Preparation Of Broad Specificity Antibodies And Development Of Emzyme Immunoassay For The Type Ⅱ Pyrethroids

Synthetic pyrethroids are used widely in agriculture, forestry, veterinary and public health applications because of their remarkably high insecticidal activities and low toxicity to mammals, although pyrethroids are thought to be safe for humans, suppressive effects on the immune system have been reported after exposure. Some pyrethroids may cause lymph node and splenic damage as well as carcinogenesis. Because of these potential damage, a sensitive,selective,and rapid method for monitoring residue levels of pyrethroids is desirable. Immunoassay is an alternative quantitative method for the detection for pesticide residue analysis and screening because of its high sensitivity,high specificity and so on. In this paper, the immunoassay technology of the type II pyrethroid insecticides was researched. The primary research results were as follows:1. Synthesis of immunogen: As the hapten of the type II pyrethroids, (RS)-cyano- 3-phenoxy benzyl (RS)-cis,trans-2,2-dimethyl-3-carboxyl-cyclopropanecarboxylate was synthesized by using oxidation with potassium permanganate. Immunogen was synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) by active ester method (DCC). The hapten was identified by nuclear magnetic resonance, IR spectrophotometry and high resolution mass spectra and the immunogen were identified by UV spectrophotometry.2. Polyclonal antibodies were generated by immunizing New Zealand white rabbits with immunogen. The titers of antiserum that was generated by immunizing with immunogen hapten-BSA had reached 1:12800. The optimal working concentrations were optimized by ELISA as follows: the dilution ratio of coated antigen was 0.5μg/ml, the dilution ratio of antiserum was 1:12000.3. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for immunogen was developed by using polyclonal antibodies. The IC-ELISA showed an I50 was 0.048μg/ml for immunogen. The optimal detection conditions that the acetone content in samples was not more than 5%. The cross-reactivity was measured for the type II pyrethroids such as cyhalothrin, cypermethrin, deltamethrin, fenvalerate and cyfluthrin. The I50s of the optimized immunoassay were 1.1553μg/ml for cypermethrin, 1.7095μg/ml for cyhalothrin, I10s were 0.0117μg/ml for cypermethrin, 0.0485μg/ml for cyhalothrin.