The Research on Enzyme-Linked Immunosorbent Assay for Imidaclothiz
|School||Nanjing Agricultural College|
|Keywords||imidaclothiz hapen polycolonal antibodies monoclonal antibodies ELISA|
For the research of immunochemistry characteristic of neonicotine pesticide, imidaclothiz was choosen as research objects in this paper. Indirect competitive enzyme-linked immunosorbent assays (ic-ELISA) were developed for the quantitative detection of imidaclothiz in the environmental and agricultural samples.Hapten was synthesized, and then covalently coupled with carrier protein BSA via active ester method or OVA by mixed anhydride reaction, respectively. The polyclonal antibodies and monoclonal antibodies were obtained by immunized New Zealand rabbits and BALB/c mice, the titres of the solutions of the corresponding antibodies protein (the coating antigens were 4mg/L) were 2.56×106 and 5.12×106, respectively.The experimental parameters, including organic solvent, ionic strength, and buffer pH were studied sequentially to improve the sensitivity of the immunoassay. The optimum parameters of ELISA procedure of polyclonal antibodies were determined as follows: concentration of coating antigen and antiserum was 0.5 mg/L and 0.4 mg/L, content of methanol was 10%, pH was 7.4, and ionic strength was 0.14 mol/L. The inhibition standard curve of imidaclothiz was obtained under the optimised conditions. The half-maximal inhibition concentration and the limit of detection were 0.18 mg/L and 0.0018 mg/L of imidaclothiz, respectively. Very low cross-reactivity was found for some structurally related compounds except imidacloprid. At different spiked levels (0.05-1mg/kg in water, soil and cabbage) recoveries of imidaclothiz were from 91.42%-113.82%, with coefficient of variations of 0.72%-8.68%.And for monoclonal antibodies the optimum parameters were determined as follows: concentration of coating antigen and antiserum was 0.65 mg/L and 0.4 mg/L, content of methanol was 10%, pH was 7.4, and ionic strength was 0.14 mol/L. Under the optimized conditions, the half-maximal inhibition concentration were 0.088 mg/L and the limit of detection were 0.018 mg/L of imidaclothiz. There were no obvious cross-reactivities with most of structural analogues of neonicotinoids insecticides except imidacloprid. The recoveries of imidaclothiz in environmental and agricultural samples, including tap water, paddy water, soil and cabbage, ranged from 80.43% to 113.83%, with coefficient of variations (CVs) of 1.08-8.92%, well within the requirements of residue detection.