Preparation And Identification Of Monoclonal Antibody For GST And Establishing Immunoassay Method Of GST

The GST protein was expressed by using a pGEX-4T expression vector. Then female Balb/c mice were immunized with the purified GST protein. According to the immunity procedure, 8 week's age Balb/c mice were immunized by injecting GST and picked blood from their tail tip after 3 days. The titer of anti-GST antibody was detected by the ELISA technique.The highest titer mouse was facilitatory immunized after they were immunized about five to seven times. The spleen's B lymphocyte of the mice has been fused with SP2/0 plasmacytoma cells after 3 days. The wells containing hybridomas secreting McAb specific for GST were got by ELISA. After they were subcloned by the limiting dilution method, expanded cultivated, repeated frozen deposit and anabiosised, four hybridoma cells which were secretive and high affinity monoclonal antibodies of anti-GST were harvested and named with2F5, 2G1, 3C7.①2F5's subtype is IgM, 2G1's subtype is IgGl, 3C7's subtype is IgG2a;②The titers of four hybridoma cells were more than 1:7×106; Compare affinity of the monoclonal antibodies are 2G1>3C7>2F5. The specificity of McAbs was tested for all lines by ELISA against GST, GST-PreS1 fusion protein.McAb of 2F5, 2G1, 3C7 cell was marked by HRP respectively. the McAb of the other two cell were selected by filling ELISA respectively. The result showed that the McAb of 2F5 cell and the McAb of 2G1 cell had the most perfect reaction. The result indicted that the 2F5, 2G1 were the aimed cells of GST determinant antigenic. Study was performanced by ELISA detection of GST with the McAb of 2F5, 2G1, The comparative study was manueuvered by the condition of the solid carrier,coated time,blocking out fluid and time, NaCl andTween-20 concentration in PBS diluted liquid, optimized combination, finally, the double antibody filled ELISA detection of GST was preliminary established. The method could be used in qualitative analyzing of GST and half-quantitative analyzing of GST with standard refered sample, the detective result was more perfective. It indicated that the method could been used in clinical, molecular biology and other corresponsive fields, meanwhile, it was the base for the next research of express diagnosis kit (immune gold badged McAb kit).