Preparation And Functional Identification Of Mouse Anti-human BAFF Monoclonal Antibody

Background:B cell activating factor(BAFF),a member of the tumor necrosis factor superfamily,was first discovered in 1999 and has two forms of membrane protein and soluble protein.BAFF co-stimulates B cell proliferation and induces immunoglobulin production in vivo,which plays a critical role in B cell activation,differentiation and survival.By binding to BAFF-R,TACI and BCMA receptors,BAFF can promote B cell differentiation,maturation and category conversion,promote humoral immune response,and participate in T cell activation.In addition to its function as an inflammatory cytokine,BAFF has several regulatory functions,such as regulating IL-10 expression.A large number of evidences show that BAFF is closely related to the occurrence and development of systemic lupus erythematosus(SLE),primary sjogren’s syndrome and other autoimmune diseases,so BAFF has become an important target for the treatment of autoimmune diseases.Monoclonal antibodies are highly uniform antibodies produced by B cells that target only a specific epitope.Antibodies can be further divided into two parts: a constant region and a variable region.Among them,the amino acid sequence in a small part of the variable region is very prone to mutation,which is called the hypervariable region.The amino acid sequence of the hypervariable region determines the specificity of the antibody binding to the corresponding antigen.This is also an important reason why antibodies produced against the same antigen still differ.Objective: To enrich the selection of biological agents for autoimmune therapy,expand the existing anti-BAFF monoclonal antibody library,prepare mouse anti-h BAFF monoclonal antibody and preliminarily verify its performance,so as to provide basis for future application.Methods: In this study,the prokaryotic expression vector of human BAFF gene was successfully constructed by molecular cloning technology,and its protein expression was optimized in E.coli BL21.The recombinant human BAFF protein with a purity of about 80% was obtained by ammonium sulfate precipitation method.The obtained recombinant human BAFF protein was injected subcutaneously into the immunized mouse,and the serum titer of the antih BAFF antibody was measured by ELISA.The spleen cells of the mice with the highest titer were obtained and the hybridoma cell lines were fused with PEG2000.Three monoclonal cell lines,6B2B12,8E6E3 and 8E6G8,were obtained by flow cytometry and ELISA for cell line conservation and expansion culture.A large number of monoclonal antibodies were produced by ascites tumor method and purified by Protein G Protein to obtain three strains of monoclonal antibodies with purity up to 99%.The dose-effect relationship between affinity and blocking was detected,and the application of Protein western blot detection,flow cytometry and immunoprecipitation was explored.Results: The three antibodies all had the affinity of immune antigen,commercial BAFF protein and natural BAFF,and 6B2B12 had the highest affinity,which was inferior to commercial antibody in flow affinity detection.The three antibody strains all had certain blocking ability,which could reduce the binding ratio of BAFF-Biotin to BAFF receptor,and the highest blocking efficiency was 8E6G8.Compared with commercial antibodies,no specific band of BAFF protein was developed in the application of western blot test.The results of immunoprecipitation showed that the three antibody strains could bind and immunoprecipitate the natural BAFF protein in PBMCs,which could be used in immunoprecipitation.Conclusion: Three strains of mouse monoclonal antibodies against human BAFF were prepared in this study,which have the natural affinity and resistance of BAFF protein,and can be used in immunoprecipitation.It provides more options for the detection and treatment of autoimmune diseases.