CN112626063B - Method for preparing hybridoma by enriching mouse plasma cells with CD138+ biomarker and application - Google Patents
Method for preparing hybridoma by enriching mouse plasma cells with CD138+ biomarker and application Download PDFInfo
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Abstract
The invention discloses a method for preparing hybridomas by enriching mouse plasma cells with CD138+ biomarkers. The method comprises the following steps. Step one, enrichment: mouse plasma cells were enriched using plasma cell surface cd138+ biomarkers. Step two, fusion: and collecting the enriched mouse plasma cells and carrying out cell electrofusion on the mouse myeloma cells. The invention utilizes the CD138+ biomarker on the surface of the plasma cells to enrich the plasma cells of the mice, thereby greatly improving the purity of the plasma cells, obtaining a large number of hybridoma cells secreting specific antibodies, and greatly improving the positive rate of the specific antibodies in the screening process. Meanwhile, the cell number of fusion of the initial cells and the mouse myeloma cells is greatly reduced, so that the number of plates is greatly reduced, and the high-throughput screening and the cost of manpower and material resources are reduced.
Description
Technical Field
The invention relates to the technical field of plasma cell and hybridoma cell-monoclonal antibody preparation, in particular to a method for preparing hybridomas by enriching mouse plasma cells with CD138+ biomarkers and application thereof.
Background
Hybridoma-monoclonal antibody technology was invented by Kohler and Milstein in 1975 and obtained from nobel physiological and medical rewards in 1984. The main process of the traditional hybridoma technology is as follows: 1) Immunizing a mouse with a specific antigen; 2) Then taking immune mouse spleen cells and mouse myeloma cells to perform cell fusion, wherein the two cells form new cells after in vitro fusion, which have the capacity of immortalizing myeloma cells and the capacity of secreting antibodies by B cells, and the fused cells are called hybridoma cells; 3) High throughput, rapid screening of large numbers of hybridomas to determine positive hybridoma cells for a specific antigen.
The hybridoma-monoclonal antibody technology platform has unique advantages, and is the most dominant and most commonly used technology platform in the field of new drug antibody research and development at present. The technology platform is mature, stable and high in flux, and the produced new drug antibodies are large in quantity, strong in specificity and high in affinity.
Monoclonal antibodies are widely used in various fields of life sciences including: diagnosis and detection of diseases, treatment of diseases, in particular in the field of tumor treatment and autoimmune diseases, etc. Monoclonal antibody drugs currently in clinical use are mainly produced and obtained from hybridoma technology platforms.
The traditional hybridoma technology still has some key technical bottlenecks, including 1) more than 90% of mixed cells directly separated from spleen and lymph nodes of an immunized mouse are interfering cells which do not secrete specific antibodies, and only plasma cells with the content of about 0.1-6% can generate the specific antibodies and form hybridomas; 2) Because of the large cell quantity used for fusion, a large number of cell culture plates (usually at least 80-100 96 well plates) need to be paved for each fusion, a large amount of consumables and expensive reagents are consumed for high-throughput screening, the working time of research personnel is prolonged, and the working intensity is extremely high; 3) Plasma cells, which have very low levels and truly secrete specific antibodies, are severely disturbed by other unrelated cells, producing large numbers of null hybridomas, greatly increasing the challenges of the development project and even leading to project failure.
Therefore, it is important to reduce the interference of other cells in the original cell mixture and to improve the availability of plasma cells that actually secrete specific antibodies. The plasma cell hybridoma fusion improves the hybridoma positive rate, greatly increases the hybridoma diversity, and has very important significance for ensuring the success of research and development projects. The quality is ensured, the number of the plates is greatly reduced, and the high-throughput screening and the cost of manpower and material resources are reduced.
The traditional mouse hybridoma cell preparation monoclonal antibody is that spleen cells and lymphocytes obtained by taking spleen and lymph node tissues of an immunized mouse through separation and grinding are subjected to cell fusion with mouse myeloma cells to obtain the mouse hybridoma cells capable of unlimited proliferation and antibody secretion. Because of the large number of cells to be cultured and screened after fusion, the process often involves a large number of cell culture plates (typically at least 80-100 96-well plates for conventional hybridomas), a large number of cell culture media, high throughput screening for a variety of screening strategies, a large number of consumables, and expensive reagents. And screening in a short time leads to the working time of research and development personnel, and the working strength is extremely high. Even some projects contain very few plasma cell components (less than 0.5%), fused specific plasma cells are severely disturbed by other mixed tissue cells, a large number of null hybridomas are generated, the challenges of research and development projects are greatly increased, and even the research and development projects fail.
There is a need in the art to address the above problems, prepare monoclonal antibodies of high quality, specificity, diversity, and improve the working efficiency of research and development personnel.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide a method for preparing hybridomas by enriching mouse plasma cells with CD138+ biomarkers. The invention utilizes CD138+ biomarker on plasma cells to carry out CD138+ positive enrichment on mouse immune tissue cells only containing 0.1-6% of plasma cells, thus obtaining 70-80% of high-purity plasma cells. More than 90% of other irrelevant cell components which do not secrete antibodies are removed, so that the interference to subsequent experiments is greatly reduced.
The second technical problem to be solved by the invention is to provide the hybridoma prepared by the method.
The third technical problem to be solved by the invention is to provide the application of the hybridoma in preparing monoclonal antibody medicines.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a method for preparing a hybridoma using cd138+ biomarker enriched mouse plasma cells, comprising the steps of:
(1) Enrichment: enriching mouse plasma cells by using a mouse CD138 positive selection kit;
(2) Fusion: collecting the enriched mouse plasma cells; centrifuging the mouse plasma cell suspension and the mouse myeloma cell suspension respectively, discarding supernatant, washing, and performing cell electrofusion according to the ratio of the mouse plasma cells to the mouse myeloma cells of 1:1-1:3;
(3) Culturing: inoculating the fused cells into a HAT culture medium to be cultured in a carbon dioxide incubator;
(4) Screening: and placing the fused cells in a carbon dioxide incubator for continuous culture, and carrying out high-throughput screening on the specific antibodies after 7-14 days.
As a preferred technical scheme of the invention, the specific enrichment method in the step (1) is as follows:
(a) Preparing buffer solution, filtering, sterilizing and then making the buffer solution be 1X 10 8 Density of cells/mL regulates immune tissue mixed cells;
(b) Adding a mouse FcR blocker and a mouse CD138 positive selection mixture into the cell suspension obtained in the step (a) according to the volume ratio of 50 mu L/mL, uniformly mixing and incubating;
(c) Adding magnetic bead beads into the cell suspension obtained in the step (b) according to the volume ratio of 50 mu L/mL, uniformly mixing and incubating;
(d) Filling the solution obtained in the step (c) with buffer solution to corresponding volumes, uniformly mixing, placing the mixture in a magnet, incubating, and pouring out irrelevant cell suspensions; and (3) washing the tube wall with buffer solution, filling corresponding volumes, uniformly mixing, placing the solution into a magnet for incubation, pouring out irrelevant cell suspension, and collecting cells enriched in the tube wall.
As a preferred embodiment of the present invention, in the step (a), the buffer contains PBS (no Ca 2+ ，Mg 2+ )，2％FBS，1mM EDTA。
As a preferred technical scheme of the invention, in the step (b), the specific method of incubation is to incubate at 2-8 ℃ for 3-10 minutes.
As a preferred technical scheme of the invention, in the step (c), the specific method of incubation is to incubate at 2-8 ℃ for 3-10 minutes.
As a preferred embodiment of the present invention, in the step (d), the incubation is performed for 5 to 10 minutes at room temperature.
As a preferred embodiment of the present invention, the cells of step (2) are electrofused at a rate of 1 to 2X 10 each before the cells are electrofused 8 The cells were treated with 1mL pronase buffer for 5-60 seconds in combination with more than 10 volumes of FBS to interfere with the response.
As a preferable technical scheme of the invention, in the step (3), the concentration of carbon dioxide in the incubator is 5%, the culture temperature is 37 ℃, and the culture time is 1-3 hours.
As a preferable technical scheme of the invention, in the step (4), the concentration of carbon dioxide in the incubator is 5%, and the culture temperature is 37 ℃.
In a second aspect of the present invention, there is also provided a hybridoma produced by the above method.
In a third aspect of the invention, there is also provided the use of said hybridoma in the preparation of a monoclonal antibody medicament.
Compared with the prior art, the invention has the beneficial effects that:
1. by utilizing the CD138+ biomarker, the purity of the plasma cells is improved from 0.1-6% of total cells of immune tissues before enrichment to 70-80% after enrichment, and more than 90% of irrelevant cell components are removed, so that the high-purity plasma cells are obtained, immune materials are not wasted, the interference to subsequent experiments is greatly reduced, a large number of hybridoma cells secreting specific antibodies are finally obtained, the positive rate of the specific antibodies in the screening process is greatly improved, and the positive rate of the hybridomas is improved by 5-10 times.
2. The high-purity plasma cells are fused with mouse myeloma cells, so that the plasma cells with antibody secretion function are fully utilized to the maximum extent, more plasma cells can form 'effective' hybridoma cells secreting antibodies, the positive rate of the hybridoma is improved by 5-10 times, and the specificity and diversity (including antibody subtype diversity and antigen binding epitope diversity) of the hybridoma are obviously improved. Greatly improves the yield and quality of positive hybridomas.
3. The original mixed cells are subjected to plasma cell enrichment, so that effective plasma cells in tissues can be fully utilized, more than 90% of irrelevant cell components are removed, the cell number of fusion of the original cells and mouse myeloma cells is greatly reduced, the number of cell fusion plating plates is further greatly reduced from at least 80-100 96 orifice plates to 10-30 96 orifice plates, the manpower, material resources and time cost of subsequent cell screening are greatly reduced to be more than 70%, and the working efficiency and the working quality of research and development staff are greatly improved.
4. The density of the cell plating of plasma cell fusion is 5-6 times lower than that of the traditional hybridoma fusion plating, and the average of about 1-2 hybridoma cell clusters in each fusion plate hole ensures the positive rate and diversity of real specific antibodies in the screening process.
Drawings
FIG. 1 is a flow chart of enrichment of mouse plasma cells with CD138+ biomarkers in example 1 of the invention.
FIG. 2 is a graph showing the ratio of the Balb/c immunized mice to the plasma cell enrichment of the spleen tissue B lymphocyte population in example 1 of the present invention.
FIG. 3 is a graph showing the ratio of the Balb/c immunized mice to the plasma cell enrichment of B lymphocyte populations of spleen tissue in example 1 of the present invention.
FIG. 4 is a comparative schematic of a conventional hybridoma of example 2 of the present invention and a plasmacytoma-positive hybridoma.
FIG. 5 is a comparative schematic of a conventional hybridoma of example 2 of the present invention and a plasmacytoma-positive hybridoma.
FIG. 6 is a comparative schematic of plate-positive hybridomas of different cell densities in conventional hybridoma fusion and plasma cell fusion according to invention example 4.
Detailed Description
The above-described aspects are further described below in conjunction with specific embodiments. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1: mouse plasma cell enrichment
The kit for enriching mouse plasma cells in the current market mainly comprises the following components: STEMCELL easy Sep.mouse CD138 Positive Selection Kit (Catalog # 18957) and Miltenyi Biotec CD138+ Plasma Cell Isolation Kit (No. 130-092-530), the former was used for plasma cell enrichment in the present invention.
Plasma cell enrichment process material:
| reagent name | Specification of specification | (Storage) |
| Mouse |
1×1mL | 2-8 |
| Dextran RapidSpheres | ||
| 1×1mL | 2-8℃ | |
| Mouse FcR Blocker | 2×0.5mL | 2-8℃ |
See: STEMCELL EasySep.Mouse CD138 Positive Selection Kit (Catalog # 18957)
FIGS. 2 and 3, spleen or spleen and lymph node tissues of different Balb/c mice after immunization were taken, respectively, and the immune tissue-mixed cells were isolated by glass rod milling, and operated as follows:
1. preparation Recommended Medium:
PBS containing (without Ca) 2+ ，Mg 2+ )，Filter sterilization with 2%FBS,1mM EDTA,0.22 μm filter.
2. According to 1X 10 with Recommended Medium 8 Density of cells/mL the immune tissue was adjusted and the mixed cell suspension was added to a clean polystyrene round bottom tube (e.g. STEMCELL catalyst # 38008).
3. For example 1X 10 as described above 8 cells/mL were added Mouse FcR Blocker and Mouse CD138 Positive Selection Cocktail sequentially at 50. Mu.L/mL to the above samples, mixed well, incubated on ice or at 2-8deg.C for 3-10 min, preferably 5 min (see first step "incubation at 2-8deg.C for 5 min" in FIG. 1).
4. Vortex RapidSpheres for at least 30 seconds and add to the sample at 50 μl/mL. Mixing, incubating on ice or at 2-8deg.C for 3-10 min, preferably 5 min (see second step "incubation at 2-8deg.C for 5 min" in FIG. 1).
5. Add Recommended Medium to volume to the corresponding volume (for <4mL sample, add to 5mL; for. Gtoreq.4 mL sample, add to 10 mL).
6. Polystyrene round bottom tubes were inserted into the magnet and incubated at room temperature for 5-10 minutes (see "place tube in magnet, room temperature for 5-10 minutes" in fig. 1).
7. Holding the magnet with the hand and gently holding the polystyrene round bottom tube wall with the thumb (round bottom tube does not remove magnet), gently pouring out the irrelevant cell suspension, repeating steps 5 and 6, washing the tube wall, again inserting the polystyrene round bottom tube into the magnet, incubating at room temperature for 5-10 minutes (see "again placing tube in magnet, incubating at room temperature for 5-10 minutes" in fig. 1), gently pouring out the cell suspension, removing the polystyrene round bottom tube from the magnet, and collecting the cells.
The enriched cells were stained by FACS to detect specific cd138+ cells. The results of the two enrichment steps show that: by utilizing the CD138+ biomarker, the purity of the plasma cells can be improved from 1.8-4.4% of total cells of immune tissues before enrichment to 70-80% after enrichment, and the purity of the plasma cells is greatly improved. The results are shown in FIGS. 2 and 3.
Example 2: mouse plasma cell fusion and screening
Spleen tissue of immunized mice is taken, after the spleen tissue is ground by a glass rod, a part of cells are treated by erythrocyte lysate and then are fused with mouse myeloma cells by traditional hybridomas (in traditional hybridomas, the steps of washing, counting, protease buffer treatment and fusion of mixed cells treated by erythrocyte lysate and mouse myeloma cells can be referred to as follows). A portion of the cells were plasma cell enriched using the Mouse CD138 positive kit (step 1 above). Cell fusion is then performed with the enriched plasma cells and mouse myeloma cells.
Plasma cell fusion process material:
the cell fusion mode is exemplified by electrofusion, and the specific steps are as follows:
1. mouse plasma cell enrichment was performed as in example 1. The plasma cells after enrichment of the polystyrene round bottom tube wall were collected with DMEM basal medium and transferred to a 15mL centrifuge tube, the cells were resuspended and counted, centrifuged at 2000rpm for 3-10 minutes.
2. The supernatant was discarded at 1-2X 10 intervals 8 Cells were treated with 1. 1mL pronase buffer for 10-60s and more than 10 volumes of FBS were added to interfere with the response. Adding DMEM basal medium to a proper volume, not exceeding the volume of the centrifuge tube, filtering by a 70-mesh filter screen to remove the lumps, centrifuging at 2000rpm for 3-10 minutes.
3. The supernatant was discarded, and the cells were washed with DMEM medium, centrifuged at 2000rpm, for 3-10 minutes.
4. The supernatant was discarded, and the cell pellet was resuspended in cell electrofusion buffer and counted. Centrifugation at 2000rpm for 3-10 min.
5. Cell electrofusion buffer was added to adjust cells to 1-8×10 6 cell/mL。
6. Cultured mouse myeloma cells were collected (log phase grown mouse myeloma cells were collected, cell suspensions were collected, counted), centrifuged at 1000rpm for 3-10 minutes.
7. The supernatant was discarded, and the cells were resuspended in DMEM basal medium, centrifuged at 1000rpm for 3-10 minutes.
8. The supernatant was discarded and the mouse myeloma cells were treated with the protease buffer by centrifugation at 1000rpm for 3-10 minutes, referring to the procedure 2 above.
9. The supernatant was discarded, and the cells were washed twice with DMEM basal medium, centrifuged at 1000rpm for 3-10 minutes.
10. The supernatant was discarded, and the cell pellet was resuspended in cell electrofusion buffer and counted. Centrifuge 1000rpm,3-10 minutes.
11. Adding cell electrofusion buffer to adjust cells to 1-10×10 6 cell/mL。
12. Cell electrofusion was performed at a ratio of plasma cells to mouse myeloma cells (1:1-1:3).
13. The fused cells were transferred to a vessel containing fresh HAT medium and the vessel was placed in a carbon dioxide cell incubator (5% co 2 ，37℃)。
14. After 1-3 hours of culture, the fused cells were spread in 96-well cell culture plates and placed in a carbon dioxide cell incubator (5% CO) 2 Culturing is continued at 37 ℃ and high-throughput screening of the specific antibody is carried out after 7-14 days.
Compared with the traditional hybridoma fusion, the plasma cell hybridoma fusion can furthest improve the availability of plasma cells, so that more plasma cells can be fused with mouse myeloma cells to form effective hybridoma cells secreting antibodies, and the positive rate of specific antibodies is improved. The hybridoma fusion of the enriched plasma cells and the mouse myeloma can prepare high-quality and specific antibodies, can remove more than 90% of irrelevant original mixed cell components, fully utilizes the effective plasma cells in tissues, greatly reduces the cell number of the fusion of the initial cells and the mouse myeloma cells, and greatly reduces the cell fusion plating number from at least 80-100 96 orifice plates to 10-30 96 orifice plates. The labor, material and time costs in the screening process are greatly reduced, and the working efficiency and the working quality are greatly improved. The results are shown in fig. 4 and 5, wherein fig. 4 represents item 1, the target antigen protein of item 1 is used for immunizing mice, fig. 5 represents item 2, the target antigen protein of item 2 is used for immunizing different mice, after different antigens are adopted for immunizing different mice in item 1 and item 2, after the immunized mice generate stronger immune response aiming at specific antigens, spleen cells and lymph node cells of the corresponding immunized mice are taken for plasma cell enrichment, and myeloma cells of the mice are electrically fused, so that a traditional hybridoma fusion cooperation control group corresponding to immune tissue cells is set.
Example 3: mouse plasma cell fusion vs traditional hybridoma fusion positive hybridoma antibody epitope binding diversity and subtype diversity comparison
1. Subtype detection of antibodies secreted by positive hybridomas screened in example 2 (corresponding to FIG. 5) showed that positive hybridoma antibodies screened by plasma cell fusion showed more subtype diversity than hybridoma antibodies screened by conventional hybridomas, and not only produced specific antibodies, but also produced antibodies with more diversity. The results are shown in Table 1.
2. The antibodies secreted by the positive hybridomas screened in example 2 (corresponding to fig. 5) were subjected to specific antigen-antibody binding epitope experiments with BMK, and the results showed that the positive hybridoma antibodies screened by plasma cell fusion exhibited more antigen-binding epitope diversity than the hybridoma antibodies screened by conventional hybridomas. The antigen binding epitopes are highly similar, the produced antibodies are relatively single, the antigen binding epitopes are diversified, the produced antibodies also show diversity, the diversity is high, and the antibodies with large difference can provide more opportunities and advantages for developing new medicines of the antibodies. The results are shown in Table 2.
Table 1: traditional hybridoma fusion and plasma cell fusion positive hybridoma subtype diversity comparison
Remarks: "/": the method is free; "+": subtype of positive hybridoma appearance;
table 2: comparison of binding diversity of antigen epitope of positive hybridoma fused with conventional hybridoma fused with vs plasma cell hybridoma
Remarks: "+": positive hybridomas are identical to BMK antigen binding epitopes; "-": positive hybridomas are different from BMK antigen binding epitopes; "NA": others;
example 4: the positive rate of different cell plating densities of traditional hybridoma fusion and mouse plasma cell fusion is compared with that of the traditional hybridoma fusion cell plating density of 8000-20000 cells/hole, 5 blocks of 96 Kong Rong plates, and 52 positive hybridomas are obtained through screening. At the same plate number, in plasma cell fusion, the cell plating density is 8000-10000 cells/hole, 470 positive hybridomas are obtained through screening, the cell plating density is 500-4000 cells/hole, and 449 positive hybridomas are obtained through screening. The results indicate that in plasma cell fusion, the low-density cell fusion plate reflects the positive rate and diversity of the true specific antibodies during the screening process. The results are shown in FIG. 6.
Claims (7)
1. A method for preparing hybridomas by enriching mouse plasma cells with cd138+ biomarkers, which is characterized by comprising the following steps:
(1) Enrichment: enriching mouse plasma cells by direct positive selection of mouse CD138 positive selection kit; the mouse CD138 positive selection kit comprises a mouse CD138 positive selection mixture, a mouse FcR blocker and magnetic beads; the specific method for enriching in the step (1) is as follows:
(a) Preparing buffer solution, filtering, sterilizing and then making the buffer solution be 1X 10 8 Density of cells/mL regulates immune tissue mixed cells;
(b) Adding a mouse FcR blocker and a mouse CD138 positive selection mixture into the cell suspension obtained in the step (a) according to the volume ratio of 50 mu L/mL, uniformly mixing and incubating;
(c) Adding magnetic bead beads according to the volume ratio of 50 mu L/mL in the step (b), uniformly mixing and incubating;
(d) Filling the solution obtained in the step (c) with buffer solution to corresponding volumes, uniformly mixing, placing the mixture in a magnet, incubating, and pouring out irrelevant cell suspensions; washing the tube wall with buffer solution, adding the buffer solution into a magnet, incubating, pouring out irrelevant cell suspension, and collecting the cells enriched in the tube wall;
(2) Fusion: collecting the enriched mouse plasma cells; centrifuging the mouse plasma cell suspension and the mouse myeloma cell suspension respectively, discarding supernatant, washing, and performing cell electrofusion according to the ratio of the mouse plasma cells to the mouse myeloma cells of 1:1-1:3; cell plating density of 500-4000 cells/well; before performing the electrofusion of the cells of step (2), the cells are fused at a rate of 1 to 2X 10 each 8 Treating mouse plasma cells and mouse myeloma cells by adding 1 to mL pronase buffer into the cells for 10-60 seconds, and adding FBS interference reaction with more than 10 times of volume;
(3) Culturing: inoculating the fused cells into a HAT culture medium to be cultured in a carbon dioxide incubator;
(4) Screening: and placing the fused cells in a carbon dioxide incubator for continuous culture, and carrying out high-throughput screening on the specific antibodies after 7-14 days.
2. The method of preparing hybridomas according to claim 1, wherein in step (a), said buffer contains PBS,2% FBS, and 1mM EDTA; the PBS is free of Ca 2+ ，Mg 2+ 。
3. The method for preparing hybridomas according to claim 1, wherein in step (b), the incubation is performed at 2-8 ℃ for 3-10 minutes.
4. The method for preparing hybridomas according to claim 1, wherein in step (c), the incubation is performed at 2-8 ℃ for 3-10 minutes.
5. The method for preparing hybridomas according to claim 1, wherein in step (d), said incubation is performed at room temperature for 5 to 10 minutes.
6. The method according to claim 1, wherein in the step (3), the carbon dioxide concentration in the carbon dioxide incubator is 5%, the culture temperature is 37℃and the culture time is 1 to 3 hours.
7. The method according to claim 1, wherein in the step (4), the carbon dioxide concentration in the carbon dioxide incubator is 5%, and the culture temperature is 37 ℃.
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