Homologous And Heterologous ELISA For Determination Of Diethylstilbestrol

"Food is the first necessity of people,and safety is the first necessity of food". Food safety is a significant question, which is related with Human health, national welfare and people's livelihood. It is important to detect residues of pesticide and veterinary drugs in the field of food safety. In this paper, homologous enzyme linked immunosorbent assay (Hm ELISA) and heterologous enzyme linked immunosorbent assay (Ht ELISA) were established for determination of diethylstilbestrol (DES) which is used proverbially for forage additive of aquatic product, poultry and livestock as veterinary drug. DES has carcinogenicity, which enriched in animal easily and resolved slowly in environment. DES worked the mischief for Human health and environment. In this experiment, three kinds of complete antigens were prepared, the PcAb of diethylstilbestrol was obtained from immunizing rabbit and depurated by salt fractionation (saturated ammonium sulfate precipitation method). Homologous and heterologous enzyme linked immunosorbent assay were established.Three kinds of carboxylated diethylstilbestrol were obtained from our laboratory, which respectively was Diethylstilbestrol hemisuccinate (DES-HS), Mono-o-carboxypropyl- diethylstilbestrol (DES-CP) and Diethlstilbestrol monocarboxymethoxyflurane (DES- MCME). They were transformed in the same position of DES, but different from the length and construction of the truss arm. After being identified by 1H nuclear magnetic resonance (1HNMR), the carboxylated DES was then coupled to BSA and OVA for the complete antigens by EDC methods, which were identified by mass spectrum (MS) and SDS-PAGE. Female rabbits of 2～2.5 kg weight were selected as laboratory animals. A small dose of medium-range immunity was carried out with the mixture of complete antigen and equal volume Freund adjuvant (FA). The first immunization was complete freund adjuvant, and the following steps were incomplete freund adjuvant. At the first immunization, every rabbit was injected with 2mg of DES-HS-BSA, the following immunizations dose were also 1mg of DES-HS-BSA. The antiserum was collected from rabbit ear veins before next immunization, and the titers were determined by indirect ELISA. When the titers reached to our experiment's need, the specificity of antiserum for the target antigen was validated by competitive ELISA. Whereafter, the rabbits were injected with 2mg of complete antigen without adjuvant. The antiserum was collected from rabbits'hearts, which was polyclonal antibody of DES. The Ab was depurated by salt fractionation (saturated ammonium sulfate precipitation method). The precipitations were identified by SDS-PAGE, WB and indirect ELISA, and the results showed that The precipitation of 20%～40% had the Ab of DES. The depurated Ab was stored at -70℃, w hich has a addition of 50% glycerol.The homologous and heterologous enzyme linked immunosorbent of DES were established. The coating antigen of homologous assay is DES-HS-BSA, as the coating antigen of heterologous is DES-CP-BSA or DES-MCME-BSA. The optimization concentration of coating antigens and antibody was determined by checkerboard ELISA. The regression curves of homologous and heterologous assay were obtained by indirect-competitive ELISA. The regression equations of DES-HS, DES-CP, DES-MCME respectively was I = 30.28 lgC+23.25 (R2=0.9768), I = 22.75 lgC+42.70 (R2=0.9289), I = 24.61 lgC+36.77 (R2=0.9289). The detection range respectively was 0.7810～74.85ng/mL, 0.1005～43.61ng/mL and 0.2082～57.10ng/mL. The sensitivity respectively was 7.646ng/mL, 2.094ng/mL and 3.448ng/mL. The lowest detection limit respectively was 0.3651ng/mL, 0.03653ng/mL and 0.08170ng/mL. In the precision experiment, The CV of within-run and between-run were all below 15%. There was no efficiency difference in CV between Hm ELISA and Ht ELISA.Six analogous compounds of DES were selected to detect the specificity of ELISA. The results showed that the CR subsequence all were bisphenol-A﹥nonylphenol﹥estradiol﹥Resorcino﹥phenol﹥diphenolic acid. And there was no efficiency difference in specificity for the analogous compound between two assays. In the accuracy test, three concentration levels of DES were added to samples (water, pork, milk, Crabs), the coefficient of recovery were all in the range form 70% to120%.In short, this experiment proved that the heterologous assay elevated sensitivity materially. There was no efficiency difference in accuracy and precision between Hm ELISA and Ht ELISA, which laid the foundation for the monitoring of pesticides and veterinary drugs residue, as well as providing a detection tool for the agriculture and Food Industries.