Production, Purification Of Monoclonal Antibodies Against BFGF And Application In ELISA

Aim: To select monoclonal antibodies of high affinity and specificity from several established anti-basic fibroblast growth factor (bFGF) hybridomas and use these purified antibodies to develop new enzyme-linked immunosorbent assay(ELISA) for bFGF. These ditection methods can be applied to the production and research of bFGF. Method: Monoclonal antibodies were purified by Protein-A affinity chromatography from ascite produced by injecting mouse with two hybridomas designated 2H2 and 2B5. Polyclonal antibody was purified with Sephadex G-200 column and DEAE-Sepharose Fast Flow ion-exchange column from anti-bFGF sera and was labled with HRP by sodium iodide method. The result of purification was analysed by SDS-PAGE and Western-blot. A sandwich enzyme immunoassay(EIA) for bFGF has been developed using 2B5 monoclonal antibody and enzyme-labled polyclone antibody. The precision and accuracy of this method was evaluated by statistic method. 2H2 monoclonal antibody has been used to established a indirect immunoassy and a competitive immunoassy for bFGF.Results and Conclusions: The optimum dilution of 1:80 of coated 2B5 monoclonal antibody is determined with method of square titration. The optimum concentration of enzyme-linked polyclone antibody is 1:50. The sensitivity of the sandwich enzyme immunoassay is up to Ing/well. As to the detection level of 100ng/well, x2 test is 32.84, and the precision is 82%. As to the detection level of lOOng/well, x2 test is 3.10, and the precision is 82.9%. 2H2 monoclonal antibody has been used to established a indirect immunoassy and a compete immunoassy for bFGF. The sensitivity of these two methods is up to 1 pg/well of bFGF.