CN114716545A - Method for improving yield of monoclonal antibody ascites - Google Patents
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- 206010003445 Ascites Diseases 0.000 title claims abstract description 70
- 238000000034 method Methods 0.000 title claims abstract description 27
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 25
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 21
- 210000004027 cell Anatomy 0.000 claims abstract description 19
- 239000002671 adjuvant Substances 0.000 claims abstract description 13
- 241000699670 Mus sp. Species 0.000 claims abstract description 12
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 11
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 201000000050 myeloid neoplasm Diseases 0.000 claims abstract description 11
- 238000011725 BALB/c mouse Methods 0.000 claims abstract description 8
- 239000006285 cell suspension Substances 0.000 claims abstract description 8
- 150000001335 aliphatic alkanes Chemical class 0.000 claims abstract description 7
- 210000000683 abdominal cavity Anatomy 0.000 claims abstract description 6
- 239000000427 antigen Substances 0.000 claims abstract description 5
- 102000036639 antigens Human genes 0.000 claims abstract description 5
- 108091007433 antigens Proteins 0.000 claims abstract description 5
- 238000007865 diluting Methods 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 claims abstract description 4
- 238000011091 antibody purification Methods 0.000 claims abstract description 4
- 239000004519 grease Substances 0.000 claims abstract description 4
- 230000003053 immunization Effects 0.000 claims abstract description 4
- 238000012216 screening Methods 0.000 claims abstract description 4
- 230000003248 secreting effect Effects 0.000 claims abstract description 4
- 210000000952 spleen Anatomy 0.000 claims abstract description 4
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- 239000002244 precipitate Substances 0.000 claims abstract description 3
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 230000003698 anagen phase Effects 0.000 claims description 3
- 230000004927 fusion Effects 0.000 claims description 3
- 210000004989 spleen cell Anatomy 0.000 claims description 3
- 150000002632 lipids Chemical class 0.000 claims description 2
- 206010070834 Sensitisation Diseases 0.000 abstract description 5
- 230000008313 sensitization Effects 0.000 abstract description 5
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 abstract description 3
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 17
- 239000007924 injection Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
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- Engineering & Computer Science (AREA)
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Abstract
The invention discloses a method for improving the yield of monoclonal antibody ascites, which comprises the following steps: (1) preparation of hybridoma cells: immunizing a female BALB/c white mouse with the required antigen and the age of 6-8 weeks, preparing a cell suspension by taking the spleen of the immunized mouse, fusing the cell suspension with a mouse myeloma cell, and screening to obtain a positive monoclonal cell secreting an antibody, namely obtaining a hybridoma cell; (2) preparing antibody ascites: taking 10-week-old male BALB/c mice, injecting the lipid-lowering alkane into the abdominal cavity, injecting the incomplete adjuvant into the abdominal cavity after 7 days, and injecting the hybridoma cells after 3 days; (3) ascites collection: 5 days after inoculating the hybridoma cells, observing and collecting ascites of the mice; (4) antibody purification: and centrifuging the collected ascites to remove precipitates, filtering to remove grease, diluting the ascites with PBS buffer solution, filtering for the second time, and purifying by a proteinA A column to obtain the purified antibody. The invention obviously improves the yield of ascites by changing the sensitization method.
Description
Technical Field
The invention relates to the technical field of monoclonal antibody, in particular to a method for improving the yield of monoclonal antibody ascites.
Background
Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone and directed against only a specific epitope, and are widely used for basic research, disease diagnosis and disease treatment in the biological field due to their high specificity. In recent years, due to the increase of the amount of monoclonal antibodies and the high production cost of monoclonal antibody preparation, the production amount cannot meet the market demand.
The existing method for preparing the monoclonal antibody has two types of in vivo and in vitro, the in vitro technology realizes the large-scale culture of the hybridoma cell of the monoclonal antibody along with the development of a cell fermentation technology, but the method has high requirements on production equipment and production environment, the equipment is expensive, the running cost of a production line is high, and the method cannot be popularized. The in vivo technology is to adopt BALB/C mouse of inbred line to produce antibody, the mouse is the same strain with myeloma cell and spleen cell mouse for fusion, so BALB/C is preferred to produce ascites, the individual difference of the mouse is small, the antibody content in the ascites is high and can reach 10mg/ml, but the yield of ascites produced by inducing the mouse by using pristane or liquid paraffin and the like is not ideal.
Therefore, it is highly desirable to provide a novel method for increasing the yield of ascites of monoclonal antibodies to solve the above problems.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for improving the yield of the monoclonal antibody ascites, which can obviously improve the yield of the monoclonal antibody ascites.
In order to solve the technical problems, the invention adopts a technical scheme that: the method for improving the yield of the monoclonal antibody ascites comprises the following steps:
(1) preparation of hybridoma cells: immunizing a female BALB/c mouse with 6-8 weeks old by using the required antigen, preparing a cell suspension by taking the spleen of the immunized mouse, fusing the cell suspension with mouse myeloma cells, and screening to obtain positive monoclonal cells secreting antibodies, namely obtaining hybridoma cells;
(2) preparing antibody ascites: taking 10-week-old male BALB/c mice, performing intraperitoneal injection of lipid-lowering alkane, performing intraperitoneal injection of an incomplete adjuvant 7 days later, and performing intraperitoneal injection of the hybridoma in the logarithmic growth phase prepared in the step (1) 3 days later;
(3) ascites collection: 5 days after inoculating the hybridoma cells, observing and collecting ascites of the mice;
(4) antibody purification: and centrifuging the collected ascites to remove precipitates, filtering to remove grease, diluting the ascites with PBS buffer solution, filtering for the second time, and purifying by a proteinA A column to obtain the purified antibody.
In a preferred embodiment of the present invention, in step (1), the mouse myeloma cell is myeloma cell SP 2/0.
In a preferred embodiment of the invention, the fusion ratio of mouse spleen cells to myeloma cells is 2: 1-5: 1.
In a preferred embodiment of the present invention, in step (2), BALB/c mice are injected intraperitoneally with lipid lowering alkane in an amount of 0.3-0.5 ml/mouse, and injected with incomplete adjuvant in an amount of 0.3-0.5 ml/mouse.
In a preferred embodiment of the present invention, in step (2), the amount of hybridoma cells injected is 0.5X 10 per mouse injection6-1.0×106And (4) cells.
In a preferred embodiment of the present invention, in step (3), ascites of the mouse is collected at most 3 times, and the amount of collected ascites is 15 to 20 ml.
In a preferred embodiment of the present invention, in step (3), the collected ascites is centrifuged at 10000rpm to 15000rpm at 4 ℃ for 15 to 30 min.
In a preferred embodiment of the present invention, in step (3), the second filtration is performed using a 0.22um syringe filter.
The invention has the beneficial effects that: the method adopts different sensitization methods to prepare the monoclonal antibody ascites, the process steps are simple and easy to operate, the reagents are easy to obtain in a laboratory, only two reagents are combined to use on the conventional sensitization method, the mice are sensitized in separate time periods, the yield of the ascites is improved, meanwhile, the titer, the stability and the like of the antibody are hardly influenced, and the method is suitable for wide popularization and application.
Drawings
FIG. 1 is a graph showing the comparison of ascites antibody titer in groups of mice injected with the same number of hybridoma cells 11 days after sensitization with pristane in group A, 3 days after injection of incomplete adjuvant (IFA) in group B, and 3 days after injection of incomplete adjuvant (IFA) in group C7 days after injection of pristane in group C.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the present invention more comprehensible to those skilled in the art, and will thus provide a clear and concise definition of the scope of the present invention.
Referring to fig. 1, an embodiment of the present invention includes:
a method for improving the yield of ascites of monoclonal antibodies comprises the following steps:
1) preparation of hybridoma cells: taking spleen after immunizing a female BALB/c mouse with 6-8 weeks old by taking ferritin as immunogen, preparing cell suspension, fusing the cell suspension with myeloma cell SP2/0 (a product sold in the market at present), culturing in HAT selective medium, cloning by adopting a limiting dilution method, screening out a positive monoclonal cell secreting antibody, carrying out expanded culture, and harvesting bone marrow hybridoma;
2) preparing antibody ascites: the method comprises the following three groups: a group of pristane (pristine), B group (IFA) incomplete adjuvant and C group (pristine + IFA) pristane + incomplete adjuvant, wherein each group comprises 20 SPF male 10-week-old mice, 0.5 ml/one of lipid-lowering alkane is injected into the abdominal cavity of the AC group, the incomplete adjuvant is injected into the B group after 7 days of injection of the AC group, 0.3 ml/one of incomplete adjuvant is injected into the C group after one week of injection of lipid-lowering alkane, and 0.8 multiplied by 10 hybridoma cells in logarithmic growth phase are injected into the abdominal cavity of the ABC group simultaneously on the 10 th day6One cell/one;
3) ascites collection: observing after inoculating the hybridoma cells for 5 days and collecting ascites of the mouse according to the expansion degree of the abdomen, wherein in the example, the ascites is collected 7 days after inoculating the hybridoma cells, and the collection frequency is not more than 3 times;
a pristane group: the ascites yield is 95%, ascites is collected for at most 3 times, the ascites yield is between 6 and 13ml, and the average ascites yield is 7 ml.
Incomplete adjuvant group B: the ascites yield is 95%, the ascites is collected for at most 3 times, the ascites yield is between 6 and 12ml, and the average ascites yield is 7.5 ml.
C norphytane + incomplete adjuvant group: the ascites yield is 100%, the ascites is collected for at most 3 times, the ascites yield is between 9 and 18ml, and the average ascites yield is 12 ml.
The specific conditions of the ascites production of the three groups of ABC mice are shown in Table 1, and as can be seen from the Table 1, the ascites production of the group A mice is 100ml, the total ascites production of the group B mice is about 110ml, and the ascites production of the group C mice is 160 ml.
TABLE 1
4) Comparing antibody ascites titer: respectively mixing the collected ascites of ABC mice, centrifuging, taking supernatant filter paper, filtering, and diluting by times: 10000. 20000, 40000, 80000, 160000, 320000, 640000, 1280000, by enzyme-linked immunosorbent assay, using antigen to coat the ELISA plate, adding the diluted ascites to the antigen-coated ELISA plate, and combining with goat anti-mouse IgG enzyme conjugate to determine the antibody titer.
As shown in Table 2, the OD values of the three experiments diluted to 1: 128000 were all 2.1 times greater than that of the negative control, and the test was positive (as shown in FIG. 1). There was no significant difference in the 3 groups of antibody titers, indicating that the three sensitization methods had no significant effect on potency.
TABLE 2
| Ascites dilution multiple | Group A (pristine) | Group B (IFA) | Group C (pristine + IFA) |
| 1∶10000 | 2.385 | 2.697 | 2.446 |
| 1∶20000 | 2.387 | 2.534 | 2.594 |
| 1∶40000 | 2.096 | 1.985 | 2.089 |
| 1∶80000 | 1.876 | 1.707 | 1.809 |
| 1∶160000 | 0.989 | 0.834 | 1.104 |
| 1∶320000 | 0.578 | 0.654 | 0.701 |
| 1∶640000 | 0.332 | 0.399 | 0.404 |
| 1∶1280000 | 0.152 | 0.156 | 0.189 |
| Negative of | 0.045 | 0.047 | 0.025 |
5) Antibody purification:
centrifuging 3 groups of mice ascites at 4 ℃ and 10000rpm for 30min, filtering by using filter paper to remove grease, then buffering and diluting by PBS for 4 times, filtering by using a 0.22um needle filter to remove impurities and bacteria, and finally purifying by using a proteinA column to obtain the antibody.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (8)
1. A method for improving the yield of ascites of monoclonal antibodies, which is characterized by comprising the following steps:
(1) preparation of hybridoma cells: immunizing a female BALB/c mouse with 6-8 weeks old by using the required antigen, preparing a cell suspension by taking the spleen of the immunized mouse, fusing the cell suspension with mouse myeloma cells, and screening to obtain positive monoclonal cells secreting antibodies, namely obtaining hybridoma cells;
(2) preparing antibody ascites: taking 10-week-old male BALB/c mice, injecting a lipid-lowering alkane into the abdominal cavity, injecting an incomplete adjuvant into the abdominal cavity after 7 days, and injecting the hybridoma cells in the logarithmic growth phase prepared in the step (1) after 3 days;
(3) ascites collection: 5 days after inoculating the hybridoma cells, observing and collecting ascites of the mice;
(4) antibody purification: and centrifuging the collected ascites to remove precipitates, filtering to remove grease, diluting the ascites with PBS buffer solution, filtering for the second time, and purifying by a proteinA column to obtain the purified antibody.
2. The method for increasing the yield of ascites using monoclonal antibodies according to claim 1, wherein in step (1), the mouse myeloma cell is myeloma cell SP 2/0.
3. The method for increasing the ascites production of a monoclonal antibody according to claim 1, wherein in the step (1), the fusion ratio of the mouse spleen cells to the myeloma cells is 2: 1-5: 1.
4. The method for increasing the ascites yield of a monoclonal antibody according to claim 1, wherein in the step (2), the BALB/c mice are intraperitoneally injected with the lipid lowering alkane in an amount of 0.3 to 0.5 ml/mouse, and the incomplete adjuvant is injected in an amount of 0.3 to 0.5 ml/mouse.
5. According to claim 1The method for increasing the yield of ascites of monoclonal antibodies, wherein in step (2), the amount of hybridoma cells injected is 0.5X 10 per mouse6—1.0×106And (4) cells.
6. The method for increasing the production of ascites of a monoclonal antibody according to claim 1, wherein in the step (3), ascites of the mouse is collected at a number of times of at most 3 times, and the amount of the collected ascites is 15 to 20 ml.
7. The method for increasing the production of ascites due to monoclonal antibody according to claim 1, wherein in the step (3), the collected ascites is centrifuged at 10000rpm to 15000rpm at 4 ℃ for 15 to 30 min.
8. The method for increasing the yield of ascites using a monoclonal antibody according to claim 1, wherein in the step (3), the secondary filtration is performed using a 0.22um syringe filter.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0380018A2 (en) * | 1989-01-23 | 1990-08-01 | Abbott Laboratories | Rat monoclonal antibodies directed against human antigens and processes for preparation thereof |
| CN101429248A (en) * | 2007-11-09 | 2009-05-13 | 复旦大学 | Anti-double-chain DNA monoclone antibody with antineoplastic activity and preparation method thereof |
| CN101560255A (en) * | 2009-04-22 | 2009-10-21 | 南京医科大学 | Anti-rabies virus monoclonal antibody and preparation method and application |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0380018A2 (en) * | 1989-01-23 | 1990-08-01 | Abbott Laboratories | Rat monoclonal antibodies directed against human antigens and processes for preparation thereof |
| CN101429248A (en) * | 2007-11-09 | 2009-05-13 | 复旦大学 | Anti-double-chain DNA monoclone antibody with antineoplastic activity and preparation method thereof |
| CN101560255A (en) * | 2009-04-22 | 2009-10-21 | 南京医科大学 | Anti-rabies virus monoclonal antibody and preparation method and application |
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