Preparation of Trifluralin Antibody and Development of Enzyme Linked Immunosorbent Assay
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Abstract:
An indirect competitive enzyme linked immunosorbent assay (ic-ELISA) was developed to rapidly detect trifluralin pesticide in agricultural or aquatic products in this paper. Hapten 2C was synthesized by the reaction of 4-chloro-3,5-dinitrobenzotrifluoride and secondary amine side chain amino group. And then hapten 2C was coupled with BSA or OVA to synthesize complete antigens by carbodiimide method. After that, polyclonal antibody PcAb-2C was obtained by immunizing New Zealand rabbits. Based on the principle of indirect enzyme linked immunosorbent assay, seven factors including dilution ratio of coating antigen and antibody, coating temperature, antibody and standard diluent were systematically optimized, and finally the indirect competitive enzyme linked immunosorbent assay was developed. Under the optimized conditions, the IC50 of this developed ic-ELISA was 54.07 ng/mL, the detection limit was 7.22 ng/mL, and the linear range was 12.56~282.62 ng/mL. The cross reaction with 20 trifluralin analogues showed good specificity (The cross reaction rate of two drugs was more than 15%). The recoveries of soybeans, soybean oil and eels were 89.8%~106.4%, and the coefficients of variation were less than 10%. The developed method was verified by gas chromatography and the recoveries were 89%~108.2% with the coefficients of variation less than 10%. The results showed a good linear relationship (R2=0.99). The above data show that the established method in this study has the advantages of high sensitivity, high specificity and high accuracy, which is suitable for the high-throughput rapid detection of trifluralin pesticide residues in agricultural (aquatic) products.
