Industrial Technique For Pesticide Rapid Test ELISA Kit

Two optimized haptens of insecticide triazophos, i.e. O-ethyl, O-(1-phenyl-1H-1,2,4-triazol-3- y1) N-(3-carboxypropyl) phosphoramidothioate (THBu) and [O-ethyl, O-(l-phenyl-1H-1,2,4-triazol-3-yl) N-(5-carboxyamyl) phosphoramidothioate (THHe), were synthesized after molecular modeling chemical studies and optimizing. And an other hapten, 2,2-dimethyl-2,3-dihydrobenzofuranyl,7-N-(3-carboxypropyl) carbamate (BFNB) one of insecticide carbofuran hatens was also synthesized. After the haptens covalently coupled onto carrier proteins BSA, the corresponding artificial antigens was prepared and further more, the corresponding monoclonal antibodies with high titers and specificities was produced by cross-tumour technique.Based on the monoclonal antibodies, Triazophos and carbofuran ELISA conditions were optimized, respectively. The optimum conditions for the ELISAs were given as follows: Costar^? lmmuno 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 0.01M ion strengths and 10% methanol was suitable for Ab-Ag binding reaction. Also. 2% degreasant milk powder in 0.01M pH7.4 phosphate-biphosphate buffer could reduce the non-specific adsorption and improve the sentivity for the ELISAs. HRP was selected as the enzyme tracer. The optimized stabilizer could remain the activities of the monoclonal antibodies at 37°C for 14days. Heterogeneous direct competitive ELISA (antibody coating format) and homogeneous direct competitive ELISA (anti-antibody coating format) were selected for further investigation for triazophos and carbofuran. respectively.Recoveries of triazophos and carbofuran fortified into different samples such as greenstuff samples, fruit samples, and environmental samples were determined by ELISA after sample matrix testing. Accuracies and precisions of the data were evaluated and finally two rapid test ELISA kits were prepared for triazophos and carbofuran, respectively. Results showed that, under optimized conditions, the detection limits of the ELISA kits were 2.66 ng/ml (IC₁₀) and 0.97 ng/ml (IC₁₀), with the linear concentrations ranged from 1.95 to 2 000 ng/ml and 0.8 to 340 ng/ml, for triazophos and carbofuran, respectively. The LOQs of the ELISA kits for different samples were close or up to the MRLs of the two insecticides in some countries or regions (parts of them exceeded the MRLs).At the present study, the haptens of triazophos, the anti-triazophos monoclonal antibody and the triazophos rapid test ELISA kit were firstly prepared in and abroad. The anti-cabofuran monoclonal antibody and its rapid test ELISA kit were firstly obtained in China. Further more, an industrialized technical system for the preparation of ELISA kit for low molecular compound such as pesticide was also firstly reported in China. The trial-manufactured ELISA kits of the two insecticides were applied by five authoritative organizations in China such as Institute for the Control of Agrochemicals of Ministry of Agricultural, Inspection and Quarantine Technic Center of Zhejiang (chemical lab), Institute for the Control of Agrochemicals of Shanghai, etc. It was proved that the industrialized technique was reliable, the quality of the productions was stable, and the technique could be industrialized.