Magnetic bead coating antigen applied to screening of IL-6 antibody

Abstract

The invention provides a magnetic bead coated antigen applied to screening of an IL-6 antibody, and particularly develops a method for coupling the antigen with magnetic beads through biotin and streptavidin, the coupling method is convenient and efficient, and the coupling has extremely high stability and is not easy to damage in the use process. In the screening process, under the action of a magnetic field, the cells generating the positive antibody can contain cells, linear magnetic beads and brighter fluorescence, so that the cells secreting the positive antibody can be rapidly screened out.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a magnetic bead-coated antigen used for screening IL-6 antibodies. Background Art

[0002] Monoclonal antibodies, abbreviated as MAbs, can recognize specific antigenic determinants and analyze antigenic structures; they can bind to viruses to diagnose or treat viral diseases. In terms of diagnosis, MAbs with good binding properties can be used to establish more sensitive and specific detection methods; in terms of treatment, MAbs are a relatively effective treatment method, but the treatment has high requirements for MAbs. Obtaining MAbs with the same properties as the treatment object is the key to treatment. With the rapid development of technical means, many mature MAb preparation methods have emerged.

[0003] Hybridoma technology has become a mature method for preparing monoclonal antibodies because of its simple operation and low cost. Conventional hybridoma methods are time-consuming, inefficient, and the antibodies obtained are mouse antibodies, which have some limitations. Transgenic mouse technology obtains transgenic mice by knocking out the endogenous heavy and light chain genes of mice and knocking in the heavy and light chain genes encoding human antibodies. Phage display technology forms a gene library by amplifying antibody encoding genes, and then displays the combined library on the surface of phage and obtains antibodies through screening, but the antibody is a random combination of heavy and light chains and its natural conformation cannot be guaranteed. Single B cell antibody preparation technology has the advantages of fully human antibodies, which can be directly used in human clinical research, short cycle, high efficiency, all-natural antibodies, and relatively mature technology. Using cell sorting and sequencing technology, only a few cells are needed to obtain the gene expressing the antibody, and then a large number of antigen-specific monoclonal antibodies can be quickly produced in a short time through in vitro expression. This technology has been successfully used to prepare a large number of human monoclonal antibodies and is widely used in the diagnosis and treatment of diseases.

[0004] In the existing antibody-producing cell screening technology, fluorescently labeled antigens are used. Because the antigen molecular weight is different, the sequence difference is large, and the active groups are different, the fluorescent labeling methods are different. Each labeling needs to explore different labeling methods, and it is not possible to use a unified method to label, which increases the workload. In addition, most of the current cell screening technologies are through fluorescent coupling antigens, and then the cells are labeled, sorted by flow cytometry, and positive B cells that produce corresponding antibodies are obtained. Finally, the antibodies produced by the positive B cells are sequenced, and then cloned and antibody expression is performed. The whole process is very cumbersome, and the later antibody expression and screening workload is huge. Therefore, those skilled in the art are committed to developing efficient antibody-producing cell screening technology to improve work efficiency. Summary of the invention

[0005] The object of the present invention is to provide a magnetic bead-coated antigen for screening antibodies and a preparation method thereof.

[0006] In a first aspect of the present invention, a method for screening cells producing a target antibody is provided, the method comprising the steps of:

[0007] (1) Provide magnetic beads coupled with antigens

[0008] The antigen specifically binds to the target antibody, and the antigen is coupled to the surface of the magnetic bead;

[0009] (2) Preparation of screening system

[0010] The cells to be screened are mixed with magnetic beads coupled with antigens and incubated. If the cells are target antibody-positive cells, the antigen and the target antibody are specifically bound;

[0011] (3) Obtaining target antibody-positive cells

[0012] A magnetic force is applied to the screening system to determine the presence of target antibody-positive cells, and the target antibody-positive cells are obtained by high-throughput cell sorting.

[0013] In another preferred embodiment, in step (2), the screening system further comprises a second antibody; preferably, the second antibody specifically binds to the target antibody; more preferably, the second antibody is labeled with a fluorescent signal.

[0014] In another preferred embodiment, the cell is a hybridoma cell; preferably a mouse hybridoma cell.

[0015] In another preferred embodiment, the second antibody is a goat anti-mouse secondary antibody labeled with fluorescein PE.

[0016] In another preferred embodiment, in step (3), the fluorescent signal is identified by a single-cell micro-droplet sorting system to perform high-throughput cell sorting to obtain the target antibody-positive cells.

[0017] In another preferred embodiment, in step (3), high-throughput cell sorting is performed by fluorescence-activated droplet sorting (FADS) based on droplet microfluidics technology to obtain the target antibody-positive cells.

[0018] In another preferred embodiment, the antigen is IL-6.

[0019] In another preferred embodiment, the step of preparing magnetic beads coupled with antigens comprises:

[0020] (1.1) Providing the antigen labeled with biotin;

[0021] (1.2) providing magnetic beads coupled with streptavidin;

[0022] (1.3) Magnetic beads coated with antigen

[0023] The biotin-labeled antigen and the streptavidin-coupled magnetic beads are mixed and incubated to prepare the antigen-coupled magnetic beads.

[0024] In another preferred embodiment, the cell is a eukaryotic cell; preferably a mammalian cell; more preferably a B cell or a CHO cell.

[0025] In another preferred embodiment, the step of preparing the biotin-labeled antigen comprises:

[0026] (a) Antigen pretreatment: Dialyze the antigen solution with PBS overnight to control the antigen concentration to 1.5-2 mg / mL;

[0027] (b) Biotin-PEG4-NHS was dissolved in DMSO at a concentration of 50 mg / mL;

[0028] (c) adding the required biotin solution to the antigen solution to be labeled at a biotin:antigen molar ratio of 40:1-5, mixing evenly, and incubating at 25° C. in the dark for 40 to 60 minutes;

[0029] (d) removing free Biotin-PEG4-NHS by dialysis to obtain the biotinylated antigen (the antigen labeled with biotin).

[0030] In another preferred embodiment, the method for coating magnetic beads with antigens comprises the steps of:

[0031] (a) Add streptavidin-coupled magnetic beads to PBST buffer to fully resuspend the magnetic beads;

[0032] (b) Magnetic separation, removal of supernatant, repeated washing three times, and resuspending in PBST buffer;

[0033] (c) Add antigen solution according to the ratio of 10 uL magnetic beads to 0.1-0.3 mg biotinylated antigen, and incubate at room temperature for 60-90 min to prepare magnetic beads coated with antigen.

[0034] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The OD560 emission spectrum of IL-6 antigen-coated magnetic beads is shown;

[0036] Figure 2The OD560 emission spectrum peak of IL-6 antigen-coated magnetic beads is shown;

[0037] Figure 3 Droplet images are shown: left – bright field, right – fluorescence;

[0038] Figure 4 The cell sorting and printing results are shown. DETAILED DESCRIPTION

[0039] Fluorescently labeled antigens are used in existing B cell screening technologies. Due to different antigen molecular weights, large sequence differences, and different active groups, fluorescent labeling methods are different. Each labeling requires exploring different labeling methods, and it is not possible to use a unified method to label, which increases the workload. In addition, most of the existing B cell screening technologies are through fluorescent coupling antigens, and then the cells are labeled, sorted using a flow cytometer, and positive B cells that produce corresponding antibodies are obtained. Finally, the antibodies produced by positive B cells are sequenced, and then cloned and antibody expression is performed. The whole process is very cumbersome, and the later antibody expression and screening workload is huge. The inventors have successfully developed a method for coupling antigens to magnetic beads through biotin and streptavidin through in-depth research and a large number of screenings. The coupling method is convenient and efficient, and this coupling has extremely high stability and is not easily destroyed during use. During the screening process, under the action of a magnetic field, cells that produce positive antibodies will contain three elements: cells, linear magnetic beads, and brighter fluorescence, so that cells that secrete positive antibodies can be quickly screened out.

[0040] The main advantages of the present invention are:

[0041] (1) The existing B cell screening technology uses fluorescently labeled antigens, which is not uniform and inefficient. The present invention uses biotin to activate the antigen, then coats the magnetic beads activated by streptavidin, and then uses fluorescently labeled secondary antibodies, which can solve the problem of inconsistent fluorescently labeled antigen methods;

[0042] (2) The existing B cell screening technology requires a lot of sequencing, molecular cloning, and antibody expression work. The present invention uses droplet microfluidics technology to obtain positive B cells, culture the cells, and use ELISA and antigen-antibody affinity detection to quickly obtain the target cell line. The screening work is greatly simplified and the workload is greatly reduced.

[0043] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where detailed conditions are not specified are usually carried out according to conventional conditions such as the conditions described in "Molecular Cloning Laboratory Guide" by Sambrook.J et al. (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0044] Example 1 IL-6 antigen coated magnetic beads

[0045] 1. Antigen Biotinylation

[0046] (1) Sample pretreatment: Dialyze the protein (Pujian Biotech recombinant human IL-6 protein, catalog number: ATMP00015HU) in PBS overnight to remove sodium azide, BSA, glycine, Tris or any other free amino components. The protein concentration was 1.5-2 mg / mL.

[0047] (2) Dissolve Biotin-PEG4-NHS (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S29248) in DMSO to a concentration of 50 mg / mL (about 85 mM) and use it freshly prepared;

[0048] (3) Add the required biotin solution to 100 uL of the protein solution to be labeled at a biotin:IL-6 antigen molar ratio of 40:1, mix well, and incubate at 25°C in the dark for 40 to 60 min;

[0049] (4) Dialysis overnight in a 3.5KD dialysis bag to remove free Biotin-PEG4-NHS to obtain biotinylated IL-6 antigen.

[0050] 2. Magnetic beads coated with antigen

[0051] (1) Place the magnetic bead bottle on a vortex oscillator for 20 seconds to resuspend the magnetic beads (Streptavidin Magnetic Bead Kit PureProteome, purchased from Sigma). Take 70uL of magnetic beads into a 1.5mL centrifuge tube and add 200uL PBST to fully resuspend the magnetic beads.

[0052] (2) Magnetic separation for 1 min, remove the supernatant, repeat washing three times, and resuspend in 100 uL PBST;

[0053] (3) Add antigen solution according to the ratio of 10 uL magnetic beads to 0.1-0.3 mg biotinylated IL-6 antigen, and incubate at room temperature for 60-90 min on a vertical mixer;

[0054] (4) Magnetic separation for 1 min, collect the supernatant to detect the protein concentration to evaluate the coupling rate, add 100 uL of the blocking buffer provided in the kit to the magnetic beads and incubate at room temperature for 30 min;

[0055] (5) Perform magnetic separation for 1 min, remove the supernatant, add 200 μL of the Cell buffer provided with the kit and incubate at room temperature for 30 to 60 min, or at 4°C overnight.

[0056] 3. Verification of magnetic bead coating effect

[0057] (1) Purified antibodies: IL-6 positive control antibody and negative control antibody of the same species were diluted with PBS to two concentrations of 0.5 μg / mL and 1 μg / mL;

[0058] (2) Pipette the coated IL-6 magnetic beads into a 1.5 mL EP tube, add 10 uL to each tube, then add 200 uL PBST, perform magnetic separation for 1 min, remove the supernatant, repeat the wash twice, and finally resuspend with PBS;

[0059] (3) Prepare the reaction system in an EP tube with 10uL magnetic bead solution + 200uL IL-6 antibody / negative control antibody diluted suspension;

[0060] (4) Add 200uL of 0.4ug / mL IL-6 ascites monoclonal antibody, 0.8ug / mL IL-6 ascites monoclonal antibody, and 0.8ug / mL homologous negative control monoclonal antibody to three tubes of magnetic beads respectively;

[0061] (5) The reaction tube was incubated at room temperature with shaking for 1 h, magnetically separated for 1 min, the supernatant was removed, and PBST was washed twice, and the supernatant was removed;

[0062] (6) Dilute the PE-goat anti-mouse fluorescent secondary antibody (purchased from Invitrogen) to a concentration of 5-10ug / mL, add 200uL of fluorescent secondary antibody to each tube of magnetic beads and resuspend, incubate at room temperature in the dark for 1h. Magnetic separation for 1min, remove the supernatant, wash twice with PBST, and resuspend in 200uL PBS;

[0063] Analyze the fluorescence intensity. Pipette 100uL of the mixed magnetic bead suspension into a 96-well plate, duplicate each sample point, and use a microplate reader to detect the emission spectrum of OD560. If the negative control is significantly lower than the positive control, and the fluorescence intensity increases with the increase of the positive control antibody concentration, the antigen-coated magnetic beads have reached the standard. The test results are as follows: Figure 1 and Figure 2 shown.

[0064] 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 positive control monoclonal antibody concentration, indicating that IL-6 coated magnetic beads can be used for the corresponding single B cell screening.

[0065] Example 2 IL-6 hybridoma cell processing and single cell sorting

[0066] 1. IL-6 Hybridoma Cell Treatment

[0067] (1) Mouse IL-6 hybridoma cells were prepared by conventional methods. The cells in negative and positive culture bottles were counted and their viability was detected. 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 the mixed cells were collected in a centrifuge tube.

[0068] (2) Centrifuge (300 x g, 3 min), discard the supernatant, add 1 mL of PBS to resuspend, gently pipette to mix, and repeat the above step once;

[0069] (3) Centrifuge, discard the supernatant, add 1 mL of PBS to resuspend, pass through a 70 μm cell strainer and collect into a new 15 mL centrifuge tube, rinse the original tube with 1 mL of PBS, rinse the strainer with 3 mL of PBS, and collect the filtrate.

[0070] (4) The cell concentration and viability were measured using a cell counter, and the cell viability was 99.2%.

[0071] (5) Centrifuge, discard the supernatant, and resuspend the cells in 1 mL of CSB-14. The cell density is now 21×10 6 cells / mL, the viability was 99.2%, and the cells were placed in an ice box for later use.

[0072] 2. Droplet Generation

[0073] (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.

[0074] (2) Take 66 μL of Beads-antigen, separate magnetically, and wash once with PBST. Resuspend the washed magnetic beads in 74.6 μL CSB-14, and add 8.3 μL of 10X PE-goat anti-mouse secondary antibody to the solution.

[0075] (3) After gently pipetting the cell solution to mix, take 50 μL of the cell solution (the total number of cells in this experiment was 1 M cells) and add 24.7 μL of CSB-14 into a centrifuge tube (the cell concentration at this time was 12.7 M cells / m, and the normal cell concentration was about 13.4 M cells / m). Add 8.3 μL of 10X PE-sheep anti-mouse secondary antibody to the solution, and λ = 0.3 when loading.

[0076] (4) Connect the EP tubes of the reagent phase and the cell phase to the DG300 cell analyzer.

[0077] (5) Set the parameter gas pressure for droplet generation: oil phase: 370 mbar (~13 μL / min), cell phase: 150 mbar (~4 μL / min), reagent phase: 150 mbar (~4 μL / min).

[0078] (6) After the droplet generation is stable (about 1 to 2 min), the generated droplets are collected using a 1.5 mL EP tube (containing 1.2 mL of universal droplet generation oil) and the temperature is controlled at 4 °C.

[0079] (7) Take a 2 μL droplet and inject it into the observation chip, add a small magnet, and observe the signal under a microscope.

[0080] After the droplets were incubated at 37°C (1 h), the signals were observed under a microscope and cells were sorted.

[0081] 3. Cell Sorting

[0082] (1) After the droplet incubation is completed, add a small amount of universal droplet generation oil to the EP tube containing the cells and place it in the droplet tube slot in the flip block.

[0083] (2) Set the configuration parameters, open the real-time camera image and real-time channel image, and adjust the parameters. The parameter adjustments are as follows:

[0084] ①Instrument control module: coaxial lamp: 1000, X motor: 5200, Y motor: 3100;

[0085] ②Channel setting module: FL2 channel, baseline: 150-200, gain: 650;

[0086] ③Laser control module: Laser 1, gain: 1800.

[0087] (3) Set the pre-sorting parameters, sheath liquid oil 1:4, sample: 6, sheath liquid oil 2:4, preparation time 100S, and click to flip. After the preparation is completed, start pre-sorting. When the droplets are stable and the parameters are adjusted, click to pause pre-sorting and start the subsequent formal sorting process (note to enter the printing interface in advance and open the syringe).

[0088] (4) Adjust the sorting parameters and set the parameters as follows:

[0089] ① Air pressure module: sheath fluid oil 1: 3.5, sample: 3.0, sheath fluid oil 2: 7.5;

[0090] ② Sorting force module: voltage: 1600, duration: 1300, delay time: 1500

[0091] ③Other settings: Real-time channel image FL Show Range: 100ms, real-time camera image: delay 1500us.

[0092] (5) Enter the printing interface and start printing cells.

[0093] 4. Cell printing and culture

[0094] (1) 96-well cell culture plates were pre-filled with 50 μl of the corresponding cell culture medium.

[0095] (2) Sheath fluid oils 1 and 3 are both 0.02ED-FC40 sheath fluid oil (droplet sorting sheath fluid oil II), and the sample phase is 0.5ED sheath fluid oil (droplet sorting sheath fluid oil). The printing parameters are adjusted, and the cell culture plate described in (1) is placed for printing.

[0096] (3) After printing is completed, the cell plate is centrifuged (500 g, 5 min, room temperature), and 100-150 ul of the corresponding cell culture medium is added as needed, and the plate is placed in a cell culture incubator (37° C., 5% CO 2 ).

[0097] (4) After culturing for about 3 days, observe the cell number and morphology under a microscope.

[0098] Experimental Results

[0099] 1. Droplet generation results

[0100] ① Take a photo after the droplet is wrapped, and the positive and negative signals are obvious (the droplet picture is as follows Figure 3 Shown: left – bright field, right – fluorescence);

[0101] ②Cell droplets account for 26% of all generated droplets (bright field, counting statistics).

[0102] 2. Cell sorting and printing results

[0103] (1) The actual positive rate was 2.55%, and the theoretical positive rate was 6.4% (calculation of positive cell rate: the number of positive cells accounted for 25% of the total number of cells, and the droplets with cells accounted for 26% of all generated droplets);

[0104] (2) The high signal of the gate was set for printing, and four 96-well cells were printed to test the subsequent cell demulsification culture effect.

[0105] Cell sorting and printing results Figure 4 shown.

[0106] 3. Cell demulsification culture and ELISA detection

[0107] (1) Cell demulsification culture results:

[0108] No contamination was found, and the cell pore rate was observed to be about 35% for each 96-well plate, with an average number of single-cell pores of about 20;

[0109] (2) ELISA test results:

[0110] A total of 84 wells were tested, 66 wells were positive (OD ≥ 2.1 * OD negative), and the positive rate was 78.5%.

[0111] Summary: The screening results of single B cells secreting IL-6 antibodies in the present invention are in line with expectations, single B cell sorting can be achieved quickly, and the positive rate and cell viability meet the requirements.

[0112] Most of the prior art is to fluorescently couple IL-6 antigen, then label the B cells immune to the corresponding antigen, then screen using flow cytometry, and finally do a lot of sequencing and molecular cloning for expression, and further screen the IL-6 target antibody. The whole process is very complicated, especially sequencing, cloning and antibody expression, and the workload is extremely large. In the present invention, IL-6 antigen-coated magnetic beads, combined with fluorescently labeled secondary antibodies, can quickly sort B cells through droplet microfluidics technology, and then the obtained IL-6 positive B cells are directly cultured, and the generated antibodies are subjected to ELISA and IL-6 antigen antibody affinity detection to obtain the target cell strain, which greatly simplifies the whole process and greatly reduces the workload.

[0113] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for screening cells producing a target antibody, characterized in that: The method comprises the steps of: (1) Provide magnetic beads coupled with antigens The antigen specifically binds to the target antibody, and the antigen is coupled to the surface of the magnetic bead; (2) Preparation of screening system The cells to be screened are mixed with magnetic beads coupled with antigens and incubated. If the cells are target antibody-positive cells, the antigen and the target antibody are specifically bound; (3) Obtaining target antibody-positive cells A magnetic force is applied to the screening system to determine the presence of target antibody-positive cells, and the target antibody-positive cells are obtained by high-throughput cell sorting.

2. The method according to claim 1, characterized in that In step (2), the screening system further comprises a second antibody, and the second antibody is labeled with a fluorescent signal; preferably, the second antibody specifically binds to the target antibody.

3. The method according to claim 2, characterized in that The cells are mouse hybridoma cells.

4. The method according to claim 3, characterized in that The second antibody is a goat anti-mouse secondary antibody labeled with fluorescein PE.

5. The method according to claim 2, characterized in that In the step (3), the fluorescent signal is identified by a single-cell micro-droplet sorting system to perform high-throughput cell sorting to obtain the target antibody-positive cells.

6. The method according to claim 1, characterized in that The antigen is IL-6.

7. The method according to claim 1, characterized in that The method for preparing magnetic beads coupled with antigens comprises the steps of: (1.1) Providing the antigen labeled with biotin; (1.2) providing magnetic beads coupled with streptavidin; (1.3) Magnetic beads coated with antigen The biotin-labeled antigen and the streptavidin-coupled magnetic beads are mixed and incubated to prepare the antigen-coupled magnetic beads.

8. The method according to claim 1, characterized in that The cell is a eukaryotic cell; preferably a mammalian cell; more preferably a B cell or a CHO cell.

9. The method according to claim 7, characterized in that The step of preparing the biotin-labeled antigen comprises: (a) Antigen pretreatment: Dialyze the antigen solution with PBS overnight to control the antigen concentration to 1.5-2 mg / mL; (b) Biotin-PEG4-NHS was dissolved in DMSO at a concentration of 50 mg / mL; (c) adding the required biotin solution to the antigen solution to be labeled at a biotin:antigen molar ratio of 40:1-5, mixing evenly, and incubating at 25° C. in the dark for 40 to 60 minutes; (d) removing free Biotin-PEG4-NHS by dialysis to obtain the biotinylated antigen (the antigen labeled with biotin).

10. The method according to claim 7, characterized in that The step of coating the magnetic beads with antigens comprises: (a) Add streptavidin-coupled magnetic beads to PBST buffer to fully resuspend the magnetic beads; (b) Magnetic separation, removal of supernatant, repeated washing three times, and resuspending in PBST buffer; (c) Add antigen solution according to the ratio of 10 uL magnetic beads to 0.1-0.3 mg biotinylated antigen, and incubate at room temperature for 60-90 min to prepare magnetic beads coated with antigen.

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