Studies On The Artificial Antigen Of Diethylstilbestrol

Diethylstilbestrol(DES) is a synthetic estrogen. It can produce the effects like natural estrogens in the bodies of human beings and animals.In feeding, it was used illegally to promote growth and feed conversion, but it can lead to cancer and deformity. So it was ruled not to use DES in feeding animal by any way all over the world. Animal and animal products could not be detected DES. The immunoassay of residual DES is one of the critical means in monitor of DES residues. The study is necessary and piror to develop DES residual immunoassay reagent box . The main results are as follows: 1.At the room temperature,the certain ratio of DES and succinate anhydride was stirred in anhydrous pyridine, and got the brown product. The analysis results of HRMS, IR , 13C-NMR and 1H-NMR showed that the separated and pured is product was the derivative of DES wanted, called as DES-HS. Its molecular weight is 368, Its molecular formula is C22H24O5, It includes the critical structure such as -OH ,–COOH and so on.2.The artificial antigen of DES was synthesized by mixed anhydride method using DES-HS and bovine serum albumin(BSA) and ovabumin(OVA) respectively. The characterization of hapten-protein conjugates were examined ultraviolet spectrophotometry(UV) and sodium doclecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), the results showed that the binding is successful. The number of hapten molecules per molecules of protein estimated by UVP Bioimaging system of labworks 4.0 Image Acquisition and Analysis software was 30～32 and 25～28 respectively.3.BSA-hapten conjugate synthesized was used as immunogen to immunized rabbits and Balb/c mice respectively and the polyclonal antisera were all emerged in rabbits and Balb/c mice. The specificity of antisera was tested by agar double-diffusion method and indirect enzyme-linked immunosorbent assay(ELISA), blocked ELISA detected the antisera titer at the same time. The results showed that the immune antisera titer of rabbits were not below 1:6000,the immune antisera titer of Balb/c mice were not below 1:12000. Rabbits antisera can react with free DES.To sum up above all,the conclusions were drawn: the preparation method of DES-HS was built,the artificial antigen of DES were synthesized successfully,the polyclonal antisera of DES were prepared using the synthetic DES-HS-BSA conjugate; the indirect ELISA method was builded to detect DES antibody with prepared DES polyclonal antibody. It established the base of preparing DES monoclonal and developing DES residual immunoassay reagent box which owned the independent knowledge authority of our country.