The Research On Enzyme-Linked Immunosorbent Assay For Bifenthrin And Cyhalothrin

we choose bifenthrin and cyhalothrin as being studied objects, developing ic-ELISA for bifenthrin and BP, and reseaching on relation between design forpyrethroid haptens and specificity of their corresponding antibodies in the first instance, For study of immunochemistry characteristic of pyrethroid insecticide.An indirectly competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the quantitative detection of bifenthrin. By synthesizing the hapten LBc starting from the metabolite of bifenthrin (2-methyl-3-phenylbenzyl alcohol), LBs ((2-methyl[1,1-biphenyl]-3-yl)methyl 3-carboxyl-2,2-dimethylcyclopropane carboxylate) and Lby ((2-methyl[1,1-biphenyl]-3-yl)carboxylic acid ), Three haptens were praparaed which were used in next step. Then, the hapten LBc and LBs was conjugated to bovine serum albumin (BSA) with CDI method to form immuno-antigen; the haptens LBc, LBs and LBy was conjugated to ovalbumin (OVA) with mixed anhydride method to form the the coating antigens.The new zealand rabbits were immunized by conjugate of LBc-BSA and LBs-BSA, and titres of anti-bifenthrin serum (5.12×10⁴, 3.2×10⁵) were determined by non-competitive indirect enzyme-linked assay procedure. A heterologous assay system using the coating antigen format was more sensitive. After optimization of the ic-ELISA conditions, such as pH values, inonic strengths, concentrations of methanol, pH of 7.5, PBS of 0.3M Na⁺, and thirty percent methanol were determined to optimum assay conditions. The IC₅₀ for bifenthrin was 2.16 mg/L, and lower detection limit (LDL) was 0.016 mg/L. Pyrethroids, such as cyhalothrin, deltamethrin, cypermethrin, fenvalerate, fenpropathrin, and the pyrethroid metabolite 3-Phenoxybenzoic acid, did not cross-react significantly in this assay. Using LBc-Ab, Lby-OVA, An ic-ELISA was developed for the quantitative detection of BP. The IC₅₀ for BP was 1.38μg/L, and lower detection limit (LDL) was 0.056μg/L. Pyrethroids and their metabolites did not cross-react at all.Two types of haptens, the earboxylated propyl amino derivative of the acid moiety (HCA1) that is the hydrolyzed product of cyhalothrin, and the carboxylated ethyl carbonyl derivative of the alcohol moiety (HCB2) were synthesized, and conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) respectively by the active ester method and the mixed anhydride method. The polyclonal antibodies (PAbs) were obtained after immuning to the New Zealand rabbits. One PAbs, which raised against the former hapten HCA1, was reactive with cyhalothrin and cyhalothric acid (IC₅₀: 33.12 mg/L, 0.95 mg/L). However, the other PAbs, which raised against the back hapten HCB2, was not particularly reactive with cyhalothrin. For developing an immunoassay for multiple residues of pyrethroid pesticides, the hapten N-2- (Carboxypropyl) carbanmoyl-(3'-phenoxyphenyl) methyl 3- (2'-Cldoro-3',3',3'-trifluoropropenyl) -2, 2-dimethylcyclopropane- carboxylate was synthesized from cyanohydrin and 3- (2'-Chloro-3',3',3'- trifluoropropenyl) -2, 2-dimethylcyclopropanecarboxylic acid via hydrolysis and esterification. The synthesis of the hapten was foundation of preparaing anti- cyhalothrin polyclonal antibodies.