Method for high-throughput synthesis of monoclonal antibody stably transfected expression cell strain

Abstract

The invention discloses a method for high-throughput synthesis of a monoclonal antibody stably transfected expression cell strain. The method comprises the following steps: S1, preparing a target antigen and immunizing an animal by adopting the target antigen; S2, after the immunization is completed, taking the animal spleen, screening out memory B cells and plasma cells by adopting a flow cytometry, and preparing a single-cell suspension; S3, carrying out amplification reverse transcription on mRNA of the single cell to synthesize cDNA, amplifying variable regions of a heavy chain and a light chain of an antibody, and respectively connecting the variable regions to a carrier; and S4, carrying out cell transfection to obtain the monoclonal antibody stably transfected expression cell strain. Compared with a traditional hybridoma cell strain, the cell strain prepared by the method disclosed by the invention is stable, the batch-to-batch production of secreted antibodies is stable, the yield is 10-50 times higher than that of the hybridoma cell strain, and the cell strain can be widely applied to industrial production.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibody preparation, in particular to a method for high-throughput synthesis of monoclonal antibody stably transfected expression cell lines. Background technique [0002] The traditional monoclonal antibody preparation method is to immunize animals such as mice or rabbits with antigens, and then perform single-cell screening by fusing spleen B cells with tumor cells (S / p20) to obtain hybridomas that secrete the target antigen. cell. The disadvantages of this traditional method are: (1) Since the fusion transformation rate of B cells and tumor cells is not 100%, many B cells that can recognize the target antigen during the fusion process cannot obtain these clones through the above method due to fusion failure; (2) The hybridoma cells themselves are relatively unstable, and the antibody expression levels of different hybridoma cells vary greatly, and the target antibody is easy to be lost aft...

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Problems solved by technology

The disadvantages of this traditional method are: (1) Since the fusion transformation rate of B cells and tumor cells is not 100%, many B cells that can recognize the target antigen during the fusion process cannot obtain these clones through the above method due to fusion failure; (2) The hybridoma cells themselves are relatively unstable, and the antibody expression levels of different hybridoma cells vary greatly, and the target antibody is easy to be lost after multiple passages; (3) Monoclonal target antibody screening after the synthesis of hybridoma cells will consume A lot of manual work, and to a certain extent, whether hybridoma cells targeting the target antigen can be obtained depends on luck, and the success rate of obtaining positive clones by traditional methods is not high

Although the above two methods have obtained cell lines stably expressing the corresponding monoclonal antibodies, since one immunogen was used to immunize one animal in the early stage, there were fewer types of spleen B cells obtained, and the success of obtaining the target antigen antibody rate is also lower

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