Development Of A Rapid Determination For Diethylstilbestrol By Enzyme-linked Immunosorbent Assay

Diethylstilbestrol (DES), an artificial estrogen, has been banned for its carcinogenesis and teratogenesis properties in most country. This study is to develop DES residual immunoassay reagent kit.Diethylstilbestrol Monocarboxymethoxyflurane was synthesized by modifying one phenolic hydroxyl group of DES molecule. The conjugations of DES-MCME with bovine serum albumin (BSA) and ovalbumin (OVA) as synthetic antigens were prepared by the mixed acid ahhdride method and the conjugation rates were analyzed by ultraviolet scanning. New Zealand white rabbits were immunized with DES-BSA. The results demonstrated that the isomerization was present in cis and trans of DES and its derivates, which was effected obviously by polar solvents.High resolution mass spectrum showed that the molecular weight of DES-MCME was 325. The optimal conjugation ratios of immunogen and coating antigen were 13 and 10. The dilute rate of antiserum could reach 102400, through an indirect enzyme-linked immunosorbent assay (ELISA). Based on this antiserum, enzyme-linked immunosorbent assay (ELISA) for the detection of DES had been developed. Several physicochemical parameters, such as incubation time, ionic strength, detergent concentration and pH of the antibody dilution were optimized. The linear regression equation of the standard curve was y = -0.2978x + 1.4756, R2 = 0.9986. The range of linearity was from 0.1 to 25 ng·ml-1. The 50% inhibition value (IC50) and the limit of detection (LOD) were 1.89 and 0.08 ng·ml-1, respectively. The specificity was evaluated by five anabolic steroids of similar structure, and none of them had significant cross-reactivity. The accuracy and precision of this assay were evaluated by means of spiked samples. When DES was spiked at levels of 1 and 5 ng·g-1, the recoveries of beef ranged from 50% to 80%, with the coefficient of variation of 3.7 - 8.6%. At the same time, the recoveries of pork liver ranged from 30% to 50%, with the coefficient of variation of 4.6 - 7.9%.It was found that the recovery and the coefficient of variation of this ELISA kit were consistent with the one from the Euro-diagnostica company in Holland, and the detection results were similar to the results obtained by the LC/MS/MS. So it could be concluded that all of the parameters of the ELISA kit we developed had reached the level of foreign standards, and it could be used as a screening method in practice.