Study On Enzyme-Linked Immunosorbent Assay And ELISA Kit For Triazophos

Two haptens of insecticide triazophos [O,O-diethyl O-(1- phenyl-1H-1,2,4-triazol-3-yl) phosphorothioate], THBu [O-ethyl, O-(1- phenyl-1H-1,2,4-triazol-3-yl) N-(3-Carboxypropyl) phosphoramidothioate] and THHe [O-ethyl, O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(5-Carboxyamyl) phosphoramidothioate], were synthesized. These haptens were then covalently coupled onto bovine serum albumin (BSA) with the CDI method and served as immunogens (THBu-BSA and THHe-BSA). The ratios of haptens and BSA conjugated were determined by UV spectrometer. Two antibodies, i.e., anti-THBu-Ab and anti-THHe-Ab, were obtained by immunizing the rabbits with THBu-BSA and THHe-BSA conjugates, Their titers were measured to be 5.12× 10⁵ and 2.56×10⁵, respectively.Comparing with different methods of enzyme-linked immunosorbent assay (ELISA), the antibody-coating direct competitive ELISA format was the best one, furthermore the anti-THBu-Ab-coating vs the HRP-THHe was the most sensitive combination. Based on this designation, effects of the factors, such as immuno-plate, coating buffer, ionic intensity, content of methanol, coating temperature, coating time, solvent and non-specificity adsorption were studied further more. The optimum condition for the ELISA was given as follows: Costar(?) Immuno plate was coated with the antibody in 0.01M phosphate-biphosphate buffer (pH7.4). The plate was incubated at 37℃ for 2 hours. The buffer with 0.02M ion strengths and 5% methanol was suitable for Ab-Ag reaction. Also, 2% defatted milk powder in 0.02M pH7.4 phosphate-biphosphate buffer could improve the sentivity for the ELISA method. Under optimized conditions, the linear regression equation for the method was discribed as y=12.622Ln(x)-2.092 (R²= 0.993). The linear concentrations ranged from 1.95 ng/ml to 1 000 ng/ml. The detection limit was 5.76 ng/ml (IC₂₀), and ICso value was 62.0 ng/ml. Some analogues of triazophos, such as, chlorpyrifos, diazinon, which were commonly used in agriculture, didn't interfere with the analysis., and the cross reactivities were less than 2 × 10^-4.The antibody-coating direct competitive ELISA format was used to determining residue of triazophos in environmental components and agricultural samples, such as, water, rice, soybeans (dry), carrots, Chinese cabbage, onions, eggplants, pears, citrus, and so on. Recoveries from spiked samples were 61.48-132.27% (except for pears), and the coefficients of variation were allbelow 25%. It was suggested that the ELISA Kit was a potential valuable analytic tool for the rapid screening and sensitive detecting of trizophos.