Preparation Of Antibody For Hexoestrol And Development Of Enzyme-linked Immunosorbent Assays

The safety of animal derived food has become a hot spot in the food safety industry, and various social circles are paying close attention to it. Hexoestrol residues are harmful to human health because of their potential carcinogenic properties and other adverse effects. Kits for the determination of hexoestrol (HES) have been researched in the paper, which is significant for food safety supervision and technique storage.The derivatized HES was coupled to bovine serum albumin (BSA), ovalbumin (OVA) and horseradish peroxidase (HRP) by the mixed anhydride methods,and thus immunogen HES-BSA, coating antigen HES-OVA and enzyme tracer HES-HRP were prepared. The coupling rates of the former two were 17.9 and 13.2 respectively by ultraviolet spectrophotometry analysis. New Zealand rabbits were Immunized with HES-BSA and the highest titer of serum was as high as 16000. Then, an indirect enzyme-linked immunosorbent assay (ELISA) and a direct ELISA were developed and optimized in many parameters, such as coating time, pH value, incubation time and so on. The limit of detection of the two methods was 0.05 ng/mL and 0.04 ng/mL, and 50% inhibitory concentrations (IC50) were 0.67 ng/mL and 0.53 ng/mL respectively. The direct ELISA could run in only about 1.6 h and the indirect assay duration was at least 4.65 h. Assay specificity was studied using six hormones (diethylstilbestrol, dienestrol, 19-nortestosterone, medroxyprogesterone, testosterone and estrone). The coefficients of variation (C.R.) of diethylstilbestrol and dienestrol were a little higher (25% and 6% each for the indirect assay,0.5% and 0.3% for the direct assay) and the C.R. of the rest were all below 0.1%. HES (1 ng/g, 5 ng/g) was added to pork, beef and fish samples and the recoveries were 68-108% by the indirect assay and 35-102% by the direct way, with coefficients of variation all below 16%. The accuracy of the methods was evaluated by comparison with instrumental analysis. The plate stability was also studied in the paper and the kits in the indirect assay were quite stable in 3 months under low temperature and 6 days under 37℃. The plates coated with antibody in direct ELISA were kept well in one month under low temperature. In general, all items of the assays have reached the requirements of detection, which makes the practical application possible.