Preparation, Identification And Preliminary Application Of Antibodies Against A Group Of Pyrethroid Insecticides

3-Phenoxybenzoic acid(PBA) was used as the generic hapten.Three coating antigens weresynthesized by Activated ester method, mixed anhydride method and6-aminocaproic acidmethod, using PBA and ovabumin(OVA), respectively. The coating antigen synthesized using6-aminocaproic acid method had the optimal identification for pyrethroid pesticides. For thepurpose of rapid screening assay, the broad specificity antibody against a group of pyrethroidinsecticides was used as materials and the ELISA method was established and optimized forpyrethroid pesticides residue in Chinese cabbage. The results showed that it was unnecessaryto premix the antigen and antibody, the optimum reaction time of antigen-antibody was45min; Chinese Cabbage matrix had a slight influence on the ELISA method and the detectionrate of the over standard and the false negative by this semi-quantitative ELISA to54samples were7.41%and1.86%, respectively. It was indicated that this semi-quantitativeELISA method could be used for the fast screening of pyrethroid pesticides residue inChinese Cabbage.The objective of this study is to generate broad specificity monoclonal antibody(mAb)against a group of pyrethroid insecticides and to identify its immunological characteristics.The generic hapten PBA was conjugated to carrier protein BSA by activated ester method,Balb/c mice were immunized with PBA-BSA. The titer of polyclonal antibody (pAb) wasdetected by indirect enzyme-linked immunosorbent assay(ELISA) after five timesimmunization. The mouse with high titer and sensitivity was selected for cell fusing. Thesplenocytes of immunized mice were fused with Sp2/0cell s and the cultural supernatants ofhybridoma cells were screened by indirect non-competitive ELISA based on the coatingantigen PBA-ovoalbumin (PBA-OVA).High sensitivity and high specificity monoclonalantibody was prepared after many times subcloning using limiting dilution method. Purifiedmonoclonal antibody were obtained after saturated ammonia sulfate precipitation and proteinG affinity column. The immunological characteristics of mAb such as titer,antibody subtypes,affinity constant and the sensitivity of pyrethroid insecticides were characterized by indirectELISA; UV and SDS-PAGE results showed that PBA-BSA artificial antigen was synthesizedsuccessfully. A hybridoma cell line (4H11) secreting anti-pyrethroid mAb had been established. The titre of ascites was up to1:6.5×106. the subtype of the mAb was IgG1; Theaffinity constant of the mAb was about2.5×107L/mol to PBA, with a IC50value of208.83ng/ml and the detection limit of21.23ng/mL to PBA. Simultaneously, beta-cypermethrin,flucythrinate, fenvalerate and cypermethrin were sensitively recognized by the mAb with theIC50were1.01,2.15,3.16,3.67μg/mL, respectively. This showed that the mAb could detectseveral pyrethroid insecticides as a broad-spectrum antibody at the same time, which laid afoundation for the application of rapid detection against pyrethroid insecticides.