Method for efficiently screening hybridoma cells secreting monoclonal antibodies and its application

Abstract

The present invention discloses a method and application of hybridoma cells that efficiently screen for secreting monoclonal antibodies, and relates to the technical field of immunology. The method of the present invention is to obtain Oleyl-PEG-NHS-antigen by coupling Oleyl-PEG-NHS with an antigen, add Oleyl-PEG-NHS-antigen to spleen cells to be screened for culture, discard the supernatant, and wash; then add a fluorescent substance-labeled anti-mouse secondary antibody for incubation, discard the supernatant, and wash; add a basal culture medium, sort the cells to be screened with a flow cytometer, obtain fluorescently labeled cells, that is, a specific plasma cell group, fuse the specific plasma cell group with myeloma cells, and use a single cell picker to pick the stained strong positive hybridoma cells under a dark field, and obtain hybridoma cells that secrete monoclonal antibodies. The method is efficient, fast, and has a high accuracy rate, and can be used for rapid screening of hybridoma cells that secrete monoclonal antibodies, so that it can be used for the efficient preparation of mouse monoclonal antibodies.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a method for efficiently screening hybridoma cells secreting monoclonal antibodies and an application thereof. Background Art

[0002] Monoclonal antibodies refer to highly uniform antibodies produced by a single cell clone that target only a specific antigen epitope. Hybridoma cells have both the characteristics of B lymphocytes that can produce monoclonal antibodies that target only a specific antigenic determinant cluster, and the characteristics of myeloma cells that can proliferate indefinitely. The establishment of hybridoma technology has created a new method for antibody preparation. Monoclonal antibodies are widely used in the fields of protein purification, in vivo tracing, tumor diagnosis and typing, detection of pathogens and antibodies, judgment of cell differentiation stages, treatment of tumors and viral infections, etc., and have broad market space and application value. Compared with rabbit and human monoclonal antibodies, mouse monoclonal antibodies are widely used, mature in technology, low in cost, and can be produced on a large scale for a long time. The selected BALB / c mouse strain is single and has a clear genetic background, so stable and consistent antibodies can be obtained for a long time.

[0003] At present, the traditional method for preparing mouse monoclonal antibodies is mainly hybridoma technology, but the traditional screening method based on limiting dilution and ELISA is very complicated and inefficient for screening positive cells. Subsequently, a patent reported that after the fusion of myeloma cells and spleen cells, the hybridoma cell clones can be first stained with specific cells, and then the specific cell clones can be manually picked to obtain specific hybridoma cell clones. Compared with the traditional method based on limiting dilution and ELISA, this method has greatly improved the screening efficiency, but it still has the following disadvantages: (1) Low fusion cell positive rate: Due to the small number of specific plasma cells in spleen cells and the low proportion of positive cells after the fusion of spleen cells and myeloma cells, the proportion of positive clones grown is small. (2) High cell consumption: Many consumables such as culture plate culture medium are used. Since the proportion of specific positive plasma cells in spleen cells is small, it is necessary to use as many spleen cells as possible and the corresponding proportion of SP2 / 0 cells for fusion, which consumes a lot of resources. (3) Long experimental cycle, easy to lose positive cell lines during operation: The fused hybridoma cell clusters are first observed under a fluorescence microscope, marked, and then manually picked. Since it is manual picking, the cell clones need to be large to be carried out, so it takes a long time (14-28 days), and it is easy to lose positive cell clusters during the picking process. Summary of the invention

[0004] In view of the problems raised by the background technology, the purpose of the present invention is to propose a method for efficiently screening hybridoma cells that secrete monoclonal antibodies, which can quickly and easily screen plasma cells that secrete specific antibodies, and directly fuse plasma cells with myeloma cells to form hybridoma cells that secrete monoclonal antibodies. This method can not only effectively reduce the number of consumed myeloma cells, but also significantly increase the proportion of positive cells with high accuracy. At the same time, it can greatly shorten the screening time of hybridoma cells, reduce the number of cloning times, and avoid the loss of positive cell lines.

[0005] Another object of the present invention is to provide a method for efficiently screening hybridoma cells that secrete monoclonal antibodies for use in the preparation of mouse monoclonal antibodies, which can significantly shorten the monoclonal antibody preparation cycle.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] A method for efficiently screening hybridoma cells that secrete monoclonal antibodies comprises the following steps:

[0008] (1) coupling Oleyl-PEG-NHS with an antigen to obtain Oleyl-PEG-NHS-antigen;

[0009] (2) After the spleen cells were washed, Olei1-PEG-NHS-antigen was added and incubated at 37°C and 5% CO 2 The cells were incubated under the conditions of , then the supernatant was discarded and the spleen cells were washed;

[0010] (3) Add the fluorescent substance-labeled anti-mouse secondary antibody to the spleen cells treated in step (2) and incubate at 37°C and 5% CO 2 The cells were incubated under the conditions of , then the supernatant was discarded and the spleen cells were washed;

[0011] (4) Sorting spleen cells by flow cytometry to select fluorescently labeled cells, which are plasma cells that secrete specific antibodies;

[0012] (5) fusing plasma cells with myeloma cells to obtain hybridoma cells;

[0013] (6) After staining the hybridoma cells according to step (2) and step (3), the hybridoma cells with strong fluorescence are picked using a single cell picker to obtain hybridoma cells that secrete monoclonal antibodies.

[0014] Furthermore, in step (1), the antigen is CDKN2A-P16 protein or PEDV-N protein.

[0015] Furthermore, in the step (2), based on a 96-well plate, the amount of the Oleyl-PEG-NHS-antigen added is 200 μg per well.

[0016] Furthermore, in the step (5), when the plasma cells are fused with the myeloma cells, the ratio of the number of plasma cells to the number of myeloma cells is 1:5 to 1:10.

[0017] Furthermore, in the step (3), the fluorescent substance is any one of fluorescein isothiocyanate, AF647 and allophycocyanin.

[0018] Furthermore, in step (3), the anti-mouse secondary antibody is a goat anti-mouse IgGFc secondary antibody.

[0019] Furthermore, the incubation time in step (2) is 30 to 180 minutes;

[0020] The incubation time in step (3) is 30 to 180 minutes.

[0021] Furthermore, in step (2) and step (3), phosphate buffer or 1640 basal culture medium is used to wash spleen cells respectively.

[0022] The above-mentioned method for efficiently screening hybridoma cells secreting monoclonal antibodies is applied in the preparation of mouse monoclonal antibodies.

[0023] The above technical solution has the following beneficial effects:

[0024] 1. This technical solution uses an amphiphilic substance (Oleyl-PEG-NHS) combined with flow sorting and a microscopic single-cell picker to quickly and easily screen plasma cells that secrete specific antibodies, and directly fuses plasma cells that can secrete specific antibodies with myeloma cells to form hybridoma cells that secrete monoclonal antibodies. This can not only effectively reduce the number of consumed myeloma cells, but also significantly increase the proportion of positive cells with an accuracy rate of more than 95%. The accuracy rate is high, and at the same time, it can greatly shorten the screening time of hybridoma cells, reduce the number of cloning times, and avoid the loss of positive cell lines.

[0025] 2. Improve the positive rate of fusion cells and reduce cell consumption. The amphiphilic substance (Oleyl-PEG-NHS) can be anchored on the phospholipid bilayer of the cell membrane. After coupling with the antigen, the antibody secreted by the plasma cell can bind to the antigen in the conjugate. Then, the fluorescent secondary antibody (i.e., anti-mouse secondary antibody labeled with fluorescent substance) is added to bind to the secreted antibody. Then, the fluorescent cells can be sorted out by flow cytometry, that is, single specific plasma cells. After the specific plasma cells are picked out from the spleen cell group by flow cytometry, only the specific plasma cells are fused with myeloma cells. The positive rate after fusion is greatly improved, and the use of spleen cells is greatly reduced. The use of myeloma cells is greatly reduced year-on-year;

[0026] 3. Shorten the experimental cycle, reduce experimental requirements, and improve the success rate of picking. The microscopic single-cell picker can directly obtain hybridoma single cells or small clones that secrete specific monoclonal antibodies. The cell culture time can be greatly shortened, which can significantly shorten the monoclonal antibody preparation cycle. At the same time, the single-cell picker can achieve fine picking of positive cells, reduce the requirements for experimental operators, and effectively avoid picking errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of a method for efficiently screening hybridoma cells secreting monoclonal antibodies according to an embodiment of the present invention;

[0028] Figure 2 This is a line graph of antibody titer in mouse serum detected by ELISA method in Example 1;

[0029] Figure 3 This is a UV-visible spectrophotometer scanning identification image of the antigen-coupled complex in Example 1;

[0030] Figure 4 A before and after comparison of a single hybridoma cell secreting a specific monoclonal antibody using a single cell picker in Example 1. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

[0032] A method for efficiently screening hybridoma cells that secrete monoclonal antibodies comprises the following steps:

[0033] (1) coupling Oleyl-PEG-NHS with an antigen to obtain Oleyl-PEG-NHS-antigen;

[0034] (2) After the spleen cells were washed, Olei1-PEG-NHS-antigen was added and incubated at 37°C and 5% CO 2 The cells were incubated under the conditions of , then the supernatant was discarded and the spleen cells were washed;

[0035] (3) Add the fluorescent substance-labeled anti-mouse secondary antibody to the spleen cells treated in step (2) and incubate at 37°C and 5% CO 2 The cells were incubated under the conditions of , then the supernatant was discarded and the spleen cells were washed;

[0036] (4) Sorting spleen cells by flow cytometry to select fluorescently labeled cells, which are plasma cells that secrete specific antibodies;

[0037] (5) fusing plasma cells with myeloma cells to obtain hybridoma cells;

[0038] (6) After staining the hybridoma cells according to step (2) and step (3), the hybridoma cells with strong fluorescence are picked using a single cell picker to obtain hybridoma cells that secrete monoclonal antibodies.

[0039] Since the number of specific plasma cells in spleen cells is small, the number of myeloma cells consumed is large, the proportion of positive cells after the fusion of spleen cells and myeloma cells is low, and the proportion of positive clones grown is small. The technical solution uses an amphiphilic substance (Oleyl-PEG-NHS) combined with flow sorting and a microscopic single cell picker to quickly and easily screen plasma cells that secrete specific antibodies, and directly fuses plasma cells that can secrete specific antibodies with myeloma cells to form hybridoma cells that secrete monoclonal antibodies. This can not only effectively reduce the number of consumed myeloma cells, but also significantly increase the proportion of positive cells, with an accuracy rate of more than 95%, and a high accuracy rate. At the same time, it can greatly shorten the screening time of hybridoma cells, reduce the number of cloning times, and avoid the loss of positive cell lines.

[0040] It is worth noting that Oleyl-PEG-NHS is an amphiphilic substance, one end of which is lipophilic and can be anchored on the phospholipid bilayer of the cell membrane and bind to the cell membrane. The PEG polymer at the other end is hydrophilic, and the -COO-NHS connected to its end is a carboxyl group activated by NHS, which can bind to the amino group on the antigen. The conjugated specific antigen conjugate is added to spleen cells. After incubation, one end of the conjugate is connected to the cell membrane. The conjugated antigen captures the antibodies secreted by plasma cells to achieve the positioning of plasma cells and wash away the unbound antigens. The antibody is combined, and an anti-mouse secondary antibody labeled with a fluorescent substance is added for incubation, and the unbound anti-mouse secondary antibody is washed away. The incubated cells are sorted by flow cytometry, and cells containing fluorescence are screened out, namely plasma cells secreting specific antibodies. The plasma cells obtained by flow cytometry are fused with myeloma cells to obtain hybridoma cells, and the hybridoma cells are then stained. The hybridoma cells with strong fluorescence are directly picked out by a single cell picker, namely hybridoma cells secreting monoclonal antibodies. The principle diagram of the method for efficiently screening hybridoma cells secreting monoclonal antibodies in this technical solution is shown in the attached figure. Figure 1 As shown. This technology can effectively screen out specific plasma cells through staining technology and flow cytometer sorting of spleen cells, improve the efficiency and positive rate of subsequent cell fusion, and can screen hybridoma cell lines that can secrete specific monoclonal antibodies within two weeks through a microscopic single-cell picker, which can effectively shorten the time for hybridoma cell lines to secrete specific monoclonal antibodies, and at the same time effectively avoid the impact of errors caused by traditional screening methods and avoid the loss of positive cell lines.

[0041] It is further explained that the method of the present technical solution for screening hybridoma cells secreting monoclonal antibodies has the following advantages and effects:

[0042] 1. Improve the positive rate of fusion cells and reduce cell consumption: Amphiphilic substances (Oleyl-PEG-NHS) can be anchored on the phospholipid bilayer of the cell membrane. After coupling with antigens, the antibodies secreted by plasma cells can bind to the antigens in the conjugate. Then, fluorescent secondary antibodies (i.e., anti-mouse secondary antibodies labeled with fluorescent substances) are added to bind to the secreted antibodies. Then, fluorescent cells can be sorted out by flow cytometry, i.e., single specific plasma cells. After specific plasma cells are picked out from the spleen cell population by flow cytometry, only specific plasma cells are fused with myeloma cells. The positive rate after fusion is greatly improved, and the use of spleen cells is greatly reduced. The use of myeloma cells is greatly reduced year-on-year;

[0043] (2) Shorten the experimental cycle, reduce experimental requirements, and improve the success rate of picking: The microscopic single-cell picker can directly obtain hybridoma single cells or small clones that secrete specific monoclonal antibodies. Since the specific plasma cells are screened at the beginning and then the cells are fused, there is no need to screen the hybridoma cells again, so that the cell culture time can be greatly shortened, which can significantly shorten the monoclonal antibody preparation cycle. At the same time, the single-cell picker can achieve precise picking of positive cells, reduce the requirements for experimental operators, and effectively avoid picking errors.

[0044] Specifically, the present technical solution for obtaining spleen cells comprises the following steps:

[0045] (1) Immunization of BALB / c mice: The antigen and Freund's complete adjuvant should be mixed and emulsified for the first immunization of mice. The antigen and Freund's incomplete adjuvant should be mixed and emulsified for each subsequent immunization. For the sprint immunization, adjuvant-free immunization should be selected.

[0046] (2) ELISA indirect method to evaluate the antibody content in mouse serum (such as Figure 2 shown).

[0047] (3) Take the spleen of the mouse, grind it and sieve it to obtain spleen cells.

[0048] Preferably, the BALB / c mice are 4-6 week old female BALB / c mice. When immunizing mice in the above step (1), the antigen dose is preferably 100 μg / mouse, the volume ratio of antigen to Freund's adjuvant is preferably 1:1, and the antigen dose is preferably 50 μg / mouse during sprint immunization. In step (3), when grinding spleen cells, a 200-mesh cell sieve is selected.

[0049] Further description, it is characterized in that, in the step (1), the antigen is CDKN2A-P16 protein or PEDV-N protein.

[0050] Specifically, in step (1), the method for coupling Oleyl-PEG-NHS to the antigen can be a conventional coupling method, wherein, when the antigen is an incomplete antigen, Oleyl-PEG-NHS is first coupled to a carrier protein and then coupled to the antigen, because an incomplete antigen cannot induce an immune response when it exists alone, that is, it does not have immunogenicity, but when it is cross-linked or combined with a macromolecular protein or a non-antigenic carrier, it can obtain immunogenicity, thereby inducing an immune response; when the antigen is a complete antigen, Oleyl-PEG-NHS can be directly coupled to the antigen.

[0051] Further explanation: in the step (2), based on a 96-well plate, the amount of the Oleyl-PEG-NHS-antigen added is 200 μg per well.

[0052] Specifically, the amount of Oleyl-PEG-NHS-antigen added to each well is 200 μg. Adding 200 μg can achieve the best effect. If too much is added, it will easily cause waste; if too little is added, it will easily lead to insufficient reaction.

[0053] As a further explanation, in the step (5), when the plasma cells are fused with the myeloma cells, the ratio of the number of plasma cells to the number of myeloma cells is 1:5 to 1:10.

[0054] It is worth noting that when the plasma cells are fused with the myeloma cells, when the cell number ratio is 1:5 to 1:10, the fusion efficiency is higher, which is more conducive to the fusion of plasma cells and myeloma cells to form hybridoma cells that secrete monoclonal antibodies, thereby increasing the formation rate of hybridoma cells and facilitating the efficient preparation of mouse monoclonal antibodies in the later stage.

[0055] Further explanation, in step (3), the fluorescent substance is any one of fluorescein isothiocyanate, AF647 and allophycocyanin.

[0056] Fluorescein isothiocyanate, whose full English name is Fluorescein Isothiocyanate, abbreviated as FITC, has the characteristics of high absorption rate, excellent fluorescence quantum yield and good water solubility. It can be used as a fluorescent tracer for antibodies and proteins. The isothiocyanate group of FITC can react with the amino terminus or primary amine of the protein, thereby achieving protein labeling including antibodies and lectins.

[0057] AF647 stands for Alexa Fluor 647, a bright far-red fluorescent dye. Probes with high fluorescence quantum yield and high photostability can detect low-abundance biological structures with high sensitivity. Alexa-fluor647 dye molecules can be attached to proteins at high molar ratios without significant self-quenching, resulting in brighter conjugates and more sensitive detection.

[0058] Allophycocyanin (APC) is a phycobiliprotein isolated and purified from blue-green algae, and is an ultra-sensitive fluorescent dye used for biological detection. Allophycocyanin has a wide absorption spectrum in a wide pH range, and it is relatively easy to select a suitable excitation wavelength to obtain efficient fluorescence emission, and there is a specific fluorescence emission peak when excited; and the absorbance and fluorescence quantum yield are very high, the fluorescence is strong and stable, and the sensitivity is high; at the same time, allophycocyanin is extracted from pure natural marine organisms, has no toxic side effects, does not contain radioactivity, and is very safe to operate and use.

[0059] Further explanation, in the step (3), the anti-mouse secondary antibody is a goat anti-mouse IgGFc secondary antibody, which can avoid false positives.

[0060] Further explanation, the incubation time in step (2) is 30 to 180 minutes;

[0061] The incubation time in step (3) is 30 to 180 minutes.

[0062] It is worth noting that the cell incubation time in step (2) will affect the amount of Oleyl-PEG4-NHS-antigens on the cell surface and the amount of antibodies secreted by plasma cells, both of which will affect the screening effect. If the incubation time is too short, the amount of Oleyl-PEG-NHS-antigens attached to the cell surface will be too small, resulting in insufficient capture of secreted antibodies, which will cause some positive cells to be missed, resulting in false negatives; if the incubation time is too long, the amount of secreted antibodies will be large and bind to non-antibody secreting cells, resulting in false positives.

[0063] The purpose of step (4) is to wait for the fluorescent-labeled anti-mouse secondary antibody to bind to the secreted antibody. If the incubation time is too short, the number of bindings may be small, which may easily lead to false negatives; if the incubation time is too long, it may lead to non-specific binding, resulting in false positive screening.

[0064] Preferably, the incubation time in step (2) is 180 min, and the incubation time in step (3) is 120 min.

[0065] As a further explanation, in step (2) and step (3), phosphate buffer or 1640 basal culture medium is used to wash spleen cells respectively.

[0066] The above-mentioned method for efficiently screening hybridoma cells secreting monoclonal antibodies is applied in the preparation of mouse monoclonal antibodies.

[0067] It is worth noting that the method for efficiently screening hybridoma cells secreting monoclonal antibodies in the present technical solution is used to prepare mouse monoclonal antibodies. Since the present technical solution initially screens specific plasma cells and then performs cell fusion, there is no need to screen hybridoma cells, so that the time for hybridoma cell culture can be greatly shortened, which can significantly shorten the monoclonal antibody preparation cycle.

[0068] The present technical solution is further described below in conjunction with embodiments.

[0069] Example 1

[0070] A method for efficiently screening hybridoma cells that secrete monoclonal antibodies. Before screening hybridoma cells that secrete monoclonal antibodies in this embodiment, it is necessary to first obtain spleen cells by immunizing mice. The method for obtaining spleen cells includes the following steps:

[0071] ① Immunize BALB / c mice. CDKN2A-P16 antigen and Freund's complete adjuvant should be mixed and emulsified for the first immunization of mice. CDKN2A-P16 antigen and Freund's incomplete adjuvant should be mixed and emulsified for each subsequent immunization.

[0072] ② The ELISA indirect method was used to evaluate the antibody content in mouse serum;

[0073] ③Take the spleen of the mouse, grind and sieve it to obtain spleen cells.

[0074] Spleen cells were obtained by the above method, and hybridoma cells secreting monoclonal antibodies were screened by the following method:

[0075] (1) coupling Oleyl-PEG-NHS with the antigen (CDKN2A-P16) to obtain Oleyl-PEG-NHS-antigen, which includes two steps: coupling Oleyl-PEG-NHS with the CDKN2A-P16 protein and identifying the Oleyl-PEG-NHS-CDKN2A-P16 protein. The specific operation method is as follows:

[0076] Conjugation of Oleyl-PEG-NHS to CDKN2A-P16 protein:

[0077] ① Weigh 10 mg of Oleyl-PEG-NHS and dissolve it in 1 mL of 15 mM PBS (pH 7.4) to obtain a 10 mg / mL stock solution;

[0078] ② Add 21.5 mg of CDKN2A-P16 protein into a glass bottle, add 1 mL of 15 mM pH 7.4 PBS to dissolve, and stir on a magnetic stirrer;

[0079] ③ Slowly add the stock solution in ① into the above glass bottle to make the final volume 2.15 mL, stir at room temperature for 3 hours to obtain Oleyl-PEG-NHS-CDKN2A-P16 protein solution;

[0080] ④ Filter and sterilize the Oleyl-PEG-CDKN2A-P16 protein solution obtained in ③ and store it at 4°C;

[0081] Identification of Oleyl-PEG-NHS-CDKN2A-P16 protein: The protein was identified by scanning with a UV-visible spectrophotometer. The antigen coupling effect was determined by observing the shift of the absorption peaks of different substances. Due to the difference in molecular weight between CDKN2A-P16, Oleyl-PEG-NHS-CDKN2A-P16 and Oleyl-PEG-NHS, Figure 3 It can be seen that the maximum absorption peak of Oleyl-PEG4000-NHS is at 260nm, the maximum absorption peak of Oleyl-PEG4000-NHS-antigen is at 276nm, and the maximum absorption peak of antigen is at 280nm. The maximum absorption peaks of different substances are different. Compared with before coupling, the maximum absorption peaks of each substance have shifted, proving that the Oleyl-PEG4000-NHS-antigen complex is successfully coupled;

[0082] (2) The isolated spleen cells were inoculated into a 96-well plate. After three days of culture, 200 μg of Olei1-PEG-NHS-antigen was added to each well and incubated at 37°C and 5% CO 2 Incubate for 180 min under the conditions of , then discard the supernatant, and wash the incubated spleen cells with 1640 basal medium;

[0083] (3) Add FITC-labeled goat anti-mouse IgG Fc to the spleen cells treated in step (2) and incubate at 37°C and 5% CO 2 Incubate for 120 min under the conditions of , then discard the supernatant and wash the spleen cells;

[0084] (4) placing the stained spleen cells obtained in step (3) into a flow cytometer for flow sorting to sort out the fluorescently labeled cells, which are plasma cells secreting specific antibodies;

[0085] (5) The plasma cells obtained in step (4) are fused with myeloma cells to obtain hybridoma cells. The operation method is as follows: the ratio of myeloma cells and plasma cells is controlled to be 1:5, mixed, centrifuged at 1000 rpm for 8 minutes, the supernatant is carefully poured out, and the supernatant is absorbed with sterile filter paper (absorbent paper), three timers (90s, 150s, 4min30s) are prepared, and the bottom of the centrifuge tube is gently tapped to ensure that the cells are dispersed for easy fusion (fingers are flicked, or the centrifuge tube is tapped on the clean bench); 1ml PEG is added to start three timers, and 1ml PEG is evenly added within 60 to 90s (the centrifuge tube needs to be rotated and tapped continuously at this time); then 9ml 1640 basal culture medium is added, and the first 1 to 2ml needs to be added at a speed of 1 minute / ml (before the second timer ends), and the subsequent 7ml needs to be added before the last timer ends (keep the cells in a shaking state); finally, the cells are incubated at 37°C for 10min; centrifuged at 1000 rpm for 5 minutes, and 5ml HAT medium (HAT medium contains 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine per 500 mL of HAT medium), and then diluted to 100 ml, the cell suspension was added to a 96-well plate, 100 ul per well, and the culture plate was placed in a cell culture incubator for culture to obtain CDKN2A-P16 hybridoma cells;

[0086] (6) After staining the hybridoma cells according to step (2) and step (3), a single cell picker is used to pick hybridoma cells with strong fluorescence to obtain hybridoma cells secreting monoclonal antibodies. The operation method is as follows:

[0087] ① After culturing the hybridoma cells for 3 days, add Oleyl-PEG-CDKN2A-P16 and incubate for 180 minutes. Then wash with HAT medium, add FITC-goat anti-mouse IgGFc and incubate for 120 minutes, and then wash with HAT medium;

[0088] ② Picking single hybridoma cells with strong fluorescence: Add the stained hybridoma cells to a 6-well cell culture plate, and use a single cell picker to collect the hybridoma cells with strong fluorescence and culture them in a 96-well cell culture plate;

[0089] ③Results: The fluorescent cells picked out by the single cell picker were cultured, and the supernatant was tested by ELISA. The single hybridoma cells with fluorescent labels picked out were hybridoma cells that could secrete monoclonal antibodies. The accuracy of hybridoma cells that could secrete monoclonal antibodies was calculated according to the following formula, and the accuracy was calculated to be over 95%.

[0090] Accuracy = number of cells that can secrete antibodies / number of cells picked.

[0091] Example 2

[0092] A method for efficiently screening hybridoma cells that secrete monoclonal antibodies. Before screening hybridoma cells that secrete monoclonal antibodies in this embodiment, it is necessary to first obtain spleen cells by immunizing mice. The method for obtaining spleen cells includes the following steps:

[0093] ① Immunize BALB / c mice. PEDV-N protein and Freund's complete adjuvant should be mixed and emulsified for the first immunization of mice. PEDV-N protein and Freund's incomplete adjuvant should be mixed and emulsified for each subsequent immunization.

[0094] ② The ELISA indirect method was used to evaluate the antibody content in mouse serum;

[0095] ③Take the spleen of the mouse, grind and sieve it to obtain spleen cells.

[0096] Spleen cells were obtained by the above method, and hybridoma cells secreting monoclonal antibodies were screened by the following method:

[0097] (1) coupling Oleyl-PEG-NHS with an antigen (PEDV N protein) to obtain Oleyl-PEG-NHS-antigen, which includes two steps: coupling Oleyl-PEG-NHS with PEDV N protein, and identifying Oleyl-PEG-NHS-PEDV N protein. The specific operation method is as follows:

[0098] Conjugation of Oleyl-PEG-NHS to PEDV N protein:

[0099] ① Weigh 10 mg of Oleyl-PEG-NHS and dissolve it in 1 mL of 15 mM PBS (pH 7.4) to obtain a 10 mg / mL stock solution;

[0100] ② Add 21.5 mg of PEDVN protein into a glass bottle, add 1 mL of 15 mM pH 7.4 PBS to dissolve, and place on a magnetic stirrer to stir;

[0101] ③ Slowly add the original solution in ① into the above glass bottle to make the final volume 2.15 mL, stir at room temperature for 3 hours to obtain Oleyl-PEG-NHS-PEDV N solution;

[0102] ④ Filter and sterilize the Oleyl-PEG-PEDV N protein solution obtained in ③ and store it at 4°C;

[0103] Identification of Oleyl-PEG-NHS-PEDV N protein: Scanning and identification by UV-visible spectrophotometer, and judging the antigen coupling effect by observing the shift of the absorption peaks of different substances. Since the molecular weights of PEDV N protein, Oleyl-PEG-NHS-PEDVN protein and Oleyl-PEG-NHS are somewhat different, the maximum absorption peak of Oleyl-PEG4000-NHS is at 260nm, the maximum absorption peak of Oleyl-PEG4000-NHS-antigen is at 276nm, and the maximum absorption peak of antigen is at 280nm. The maximum absorption peaks of different substances are different. Compared with before coupling, the maximum absorption peaks of each substance have shifted, proving that the Oleyl-PEG4000-NHS-antigen complex is successfully coupled;

[0104] (2) The isolated spleen cells were inoculated into a 96-well plate. After three days of culture, 200 μg of Olei1-PEG-NHS-antigen was added to each well and incubated at 37°C and 5% CO 2 Incubate for 180 min under the conditions of , then discard the supernatant, and wash the incubated spleen cells with 1640 basal medium;

[0105] (3) Add AF647-labeled goat anti-mouse IgG Fc to the spleen cells treated in step (2) and incubate at 37°C and 5% CO 2 Incubate for 120 min under the conditions of , then discard the supernatant and wash the spleen cells;

[0106] (4) placing the stained spleen cells obtained in step (3) into a flow cytometer for flow sorting to sort out the fluorescently labeled cells, which are plasma cells secreting specific antibodies;

[0107] (5) The plasma cells obtained in step (4) are fused with myeloma cells to obtain hybridoma cells. The operation method is as follows: the ratio of myeloma cells and plasma cells is controlled to be 1:10, and the mixture is centrifuged at 1000 rpm for 8 minutes. The supernatant is carefully discarded and the supernatant is absorbed with sterile filter paper (absorbent paper). Three timers (90 s, 150 s, 4 min 30 s) are prepared. The bottom of the centrifuge tube is gently tapped to ensure that the cells are dispersed for easy fusion (fingers are used, or the centrifuge tube is tapped on the clean bench); 1 ml of PEG is added to start three timers, and 1 ml of PEG is evenly added within 60 to 90 s (the centrifuge tube needs to be rotated and tapped continuously at this time); then 9 ml of 1640 basal culture medium is added, and the first 1 to 2 ml needs to be added at a rate of 1 min / ml (before the second timer ends), and the next 7 ml needs to be added before the last timer ends (keep the cells in a shaking state); finally, the cells are incubated at 37°C for 10 min; centrifuged at 1000 rpm for 5 minutes, and 5 ml HAT medium, then diluted to 100 ml, the cell suspension was added to a 96-well plate, 100 ul per well, and the culture plate was placed in a cell culture incubator to obtain PEDV N protein hybridoma cells;

[0108] (6) After staining the hybridoma cells according to step (2) and step (3), a single cell picker is used to pick hybridoma cells with strong fluorescence to obtain hybridoma cells secreting monoclonal antibodies. The operation method is as follows:

[0109] ① After culturing the hybridoma cells for 3 days, add Oleyl-PEG-PEDV N and incubate for 180 minutes. Then wash with HAT medium, add FITC-goat anti-mouse IgGFc and incubate for 120 minutes, and then wash with HAT medium;

[0110] ② Picking single hybridoma cells with strong fluorescence: Add the stained hybridoma cells to a 6-well cell culture plate, and use a single cell picker to collect the hybridoma cells with strong fluorescence and culture them in a 96-well cell culture plate;

[0111] ③Results: The fluorescent cells picked out by the single cell picker were cultured, and the supernatant was tested by ELISA. The single hybridoma cells with fluorescent labels picked out were hybridoma cells that can secrete monoclonal antibodies. The accuracy rate of hybridoma cells that can secrete monoclonal antibodies was calculated by the accuracy rate calculation formula in the embodiment, and the accuracy rate reached more than 95%.

[0112] The technical principle of the present invention is described above in conjunction with specific embodiments. These descriptions are only for explaining the principle of the present invention and cannot be interpreted as limiting the scope of protection of the present invention in any way. Based on the explanations herein, those skilled in the art can associate other specific implementations of the present invention without paying creative labor, and these methods will fall within the scope of protection of the present invention.

Claims

1. A method for efficiently screening hybridoma cells secreting monoclonal antibodies, characterized in that: The following steps are involved: (1) coupling Oleyl-PEG-NHS with an antigen to obtain Oleyl-PEG-NHS-antigen; (2) After the spleen cells were washed, Olei1-PEG-NHS-antigen was added and incubated at 37°C and 5% CO2 for 180 min, then the supernatant was discarded and the spleen cells were washed; (3) adding a fluorescently labeled anti-mouse secondary antibody to the spleen cells treated in step (2), incubating for 120 min at 37° C. and 5% CO 2 , then discarding the supernatant and washing the spleen cells; (4) Sorting spleen cells by flow cytometry to select fluorescently labeled cells, which are plasma cells that secrete specific antibodies; (5) fusing the plasma cells with myeloma cells to obtain hybridoma cells, wherein the ratio of the number of plasma cells to the number of myeloma cells is 1:5 to 1:10; (6) After staining the hybridoma cells according to step (2) and step (3), the hybridoma cells with strong fluorescence are picked using a single cell picker to obtain hybridoma cells that secrete monoclonal antibodies; The steps to obtain spleen cells are as follows: (1) Immunize 4-6 week old female BALB / c mice. For the first immunization, the antigen and Freund's complete adjuvant in a volume ratio of 1:1 are mixed and emulsified, and the antigen dose is 100 μg / mouse. For each subsequent immunization, the antigen and Freund's incomplete adjuvant are mixed and emulsified, and the antigen dose is 100 μg / mouse. For the sprint immunization, no adjuvant immunization is selected, and the antigen dose is 50 μg / mouse. (2) ELISA indirect method to assess antibody levels in mouse serum; (3) The spleen of the mouse was taken and grinded through a 200-mesh cell sieve to obtain spleen cells.

2. The method for efficiently screening hybridoma cells secreting monoclonal antibodies according to claim 1, characterized in that: In the step (1), the antigen is CDKN2A-P16 protein or PEDV-N protein.

3. The method for efficiently screening hybridoma cells secreting monoclonal antibodies according to claim 2, characterized in that: In the step (2), based on a 96-well plate, the amount of the Oleyl-PEG-NHS-antigen added is 200 μg per well.

4. The method for efficiently screening hybridoma cells secreting monoclonal antibodies according to claim 1, characterized in that: In the step (3), the fluorescent substance is any one of fluorescein isothiocyanate, AF647 and allophycocyanin.

5. The method for efficiently screening hybridoma cells secreting monoclonal antibodies according to claim 4, characterized in that: In the step (3), the anti-mouse secondary antibody is a goat anti-mouse IgGFc secondary antibody.

6. The method for efficiently screening hybridoma cells secreting monoclonal antibodies according to claim 1, characterized in that: In the step (2) and the step (3), phosphate buffer or 1640 basal medium is used to wash spleen cells respectively.

7. Use of the method for efficiently screening hybridoma cells secreting monoclonal antibodies according to any one of claims 1 to 6 in preparing mouse monoclonal antibodies.

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