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Method for improving effective cell fusion
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A cell and fusion cell technology, applied in the direction of fusion cells, cells modified by introducing foreign genetic material, etc., can solve the problem of not giving
Inactive Publication Date: 2017-08-01
SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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Although this patented method is helpful for immunization to obtain a good effect, its use is mainly as an adjuvant, and there is no technical solution for how to increase the number of effective cell lines obtained by screening after fusion. Therefore, it is necessary to improve the cell fusion method
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Embodiment 1
[0051]Example 1: Determination of the lowest dose when mice produce an obvious immune response to LPS
[0052] 1) Take 6 centrifuge tubes, dilute LPS with PBS according to 0mg / ml, 0.5mg / ml, 1mg / ml, 2mg / ml, 3mg / ml, 4mg / ml, 1ml for each tube;
[0053] 2) Filter and sterilize the upper liquid through a 0.22 μm filter, and collect 0.8 ml respectively;
[0054] 3) Take 18 BALB / c female mice with a body weight of 25 ± 10g, divide them into 6 groups at random, inject 0.1ml (0.2ml) of the above-mentioned dilutions into each group, and conduct 3 parallel experiments for each dilution;
[0055] 4) Observe the situation of the mice, and record the lowest dose of LPS used when the mice produce an obvious immune response
[0056] The result is as follows
[0057]
Embodiment 2
[0058] Example 2: Preparation of monoclonal antibody according to the dose of 0.5 mg / ml co-immunostimulated mice determined in Example 1
[0059] 1) First immunization: Dissolve 50 μg of antigen in 150 μl PBS, then add 150 μl Freund’s complete adjuvant, and stir mechanically for 10 minutes to make the mixture milky, take a drop in water, do not disperse, and meet the emulsification requirement;
[0060] 2) Second immunization: Dissolve 25 μg of antigen in 150 μl of PBS, add 150 μl of Freund’s incomplete adjuvant, and stir mechanically for 10 minutes to make the mixture milky, take one drop in water, and do not disperse to meet the emulsification requirement;
[0061] 3) Three days after the second immunization, the blood was collected by docking the tail, and detected by indirect enzyme-linked immunosorbent assay, and the titer reached 16,000. The method is as follows:
[0089] Example 3: According to the dose 1 mg / ml determined in Example 1, co-immune stimulated mice to prepare monoclonal antibodies
[0090] 1) First immunization: Dissolve 50 μg of antigen in 150 μl PBS, then add 150 μl Freund’s complete adjuvant, and stir mechanically for 10 minutes to make the mixture milky, take a drop in water, do not disperse, and meet the emulsification requirement;
[0091] 2) Second immunization: Dissolve 25 μg of antigen in 150 μl of PBS, add 150 μl of Freund’s incomplete adjuvant, and stir mechanically for 10 minutes to make the mixture milky, take one drop in water, and achieve emulsification without dispersion;
[0092] 3) Three days after the second immunization, the blood was collected by docking the tail, and detected by indirect enzyme-linked immunosorbent assay, and the titer reached 16,000. The method is as follows:
[0093] Ⅰ Dock the tail of the mouse to collect blood, use a surgical operation to cut the tail of the mouse 1-2 mm, and take ...
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Abstract
The invention provides a method for improving effective cell fusion. The method comprises: (1), measuring the immunization dosage of an experimental animal to LPS, (2) monitoring the antigen immunization effect of the experimental animal, and (3) performing stimulating immunization to the experimental animal which accords with the immunization condition with LPS. According to experiment statistics, the method has obvious advantage that 24 cell strains can be obtained through fusion of three times, compared with 3 cell strains obtained through fusion in the prior art. The probability of screening of a target cell strain through the method is 8 times of that of conventional technology. The method makes a notable progress. The method can be used for screening and fusing an antibody, such as a monoclonalantibody, is beneficial for increasing the quantity of antibody-secreting cell strains and is beneficial for screening of a proper antibody during antibody base establishment. When an antibody is produced through in-vitro culture, growth of cell strains can be boosted, the time can be shortened, and the production cost is reduced. The method is suitable for industrial production and has great application value.
Description
technical field [0001] The invention relates to biotechnology, in particular to a method for increasing effective fusion cells. Background technique [0002] In conventional immunization and cell fusion, because the number of plasma cells targeting the target antigen is small, a large number of nonsense B lymphocytes in the fusion are fused with the mouse myeloma cell line (SP2 / 0-Ag14, SP2 / 0 for short) , which increases the workload of screening target cells and affects the acquisition of target cell lines. At the same time, under natural circumstances, the target cell line is often at a disadvantage in the competition between the target cell line and the miscellaneous cell line. Therefore, the target cell line is obtained through one fusion Little to nothing. This increases the number of immunizations, prolongs the experimental time, is not conducive to animal protection, and increases the cost of cell fusion to obtain effective cell lines. The patent 200980160301.8 appli...
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IPC IPC(8): C12N5/16
CPCC12N5/16
Inventor 阮星张海涛孙卫兵张跃建
Owner SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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