen Biotinylation (1) Dissolve Biotin-PEG4-NHS in DMSO, add the required biotin solution to the protein solution to be labeled according to the biotin:IL-6 antigen molar ratio of 40:1, and incubate at 25°C in the dark for 40~60 min; (2) Overnight dialysis with a 3.5KD dialysis bag to remove free Biotin-PEG4-NHS and biotinylated IL-6 antigen.

[0051] 1.2 Magnetic bead coating antigen (1) Take 70 μL of magnetic beads (containing streptavidin) into a 1.5 mL centrifuge tube, add 200 μL of PBST to fully resuspend the magnetic beads; repeat the washing three times, and resuspend in 100 μL of PBST; (2) Add antigen solution according to the ratio of 0.1~0.3mg biotinylated IL-6 antigen to 10uL magnetic beads, and incubate at room temperature for 60~90min on a vertical mixer; (3) Remove the supernatant, add 100uL of Blocking buffer to the magnetic beads and incubate at room temperature for 30 min; remove the supernatant, add 200uL of Cell buffer and incubate at room temperature for 30~60 min, or incubate at 4℃ overnight.

[0052] 1.3 PE-labeled goat anti-mouse secondary antibody

[0053] (1) PE and LC-SMCC were coupled at a mass ratio of 2:1, with PE concentration above 1 mg / mL, and incubated at 25℃, 600 rpm / min for 70 min with shaking. (2) Use PBS for ultrafiltration to completely remove free LC-SMCC from PE-SMCC; (3) Adjust the concentration of the secondary antibody to 1~2 mg / ml, the concentration of TCEP to 5~10 mM / L, and incubate at 25℃, 600 rpm / min with shaking for 10~20 min.

[0054] (4) Add 5 times the volume of 65% ammonium sulfate solution to the reduction product, let stand in an ice bath for 10 min, centrifuge at 13000 rpm and 4℃ for 10 min, remove the supernatant, and reconstitute the precipitate with PBS at an 80% recovery rate.

[0055] (5) The reduced antibody is coupled with fluorescent protein PE-SMCC at a ratio of 1:1 between the secondary antibody and PE-SMCC (based on the mass of PE). The mixture is then incubated at 20°C and 600 rpm for 90-120 min with shaking.

[0056] (6) Add 0.5 mg glycine and 0.1 mg ethyl maleimide blocking agent, and incubate at 25°C and 600 rpm for 90-120 min with shaking.

[0057] (7) The conjugate product was purified by molecular sieve. The purification buffer was PBS. The first elution peak was collected and concentrated, which was the PE-goat anti-mouse secondary antibody conjugate product.

[0058] (8) Validation of the efficacy of PE-labeled goat anti-mouse secondary antibody See results Figure 1 and Figure 2 .like Figure 1 The PE-labeled goat anti-mouse secondary antibody conjugation rate (OD499 / OD280) was 1.84. Figure 2 The results of coupling rate and fluorescence spectroscopy showed that the PE-labeled goat anti-mouse secondary antibody had a good effect.

[0059] 1.4 Verification of the effect of magnetic bead coating (1) Purification of antibodies: IL-6 positive control antibody and negative control antibody of the same species were diluted with PBS to two concentrations: 0.5ug / mL and 1μg / mL. (2) Add 10 μL of IL-6 magnetic beads to each tube, then add 200 μL of PBST, repeat the washing twice, and finally resuspend in PBS; (3) Prepare the reaction system in an EP tube by diluting the suspension with 10 μL of magnetic bead solution + 200 μL of IL-6 antibody / negative control antibody; (4) Add 200 μL each of 0.4 μg / mL IL-6 ascites antibody, 0.8 μg / mL IL-6 ascites antibody, and 0.8 μg / mL homologous negative control antibody to the three magnetic beads respectively; wash twice with PBST and remove the supernatant; (5) Dilute the PE-goat anti-mouse secondary antibody to 5-10 μg / mL, add 200 μL of fluorescent secondary antibody to each magnetic bead tube for resuspending, and incubate at room temperature in the dark with shaking for 1 h. Perform magnetic separation for 1 min, remove the supernatant, wash twice with PBST, resuspend in 200 μL of PBS, and analyze the fluorescence intensity. See the results below. Figure 3 and Figure 4 .

[0060] (6) The results showed that the fluorescence intensity of the negative control was significantly lower than that of the IL-6 monoclonal antibody positive control, and the fluorescence intensity increased with the increase of the concentration of the positive control monoclonal antibody, indicating that IL-6 coated magnetic beads can be used for screening corresponding single B cells.

[0061] Example 2: IL-6 hybridoma cell treatment and single-cell sorting 2.1 IL-6 hybridoma cell treatment (1) Cell count and viability detection in negative and positive culture flasks. The viability of negative cells was 99.52% and the viability of positive cells was 99.11%. The cells were mixed at a ratio of negative cells to positive cells of 3:1 and collected in centrifuge tubes.

[0062] (2) Centrifuge, discard the supernatant, add 1 mL PBS to resuspend, pass through a 70 μm cell filter to collect into a new 15 mL centrifuge tube, rinse the original tube with 1 mL PBS, rinse the filter with 3 mL PBS, collect the filtrate, and calculate the cell viability to be 99.2%.

[0063] (3) The cells were resuspended in 1 mL of CSB-14. At this time, the cell density was 21 × 106 cells / mL and the viability was 99.2%.

[0064] 2.2 Droplet formation (1) Take 15 μL of 0.5 mg / mL PE-goat anti-mouse secondary antibody and add 85 μL of CSB-14 to prepare a 500 nM 10X PE-goat anti-mouse secondary antibody stock solution.

[0065] (2) Take 66µL of Beads-antigen, wash the magnetic beads and resuspend them in 74.6µL of CSB-14, and then add 8.3µL of 10X PE-goat anti-mouse secondary antibody.

[0066] (3) Take 50µL of cell solution (total number of cells in this experiment is 1M cells) and add 24.7µL of CSB-14 to a centrifuge tube. Add 8.3µL of 10X PE-goat anti-mouse secondary antibody to the solution. When loading the sample, λ=0.3.

[0067] (4) Connect the reagent phase and cell phase EP tubes to the DG300, set the parameters, and after the droplet generation is stable, collect the generated droplets with a 1.5mL EP tube (containing 1.2mL of general droplet generation oil) and control the temperature at 4℃.

[0068] (5) After incubating the droplets at 37°C for 1 hour, observe the signal under a microscope and sort the cells.

[0069] 2.3 Cell sorting (1) After the droplet incubation is complete, add a small amount of universal droplet generating oil to the EP tube containing cells and place it into the droplet tube slot in the flip block.

[0070] (2) Set the configuration parameters, enter the printing interface, and start printing cells.

[0071] 2.4 Cell Printing and Culture (1) 50 μL of the corresponding cell culture medium was added to the 96-well cell culture plate beforehand.

[0072] (2) Adjust the printing parameters and print. After printing, centrifuge the cell plate (500g, 5min, room temperature), add the appropriate cell culture medium as needed, and place it in a cell culture incubator (37℃, 5% CO2).

[0073] (3) After culturing for about 3 days, observe the number and morphology of cells under a microscope.

[0074] Example 3, Experimental Results 3.1 Droplet formation results ① The image was taken after being encapsulated by a droplet, and the positive and negative signals were clearly visible (left - bright field, right - fluorescence). ② Cell droplets account for 26% of all generated droplets (bright field, count statistics); See results Figure 5 .

[0075] 3.2 Cell sorting and printing results See Figure 6 and Figure 7 .

[0076] (1) The positive rate on the machine was 2.55%, and the theoretical positive rate was 6.4% (the positive cell rate was calculated as follows: the number of positive cells accounted for 25% of the total number of cells, and the number of droplets with cells accounted for 26% of all droplets generated) (as shown in the figure); (2) Set the high signal of the gate for printing and print 4 96-well cells to test the effect of subsequent cell demulsification culture.

[0077] 3.3 Cell demulsification culture and ELISA detection (1) Cell demulsification culture results: No contamination was found. In each 96-well plate, the cell porosity was about 35%, and the average number of cells per well was about 20. (2) ELISA test results: A total of 84 wells were tested, and 66 wells were positive (OD≥2.1). (OD negative), positive rate 78.5%.

[0078] In summary, the screening results of single B cells secreting IL-6 antibodies in this invention meet expectations, enabling rapid single B cell sorting with satisfactory positivity and cell viability.

[0079] Those skilled in the art will understand that the above embodiments are specific implementations of this application, and in practical applications, various changes can be made in form and detail without departing from the spirit and scope of this application.

Claims

1. A method for screening single B cells, characterized in that, The method includes the following steps: Step 1: Biotinylate the IL-6 antigen to obtain the biotinylated IL-6 antigen. Step 2: Coat the biotinylated IL-6 antigen with immunomagnetic beads to obtain immunomagnetic beads coated with IL-6 antigen. Step 3: Polyorubin is conjugated with secondary antibody to obtain PE-secondary antibody conjugation product; Step 4: Mix the treated IL-6 hybridoma cells with the PE-secondary antibody conjugation product, then add the biotinylated IL-6 antigen-coated immunomagnetic beads, and wait for droplet formation; and Step 5: Incubate the droplets and perform cell sorting.

2. The method according to claim 1, characterized in that, In step 1, the biotinylation of the IL-6 antigen includes the following steps: mixing the solution containing dissolved Biotin-PEG4-NHS with the IL-6 antigen solution and then incubating.

3. The method according to claim 1, characterized in that, In step 1, after adding biotin solution to the IL-6 antigen solution to be labeled, the incubation temperature is approximately 25°C, and the incubation time is approximately 40-60 minutes. Preferably, the incubation is carried out at 25°C in the dark for 40-60 minutes to obtain the biotinylated IL-6 antigen.

4. The method according to claim 1, characterized in that, In step 2, the step of coating the biotinylated IL-6 antigen onto the immunomagnetic beads includes the following steps: taking magnetic beads containing streptavidin, resuspending them, adding a solution of the biotinylated IL-6 antigen, and incubating them to obtain the biotinylated IL-6 antigen-coated immunomagnetic beads.

5. The method according to claim 1, characterized in that, Step 3, the conjugation of phycoerythrin (PE) with secondary antibody includes the following steps: PE is coupled with a crosslinking agent to obtain PE-crosslinking agent; Then the secondary antibody and reducing agent are mixed and reacted to obtain the reduced secondary antibody; Then, the reduced secondary antibody is coupled with a PE crosslinking agent, and a blocking agent is added for blocking, thus obtaining the desired result.

6. The method according to claim 5, characterized in that, In step 3, when coupling PE and crosslinking agent, the coupling is carried out according to a feeding ratio of PE to crosslinking agent of 2:1 by mass.

7. The method according to claim 6, characterized in that, Step 3, the coupling of the reduced secondary antibody with the PE crosslinking agent, includes the following steps: coupling reaction at a mass ratio of approximately 1:1 between the reduced secondary antibody and the PE crosslinking agent. Preferably, the reduced secondary antibody and the PE crosslinking agent are reacted at 20-25°C for 50-80 min or at 18-20°C for 80-100 min to obtain the desired result.

8. The method according to claim 1, characterized in that, Step 4, the mixing of the treated IL-6 hybridoma cells with the PE-secondary antibody conjugate product, includes the following steps: mixing the PE-secondary antibody conjugate product with immunomagnetic beads coated with IL-6 antigen to form a reagent phase; then mixing the treated IL-6 hybridoma cells with the PE-secondary antibody conjugate product to form a cell phase; and injecting the reagent phase and the cell phase into a microfluidic chip system (e.g., a DG300 microfluidic chip system) to generate droplets. Preferably, in step 4, the desired encapsulation efficiency λ = 0.

3.

9. The method according to claim 1, characterized in that, In step 5, incubating the droplets includes the following steps: placing the droplets at 37°C for about 1 hour to allow the secreted antibodies to bind to the antigens on the magnetic beads within the droplets, which are then labeled by PE-secondary antibody to form a fluorescent signal.

10. The method according to claim 1, characterized in that, In step 5, droplets are observed using a microscope to identify and select those droplets that can emit specific fluorescent signals, and then sort them to obtain single B cells.

11. A method for preparing an antibody, characterized in that, The method comprises culturing mono-B cells obtained by any one of claims 1-10 to induce antibody secretion.

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