Study On Synthesis Of Diethylstibestrol Artificial Antigen And Development Of ELISA

Diethylstibestrol(DES) is an artificial estrogen. It has a good effect in improving the metabolism of protein, the growth of the animal and decreasing the fat, so was used in the feeding of the animal. The International Agency for Research on Cancer(IARC)have found: DES can lead to cancer, and can residues in animal products, such as liver, meats, egg and milk, and hazard health through food chain. So it was ruled not to use DES in feeding animal by any way all over the world. Animals and animal products could not be detected DES.This article is divided into two parts, In this study, we improve the general applicability of this method by synthesizing haptens for diethylstilbestrol. The synthesis of innovative succinate and 4-bromine-ethyl butyrateby derivatives of diethylstilbestrol. Haptens was identified as successful by IR, mass spectrometry and nuclear magnetic resonance spectroscopy identification. Then, Bovine serum albumin(BSA) and egg serum albumin(OVA) were used as two protein carriers respectively to couple with the hapten DSE by active esters method and mixed-anhydride method. The complete artificial antigens acted as both immune antigen and coating antigen in ciELISA respectively. Selected a few of immunal programs to immunize the New Zealand rabbits to genenerate specific antiserum.The second part of the study establishing the indirect competitive Enzyme-linked. Test methods and results are as follows:artifical antigen, used as immune antigen, is made by DES and protein carrier (BSA) through the improved active esters method and mixed-anhydride method. The coating antigen is synthesized by using the same methods. The UV scanning atlas, SDS-PAGE analysis and animal immune tests proved the success of coupling. The antiserum of DES is obtained from immunized New Zealand rabbits of by the normal immune process. Check specialties of antiserum by Western Blot and ciELISA. The the active esters method is better than the the mixed-anhydride method. Thus, the antiserum is obtained by it in the later ELISA experiment. Immunosorbent assay method for detecting trace element of DES. The most suitable working condition of ciELISA is obtained by several grid tests which are used to calculate the most optimal concentration and dilution of the coating antigen and antiserum, and the results are 2.5μg/mL and 1:6400 relatively. The standard curve of ciELISA is also established and the curve indicates that the lowest detection limit is 8.4ng/mL. The regression equation of the curve is y=-2.6679x+3.0589 (R2=0.9969). In the context of the standard curve 0ng/-mL100ng/mL showed a favorable linearity relation.The accuracy of the established ELISA protocol can be determined within one batch. The coefficient of variation within one batch is 2.57%. The average rate of recovery of Pork by ciELISA is 109.41%. In the range of concentration can be determined, Not found cross-reactivity between antibody and Three other estrogen including Dienestrol, Dienesterol Diacetate, hexestrol. The experimental results show that the indirect ELISA method has a high specificity and selectivity of drug residues in samples for rapid detection of diethylstilbestrol.