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Culture medium for preparing hybridoma cell clone of monoclone antibody and preparation method

A hybridoma cell and monoclonal antibody technology, which is applied in the field of hybridoma cell cloning medium and preparation for the preparation of monoclonal antibodies, and can solve the problems of large differences in the number of peritoneal macrophages, unfavorable standardization of the cloning process, and individual differences in animals and other issues, to achieve the effect of standardization, easy operation, and elimination of influence

Inactive Publication Date: 2009-08-19
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process needs to be prepared before cell cloning, and the number of peritoneal macrophages prepared by different operators varies greatly, and is affected by individual differences in animals, which is not conducive to the standardization of the cloning process, and also increases the chance of contamination

Method used

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  • Culture medium for preparing hybridoma cell clone of monoclone antibody and preparation method
  • Culture medium for preparing hybridoma cell clone of monoclone antibody and preparation method
  • Culture medium for preparing hybridoma cell clone of monoclone antibody and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] A preparation process for a culture medium for hybridoma cell cloning, comprising the following steps:

[0036] 1) Preparation of 1640 serum-free medium

[0037] 10.4 grams of RPMI1640 (dry powder) (manufactured by GIBCO), 3.574 grams of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, Shanghai Sangong, analytically pure), and 2 grams of NaHCO3 (Beijing Chemical Plant, analytically pure) were deionized 900 ml of water, adjust the pH value to 7.0, then dilute to 1000 ml, filter and sterilize with a 0.22 μm filter membrane, and pack aseptically for later use;

[0038] 2) Preparation of 100 times concentrated double antibody

[0039] Dissolve 1,000,000 units of penicillin and 1,000,000 units of streptomycin in 100ml of deionized water in turn, filter and sterilize with a 0.22μm filter membrane to make a double antibody solution, and store in -20°C for later use;

[0040] 3) Preparation of 1640 complete medium

[0041] Take 79 ml of the 1640 serum-free medium prepared...

Embodiment 2

[0059] The comparison between the culture medium for hybridoma cell cloning and conventional medium containing different proportions of supernatant of subcultured cells

[0060] 1. Prepare the BALB / c peritoneal macrophage suspension in the same manner as step 1 of the test example and add it to a 96-well plate.

[0061] 2. The configuration of the culture medium for hybridoma cell clones in different proportions and added to the 96-well plate The medium configured in step 7 of the embodiment was added to the 96-well culture plate in an amount of 100 μl per well. There are 32 repeat holes.

[0062] 3. Apply the medium configured in step 7 of the embodiment to dilute the SP2 / 0 cells in the logarithmic growth phase to 10 cells / ml, then add to the culture plate containing the same medium, 100 μl per well, place at 37 Cultivate under the condition of 5% CO2. On day 6 and day 9, 100 μl of the same medium per well was replaced, and on day 12, the diameters of cell colonies formed u...

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Abstract

The invention provides a culture medium for hybrid tumor cell clone and for preparing monoclonal antibodies and a preparation method thereof; RPMT 1640 complete media are prepared by conventional method, and then mixed with cell-free culture supernatant of the cells with certain proportions, such as BHK-21, CRFK, Hela and the like; the volume ratio of the complete media to each cell supernatant is (7-1):1. The advantages of the invention are as follows: due to the adoption of the inventive culture medium for hybrid tumor cell clone, the link in which BALB / c mouse peritoneal macrophage is used as feeder cells in the conventional hybrid tumor cells clone process is completely left out, which greatly simplifies clone operations and obviously increases the growth speed of the cloned cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular provides a culture medium for cloning tumor cells for preparing monoclonal antibodies and a preparation method. Background technique [0002] With its high specificity and sensitivity, monoclonal antibodies are widely used in the fields of immunological detection, affinity purification of target proteins and tumor treatment. At present, in the process of monoclonal antibody preparation, 1640 complete medium is applied to positive hybridoma cell wells and cloned by limiting dilution method. Under this condition, the cloned cells grow slowly. In order to promote the rapid proliferation of single cells, the normal BALB / c peritoneal macrophage suspension is usually prepared first before cloning, and then 2×10 4 ~2×10 5 Cells / well are added to the plate to be cloned. This process needs to be prepared before cell cloning, and the number of peritoneal macrophages prepared by different o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/12
Inventor 张茂林段铭关振宏王心蕊陈启军柳增善
Owner JILIN UNIV
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