CN114350606A - Method for enriching rat plasma cells and establishing plasma cell hybridoma - Google Patents
Method for enriching rat plasma cells and establishing plasma cell hybridoma Download PDFInfo
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- CN114350606A CN114350606A CN202210003945.3A CN202210003945A CN114350606A CN 114350606 A CN114350606 A CN 114350606A CN 202210003945 A CN202210003945 A CN 202210003945A CN 114350606 A CN114350606 A CN 114350606A
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- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
The invention discloses a method for enriching rat plasma cells and establishing plasma cell hybridomas, which comprises the following steps: s1, developing a specific anti-rat CD138 monoclonal antibody by using a hybridoma technology with rat CD138 as a target spot; s2, enriching rat plasma cells by using an anti-rat CD138 antibody, wherein the anti-rat CD138 antibody contains EZ-Link NHS-PEG 4-biotin; s3, treating the mouse myeloma cells and the rat plasma cells, and performing cell electrofusion on the treated mouse myeloma cells and the rat plasma cells; s4, transferring the fused cells to HAT screening medium in CO2Culturing for 1-2 hours in an incubator, then spreading the culture medium in a 96-hole cell culture plate for culturing, and carrying out hybridoma 7-14 days laterAnd (4) screening antibodies. The method provided by the invention has high enrichment degree, improves the rat plasma cell hybridoma fusion technology, improves the hybridoma antibody positive rate by 10-20 times, greatly improves the fusion efficiency and the bioavailability of the rat plasma cells, has low plate laying quantity, and saves the cost of manpower and material resources.
Description
Technical Field
The invention relates to the fields of anti-rat CD138 antibody development, rat plasma cell enrichment application, new rat hybridoma monoclonal antibody drugs and new rat single cell antibody drug research and development, and particularly relates to a method for enriching rat plasma cells and establishing plasma cell hybridomas.
Background
Hybridoma technology was invented by Kohler and Milstein in 1975 and thus acquired a prize in nobel physiology and medicine in 1984. The technology utilizes spleen cells of an immunized mouse to be fused with myeloma cells to generate hybridoma cells which stably secrete antibodies. Hybridoma monoclonal antibodies are widely used in the life science fields such as disease diagnosis, disease treatment, and biological applications due to their high specificity and high affinity. Currently, therapeutic antibodies approved by the U.S. Food and Drug Administration (FDA) are produced primarily by hybridoma technology.
The main process of the traditional hybridoma technology comprises the following steps: 1) animal immunization: immunizing a mouse with a specific antigen and an adjuvant; 2) cell fusion and culture: separating a large number of spleen cells from an immune mouse and directly carrying out cell fusion with mouse myeloma cells to form a large number of hybridoma cells, wherein the hybridoma cells have the capability of stably secreting specific antibodies by plasma cells and the capability of infinitely proliferating the myeloma cells;
3) high-throughput screening: and (3) carrying out high-throughput antibody screening on a large number of hybridoma cells to finally obtain the positive hybridoma cell strain with specific binding.
Plasma cells, as terminally differentiated B lymphocytes, produce large amounts of specific antibodies, and are critical for the body's clearance of pathogen infection and anti-tumor. The core principle of hybridoma technology is to fuse plasma cells secreting antibodies with myeloma cells to produce hybridoma cells that can proliferate indefinitely and secrete antibodies stably. Since the traditional hybridoma technology is the fusion of primary mixed B cells with very low plasma cell content with myeloma cells, a large number of "null hybridomas" are produced. Therefore, the isolation of high-purity plasma cells secreting specific antibodies is of great importance for the development of new hybridoma monoclonal antibody drugs.
The mature plasma cell surface has unique and marked CD138 protein expression. Both pre-B cells and immature B cells express CD138 protein in bone marrow, whereas circulating B cells and peripheral B cells lose CD138 protein, eventually the plasma cells re-express CD138 protein. Thus, the CD138 protein serves as a plasma cell surface specific biomarker in blood cells. Currently, there is no specific anti-rat CD138 monoclonal antibody for rat plasma cell enrichment.
To date, the FDA has approved over 100 therapeutic antibody drugs, with 84% of new antibody drugs coming from hybridoma technology. However, the existing hybridoma technology still has the following key technical bottlenecks: 1. the quantity of mixed B cells of the spleen and the lymph node of the rat is large, but the content of plasma cells which really secrete the antibody is extremely low and only accounts for 0.1-6%; 2. a plurality of unrelated cells are fused with mouse myeloma cells to generate a plurality of 'null hybridoma' cells; 3. large numbers of mixed B cells are required for each fusion, resulting in large numbers of traditional fusion plates (at least 80-100 96-well cell culture plates). Therefore, the high-throughput screening of the antibody has large workload, and extremely high manpower, material resources, time and capital costs; 4. the large number of "null hybridomas" that are generated, severely interfering with plasma cell hybridomas that truly secrete specific antibodies, greatly increases the challenges of new drug antibody screening and specific antibody generation, even leading to eventual failure of the project.
Therefore, it is necessary to develop a method for enriching rat plasma cells and establishing plasma cell hybridomas, which has high enrichment purity, fewer irrelevant hybrid cells and ineffective hybridoma cells, is easy to implement, convenient to operate and low in plating amount.
In order to solve the problems, the invention successfully develops the specific anti-rat CD138 monoclonal antibody by using the hybridoma technology and taking rat CD138 as a target point. The antibody is specifically combined with a rat plasma cell surface CD138+ biomarker, so that the rat plasma cells with higher purity are successfully separated from a large amount of immune mixed B cells for the first time, and the interference of a large amount of unrelated hybrid cells in initial cells is reduced. The invention successfully establishes a high-purity rat plasma cell hybridoma technology for the first time, utilizes the effective plasma cells to the utmost extent, greatly improves the positive rate of hybridoma antibodies, obtains more specific hybridoma antibodies, further greatly enriches the diversity of the hybridoma antibodies, and leaps the high yield to the high-quality antibodies ensure the success of new antibody drug development projects. Meanwhile, the number of fusion plates and high-throughput screening work are greatly reduced, and a large amount of manpower, material resources, time and capital cost are saved.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for enriching rat plasma cells and establishing plasma cell hybridomas, which comprises the following steps:
s1, development of anti-rat CD138 monoclonal antibodies: BALB/C mice are immunized by rat CD138 DNA plasmids, and specific anti-rat CD138 monoclonal antibodies are prepared by a hybridoma technology.
S2, enrichment: enriching rat plasma cells by using an anti-rat CD138 antibody and a biotin positive selection kit, wherein the anti-rat CD138 antibody contains EZ-Link NHS-PEG 4-biotin, and the EZ-Link NHS-PEG 4-biotin is used for marking anti-rat CD138 antibody;
s3, fusion: using a protease buffer to treat mouse myeloma cells, collecting enriched rat plasma cells, carrying out pretreatment, and carrying out cell electrofusion on the treated mouse myeloma cells and the rat plasma cells according to a ratio of 1: 1-1: 3;
s4, culturing and screening: and transferring the fused cells to an HAT screening culture medium, culturing for 1-2 hours in a carbon dioxide incubator, then paving the fused cells in a 96-well cell culture plate, continuing culturing for 7-14 days, and screening the hybridoma antibody after the culture is finished.
Specifically, in S2, the enriching comprises the following steps:
1) preparing rat immune mixed B cells, and preparing an enrichment buffer solution, wherein the enrichment buffer solution is PBS (phosphate buffer solution) which does not contain calcium ions and magnesium ions, and 2% FBS (FBS) and 1mM EDTA are added into the PBS;
2) resuspending rat immune mixed B cells by using a prepared enrichment buffer solution to obtain a cell suspension;
3) adding a rat FcR blocker into the cell suspension, fully and uniformly mixing, adding an anti-rat CD138 antibody, fully and uniformly mixing, and incubating at room temperature for 15 minutes;
4) adding enrichment buffer solution, centrifuging for 5 minutes, discarding supernatant, and adjusting to initial volume;
5) adding Biotin Selection Cocktail, fully and uniformly mixing, and incubating for 10-20 minutes at room temperature;
6) adding RapidSpheres magnetic beads, and incubating for 5-15 minutes at room temperature;
7) using an enrichment buffer solution to fix the volume of a sample, fully and uniformly mixing the sample, inserting the sample into a magnet, standing the sample at room temperature for 2-8 minutes, and pouring out irrelevant cell sap;
8) and 7) repeating the step 7), and finally collecting the plasma cells enriched on the tube wall.
Specifically, the addition amount of the rat FcR blocker is 50-200 mu L of rat FcR blocker added into each milliliter of cell suspension.
Specifically, the addition amount of the EZ-Link NHS-PEG 4-biotin is 20-100 mu L of EZ-Link NHS-PEG 4-biotin added into each ml of cell suspension.
Specifically, the amount of the Biotin Selection Cocktail added is 100. mu.L of the Biotin Selection Cocktail per ml of the cell suspension.
Specifically, the addition amount of the RapidSpheres magnetic beads is 50 μ L of RapidSpheres magnetic beads per ml of cell suspension.
Specifically, the volume fixing of the sample by using the enrichment buffer solution refers to that the volume is fixed to 5mL when the sample volume is less than 1 mL; when the sample amount exceeds 1mL, the volume is adjusted to 10 mL.
Specifically, in S3, the treatment of mouse myeloma cells with a protease buffer is performed every 1X 108And (3) adding 1mL of pronase buffer into the cell to process the mouse myeloma cells for 10-30 s, and adding 10 times of volume of fetal calf serum to stop the reaction, wherein the mouse myeloma cells are the mouse myeloma cells in the logarithmic growth phase.
Specifically, in S3, the pretreatment is performed by resuspending rat plasma cells in a DMEM basal medium and then centrifuging the rat plasma cells at 2000rpm for 5 minutes.
Specifically, in S4, the carbon dioxide incubator is set to a culture condition of 37 ℃ and CO2The concentration was 5%.
The invention also provides the rat plasma cell hybridoma prepared by the method.
The invention also provides application of the rat plasma cell hybridoma in research and development of monoclonal antibody medicaments.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, an EZ-Link NHS-PEG 4-biotin labeled anti-rat CD138 antibody is used, a specific anti-rat CD138 monoclonal antibody is developed for enriching rat plasma cells for the first time, the enrichment purity of the rat plasma cells is greatly improved, the purity of the rat plasma cells is improved to 54% -66% from 5.6% -6.3% with a very small amount, the purity of the plasma cells is improved by nearly 10 times, meanwhile, a large amount of irrelevant hybrid cells are removed, and the interference of 'ineffective hybridomas' is greatly reduced.
2. The invention carries out cell fusion by utilizing high-purity rat plasma cells, makes full use of the plasma cells secreting antibodies in the lymph and spleen cells of rats to the utmost extent, enables the plasma cells to form 'effective hybridoma cells' as much as possible, obtains a large number of positive hybridomas, improves the positive rate of the hybridomas by 10-20 times, further improves the diversity of the hybridoma antibodies, generates high-yield and high-quality hybridoma antibodies, and provides more possibility for the successful research and development of hybridoma antibody medicaments.
3. The rat plasma cell enrichment method can remove most irrelevant hybrid cells, so that the formation of ineffective hybridomas is reduced, the number of fused cells is reduced, the plate laying number of the fused cells is greatly reduced, the traditional method needs about 80-100 96 pore plates, the plate laying number of the 96 pore plates can be reduced to 10-30 by the method, the downstream high-throughput antibody screening work is greatly reduced, and a large amount of labor and material cost is saved.
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FIG. 1 is a flow chart of the process for enriching rat plasma cells in the second embodiment of the present invention;
FIG. 2 is a graph showing the ratio of plasma cells of B lymphocyte population before and after enrichment in lymph and spleen tissue of SD rat according to item one of the second embodiment of the present invention;
FIG. 3 is a graph showing the ratio of plasma cells of B lymphocyte population before and after enrichment in lymph and spleen tissue of SD rat according to item two of the present invention;
FIG. 4 is a comparison of the conventional hybridoma and rat plasma cell hybridoma antibody screening of item one in the third embodiment of the present invention;
FIG. 5 is a comparison of the conventional hybridoma and rat plasma cell hybridoma screening of item two in example three of the present invention;
FIG. 6 is a comparison of the conventional hybridoma and rat plasma cell hybridoma screening for item three of example three of the present invention;
FIG. 7 is a graph showing the comparison of the competitive effect of the conventional hybridoma antibody of item three of the third example of the present invention with that of the rat plasma cell hybridoma antibody.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Materials, instruments, reagents and the like used in the following examples are commercially available unless otherwise specified. The technical means used in the examples are conventional means well known to those skilled in the art, unless otherwise specified.
EXAMPLE-preparation of specific anti-rat CD138 monoclonal antibody
In a specific embodiment, a rat CD138 is taken as a target, and a hybridoma technology is adopted to prepare a specific anti-rat CD138 monoclonal antibody. In blood cells, CD138 serves as a plasma cell specific biomarker. The CD138 antibody is specifically combined with the CD138 protein on the surface of the plasma cell, the plasma cell secreting the antibody can be separated from a large number of lymph and spleen cells, and the plasma cell is subjected to hybridoma fusion, so that the positive rate and diversity of the hybridoma antibody are greatly improved. However, rats are an important platform for hybridoma antibody technology, and no specific anti-rat CD138 antibody is used for enriching high-purity rat plasma cells at present, so that the traditional rat hybridoma technology can only use low-purity plasma cells for fusion to generate a large amount of 'ineffective hybridomas', the positive rate and diversity of rat hybridoma antibodies are seriously influenced, and even the failure of antibody new drug development is caused. Therefore, the development of the specific anti-rat CD138 antibody has very important significance for the development of rat hybridoma monoclonal antibody drugs.
The preparation process of the anti-rat CD138 antibody comprises the following materials:
the experimental steps for preparing the anti-rat CD138 antibody are as follows:
1) animal immunization and antibody serum titer detection: 4 BALB/C mice were immunized with rat CD138 DNA plasmid (pCAGGS-rCD 138) at the immunization dose: 20-400 mu g/mouse, and the immune approach is as follows: intramuscular, subcutaneous and tail vein kinetic injections, administered once every two weeks.
2) According to the result of the antibody titer of the mouse serum, a BALB/C mouse with higher antibody titer is selected for hybridoma fusion.
3) Lymph nodes and spleen were isolated from immunized mice and ground into a cell homogenate.
4) B lymphocytes were processed according to the standard cell electrofusion procedure of this experiment and fused with mouse myeloma cells.
5) Hybridoma cells were cultured in HAT screening medium and placed in a 5% CO2 cell incubator at 37 ℃.
6) After 7-14 days of cell culture, positive hybridoma antibodies were identified by ELISA and FACS methods.
7) And selecting positive hybridoma cells, carrying out subcloning, culturing for 7-14 days, taking cell supernatant, carrying out ELISA and FACS detection, and finally screening out hybridoma monoclonal cells capable of stably secreting antibodies.
8) Culturing the positive hybridoma monoclonal cells for 7-14 days, harvesting supernatant, purifying antibody affinity by Protein A, and verifying antibody specificity by ELISA and FACS methods.
9) The purified anti-rat CD138 monoclonal antibody was biotinylated according to the Pierce-21329 biotin labeling kit procedure, and then the antibody specificity was verified by ELISA.
A mouse CD138 is taken as a target spot to immunize a BALB/C mouse, and a specific anti-mouse CD138 hybridoma monoclonal antibody is developed. The specific binding of purified anti-rat CD138 antibody to rat CD138 protein and rat CD138 stable cell lines was verified at the protein and cell levels, respectively. After the anti-rat CD138 antibody is labeled by biotin, the specific binding with rat CD138 protein is identified by ELISA, and the result shows that the anti-rat CD138 antibody has the characteristics of high specificity and strong binding force.
EXAMPLE two enrichment of rat plasma cells
In a specific embodiment, the biotin-labeled anti-rat CD138 antibody of example one is used for enrichment of rat plasma cells. The biotin-labeled anti-rat CD138 antibody is specifically combined with rat plasma cells secreting the antibody, and is combined with streptavidin by high affinity, and finally, the rat plasma cells are enriched by a magnetic bead adsorption method. The invention relates to a kit for enriching rat plasma cells, which comprises the following components: STEMCELL, EasySepTM Biotin Positive Selection Kit II(Catalog#17683)。
The rat plasma cell enrichment process materials are as follows:
the experimental steps for enrichment of rat plasma cells are as follows:
1) plasma cell enrichment buffer was prepared by adding 2% FBS and 1mM EDTA to calcium magnesium ion free PBS and filter sterilized with a 0.22 μm filter.
2) Spleen and lymph node tissues were isolated from immunized rats, then ground into cell homogenate with a glass grinder, filtered through a 70 mesh screen to remove large cell clumps and impurities, mixed well and counted, centrifuged at 2000rpm, and the supernatant was discarded for use.
3) Resuspending the immune-mixed B cells with plasma cell enrichment buffer to adjust the cell density to 1X 108cells/mL, mixed well and then transferred to a polystyrene round bottom tube.
4) Adding 50-200 mu L/mL Rat FcR Blocker into the cell suspension, and fully and uniformly mixing. And then adding 20-100 ug/mL biotin-labeled anti-mouse CD138 antibody, fully mixing uniformly to avoid bubbles, and incubating at room temperature for 10-20 min, preferably 15 min.
5) A volume of plasma cell enrichment buffer was added, centrifuged at 2000rpm for 5 minutes, the supernatant discarded and adjusted to the initial volume.
6) Adding 100 mu L/mL Biotin Selection Cocktail, mixing well to avoid bubbles, and incubating for 10-20 min, preferably 15 min at room temperature.
7) The RapidSpheres 30s were vortexed thoroughly to evenly distribute the particles. Then, 50. mu.L/mL of magnetic beads are added, mixed well, and incubated at room temperature for 5 to 15 minutes, preferably 10 minutes.
8) Supplementing the plasma cell enrichment buffer solution, and fixing the final volume of the sample (the sample is less than 1mL, and the fixed volume is 5 mL; the sample is more than or equal to 1mL, the volume is determined to be 10mL), and the mixture is fully mixed. The sample tube is then inserted into the magnet, allowed to stand at room temperature for 2-8 minutes, preferably 5 minutes, and the cell supernatant is decanted.
9) Step 8) is repeated once to remove non-specifically bound hybrid cells. Cell counts before and after enrichment were finally collected and plasma cell purity was checked by FACS method.
CD138+ rat plasma cells were enriched from a large number of immune tissue cells using the biotin-labeled anti-rat CD138 antibody of example one, and then the purity of CD138+ plasma cells before and after enrichment was examined by FACS method. As shown in FIGS. 2 to 3, the plasma cell enrichment effect of rats was shown for two items. The result shows that the rat plasma cell enrichment method provided by the invention can separate high-purity rat CD138+ plasma cells, so that the purity of the rat plasma cells is improved from 5.6-6.3% to 54-65.9%, and the purity of the rat plasma cells is greatly improved.
EXAMPLE three rat plasma cell fusions and screens
In one embodiment, spleen and lymph node tissues are isolated from immunized SD rats and ground to a cell homogenate using a glass grinder. Dividing cells into two parts, wherein one part of cells is used for traditional hybridoma fusion, red blood cells are removed by treatment of red blood cell lysate, and then the cells are fused with mouse myeloma cells; another part of the cells was enriched for rat plasma cells according to the plasma cell enrichment method of example two, and finally the enriched rat plasma cells were fused with mouse myeloma cells.
The rat plasma cell fusion and screening steps are as follows:
1) rat plasma cells were isolated according to the enrichment method in example two. Resuspend rat plasma cells in DMEM basal medium and transfer to 15ml centrifuge tube, mix well and count, 2000rpm, centrifuge for 5 minutes, ready for use.
2) Collecting mouse myeloma cells in logarithmic phase, collecting cell suspension, mixing well and counting, centrifuging at 1000rpm for 5 min, and discarding supernatant.
3) At 1 × 108Treating mouse myeloma cells for 10-30 s by using cell/mL pronase buffer, adding a certain volume of fetal calf serum to stop enzyme reaction, adjusting the volume to a specific volume by using a DMEM basic culture medium, removing impurity blocks by using a 70-mesh screen, centrifuging at 1000rpm for 5 minutes, and discarding supernatant for later use.
4) Resuspend mouse myeloma cells in DMEM basal medium, 1000rpm, centrifuge for 5 minutes, discard the supernatant, and use.
5) Resuspending rat plasma cells and mouse myeloma cells with electrofusion buffer, mixing well and counting, centrifuging for 5 min, and discarding supernatant.
6) The rat plasma cells and the mouse myeloma cells are subjected to cell electrofusion experiments according to the cell density ratio of 1: 1-1: 3.
7) The fused cells were transferred to fresh HAT medium and left at 37 ℃ with 5% CO2Culturing in a cell culture box.
8) After culturing for 1-2 hours, the fused cells were plated on a 96-well plate, and then placed at 37 ℃ with 5% CO2Culturing in an incubator, and screening hybridoma antibodies 7-14 days later.
Rat plasma cell hybridoma fusion takes rat plasma cells as fusion materials, and utilizes plasma cells secreting antibodies to the maximum extent to generate a large amount of 'effective hybridomas', so that the positive rate of the hybridomas is greatly improved. As shown in FIGS. 4 to 6, the screening comparison of the traditional hybridoma and the rat plasma cell hybridoma antibodies of the three experimental projects shows that the fusion positive rate of the rat plasma cell hybridoma is improved by 10 to 20 times. Meanwhile, most irrelevant hybrid cell components are removed, the number of fusion plates is greatly reduced, the number of the fusion plates is reduced from 80-100 96 traditional well plates to 10-30 96 traditional well plates, the labor cost, the material cost and the time cost in cell culture and high-throughput antibody screening work are greatly reduced, and the work efficiency and the work quality of research and development personnel are greatly improved.
EXAMPLE three comparison of competitive effects of antibodies generated by fusion of rat plasma cells and conventional hybridomas provided by the present invention
Based on item three in example three, the hybridoma antibody and BMK compete for specific binding of the receptor and the ligand, respectively, and the comparison result of the antibody competition effect is shown in fig. 7, it can be seen that the positive hybridoma antibody generated by rat plasma cell fusion has a higher competition effect, can effectively block the binding of the receptor and the ligand, and has a competitive inhibition rate of more than 60%, which is better than the blocking effect of BMK1 (43%). While the traditional hybridoma generates hybridoma antibody, the competitive inhibition rate is 10-20%. Therefore, the rat plasma cell fusion generates more functional hybridoma antibodies, and provides more opportunities and advantages for research and development of new antibodies and drugs.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (12)
1. A method for enriching rat plasma cells and establishing plasma cell hybridomas is characterized by comprising the following steps:
s1, development of anti-rat CD138 monoclonal antibodies: rat CD138 is taken as a target spot, and a hybridoma technology is adopted to prepare a specific anti-rat CD138 monoclonal antibody.
S2, enrichment: enriching rat plasma cells by using an anti-rat CD138 antibody, wherein the anti-rat CD138 antibody comprises EZ-Link NHS-PEG 4-biotin, and the EZ-Link NHS-PEG 4-biotin is used for marking the anti-rat CD138 antibody;
s3, fusion: using a protease buffer to treat mouse myeloma cells, collecting enriched rat plasma cells, carrying out pretreatment, and carrying out cell electrofusion on the treated mouse myeloma cells and the rat plasma cells according to a ratio of 1: 1-1: 3;
s4, culturing and screening: and transferring the fused cells to an HAT screening culture medium, culturing for 1-2 hours in a carbon dioxide incubator, then paving the fused cells in a 96-well cell culture plate for continuous culture, and screening hybridoma antibodies 7-14 days later.
2. The method for enriching rat plasma cells and establishing plasma cell hybridomas according to claim 1, characterized in that in S2, said enrichment comprises the following steps:
1) preparing rat immune mixed B cells, and preparing an enrichment buffer solution, wherein the enrichment buffer solution is PBS (phosphate buffer solution) which does not contain calcium ions and magnesium ions, and 2% FBS (FBS) and 1mM EDTA are added into the PBS;
2) resuspending rat immune mixed B cells by using a prepared enrichment buffer solution to obtain a cell suspension;
3) adding a rat FcR blocker into the cell suspension, fully and uniformly mixing, adding a biotin-labeled anti-rat CD138 antibody, fully and uniformly mixing, and incubating at room temperature for 10-20 minutes;
4) adding enrichment buffer solution, centrifuging for 5 minutes, discarding supernatant, and adjusting to initial volume;
5) adding Biotin Selection Cocktail, mixing, and incubating at room temperature for 10-20 min;
6) adding RapidSpheres magnetic beads, and incubating for 5-15 minutes at room temperature;
7) using enrichment buffer solution to fix the volume of the sample, fully and uniformly mixing the sample, inserting the sample into a magnet, standing the sample for 2 to 8 minutes at room temperature, and pouring out cell supernatant;
8) repeating the step 7), and finally collecting the enriched rat plasma cells.
3. The method for enriching rat plasma cells and establishing plasma cell hybridomas according to claim 2, wherein said rat FcR blocker is added in an amount of 50 to 200 μ L rat FcR blocker per ml cell suspension.
4. The method for enriching rat plasma cells and establishing plasma cell hybridomas as claimed in claim 1, wherein the EZ-Link NHS-PEG 4-biotin is added in an amount of 20-100 μ L EZ-Link NHS-PEG 4-biotin per ml cell suspension.
5. The method for enriching rat plasma cells and establishing plasma cell hybridomas according to claim 2, wherein the amount of the Biotin Selection Cocktail added is 100 μ L of Biotin Selection Cocktail per ml of cell suspension.
6. The method for enriching rat plasma cells and establishing plasma cell hybridomas according to claim 2, wherein said RapidSpheres magnetic beads are added in an amount of 50 μ L per ml of cell suspension.
7. The method for enriching rat plasma cells and establishing plasma cell hybridomas according to claim 2, characterized in that the volume fixing of the sample by using the enrichment buffer solution is that when the sample volume is less than 1mL, the volume fixing is performed to 5 mL; when the sample amount exceeds 1mL, the volume is adjusted to 10 mL.
8. The method for enriching rat plasma cells and establishing plasma cell hybridomas as claimed in claim 1, wherein said treating mouse myeloma cells with protease buffer in S3 is performed every 1X 108And (3) adding 1mL of pronase buffer into the cell to process the mouse myeloma cells for 10-30 s, and adding 10 times of volume of fetal calf serum to stop the enzyme reaction, wherein the mouse myeloma cells are the mouse myeloma cells in the logarithmic growth phase.
9. The method for enriching rat plasma cells and establishing plasma cell hybridomas according to claim 1, wherein the pretreatment in S3 is performed by resuspending rat plasma cells in DMEM basal medium and centrifuging the rat plasma cells at 2000rpm for 5 minutes.
10. The method for enriching rat plasma cells and establishing plasma cell hybridomas according to claim 1, wherein the culture conditions of said carbon dioxide incubator are set at 37 ℃ and CO 42The concentration was 5%.
11. A rat plasma cell hybridoma prepared by the method of any one of claims 1-10.
12. Use of the rat plasma cell hybridoma of claim 11 in the development of new monoclonal antibody drugs.
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