Study On Bispecific Monoclonal Antibody To Pesticide Imidacloprid And Parathion-Methyl

Insecticide imidacloprid and parathion-methyl have been widely used in agricultural production. Imidacloprid is a new neonicotinoid insecticide and use to control the pests of sucking mouthparts on the rice, corn and other crops. Parathion-methyl is one of organophosphate pesticides and uses to control the pests on the apple, cotton and other crops. Due to the improper use of pesticide imidacloprid and parathion-methyl, they have seriously jeopardized the environmental quality and human health. Hence, it was imperative to developed rapid, economical and sensitive assays for two pesticides residue. The enzyme-linked immunosorbent assay (ELISA) plays an important role in the field of pesticide residue because of its cheaper, simpler, more suitable for on-site monitoring and screening of a large number of samples than traditional instrument analysis.We successfully obtained anti-imidacloprid monoclonal antibodies using hybridoma technology at first. On this basis, we obtained the bispecific monoclonal antibodies to pesticide imidacloprid and parathion-methyl using hybrid hybridomas. And then we established ELISA test system based on these two antibodies. Specific methods and results were as follows:In this study, according to molecular modeling and charge distribution, two haptens 1-[(6-Carboxylethylthio-3-pyridinyl) methyl]-N-nitro-imidazolidinimine (named as H1) and 1-[(6-Chloro-3-pyridinyl) methyl]-3-carboxylpropyl-N-nitro-2-imidazolidinimine (termed as H2) were synthesized, and then the haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA) for immunogen (H1-BSA) and coating antigen (H2-OVA) respectively by NHS ester method. BALB/c mice were immunized with H1-BSA conjugate. We obtained two hybridoma cell lines 2F11/A9 and 2G6/G12 secreting antibody specific for imidacloprid from the conventional hybridoma technology. The result showed that the subtypes of obtained monoclonal antibodies were IgG3 and IgG1, respectively, and the dissociation constant of antigen-antibody were 1.1×10-10 mol/L and 3.4×10-9 mol/L. Then, we developed an ELISA assay based on monoclonal antibodies secreted by 2F11/A9. The effects of organic solvent, ionic strength, pH on the antigen-antibody reaction were evaluated. The results showed that 5% acetone in PBST,0.2 M of ionic strength and 8.0 of pH were determined as the optimum conditions. Under the optimized conditions, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) indicated the IC50 value of 5.3 ng/mL with detection limit of 1.1 ng/mL. There was no apparent cross reactivity with six analogous compounds. The recoveries obtained by standard imidacloprid addition to real samples, including tap water, soil and Chinese cabbages were all from 85.5% to 118.0%, CV 1.0%～4.7%.In order to obtain the hybrid-hybridoma cells, the hybridoma 2F11/A9 producing monoclonal antibodies to imidacloprid which selected by 5-bromo-2-deoxyuridine (5-BrdU) which make the cells lacking of thymidine kinase (TK) and the hybridoma producing monoclonal antibodies to parathion-methyl (preparation of laboratory previously) which selected by 8-azaguanine (8-AG) which make the cells lacking of hypoxanthine-guanine phosphoribosyl transferase (HGPRT). A trioma cell line (LG-D6) and a quadroma cell line (LG-A2) which secreted bispecific monoclonal antibodies (BsMcAb) against imidacloprid and parathion-methyl were obtained by cell fusion. The BsMcAb was produced by LG-D6 ascite. Then the optimal conditions for the ELISA were established based on the optimization of buffer condition. From the sensitivity results, the detection limit of imidacloprd and parathion-methyl were 69.8 ng/mL and 1.9 ng/mL respectively. There was no apparent cross reactivity with the analogous compounds except for imidaclothiz, parathion and fenitrothion. The recoveries obtained by standard imidacloprid or parathion-methyl addition to water samples were all from 87.4% to 104.0%, indicating that the established immunoassay method is accurate and reliable.Through the above experimental study, an ELISA assay for imidacloprid residue was established. A technique system for producing bispecific monoclonal antibody based on hybrid-hybridoma and its ELISA method was established successfully. It laid a solid foundation for research and development of products for immunoassay.