The Preparation And Identification Of Estrone Sulfate Polyclonal And Monoclonal Antibodies

Estrone sulfate (estrone-3-sulfate) referred to E1S, an estrogen derivative, is widely used in early pregnancy diagnosis of domestic animal. In this study, complete antigens of estrone sulfate was synthesized; polyclonal antibodies and monoclonal antibodies of estrone sulfate were prepared and identified. Following major elements:1) The synthesis of estrone sulfate antigensestrone sulfate hapten synthesized by estrone 3-sulfate sodium salt and identified by mass spectrometry,was respectively coupled with BSA and OVA through the carbodiimide method to obtain the complete antigens.Results showed that the concentrations of immunogen estrone sulfate-BSA and the the estrone sulfate-OVA were 9.29mg/ml and 4.68mg/ml.The couping ratios of estrone sulfate-BSA and estrone sulfate-OVA were 20.4:1,and 15.2:1, respectively.Identification of complete antigens by using ultraviolet method and SDS-PAGE electrophoresis method proved the success of coupling.2) The preparation of estrone sulfate monoclonal antibodiesComplete antigens was used for immunization to rabbits, the optimal reaction conditions of the indirect ELISA method was determined, the optimal concentration of antigen was 8000,optimum working concentration of A rabbit was 81000, best work concentration of B Rabbit was 27000. Polyclonal antibodies were purified by Ammonium sulfate precipitation method, and the purity detected by SDS-PAGE electrophoresis method was improved significantly.3) The preparation of estrone sulfate polyclonal antibodiesThe best conditions of mice ELISA response was determined, and the best dilution of serum was 1:1000, the best package concentration of the package is 6ug/ml.The mice which had the best antibody specificity in serum was selected,8# was choosed as the preferred integration,and 1# and 10# as the backup. Ascites monoclonal antibody obtained was purified and verified by SDS-PAGE,which showed the purity was markedly improved.