Development And Application Of Immunoassay Techniques For Pesticide Residues

Immunological analysis technique was developed and employed efficiently for the pesticide residue screening in different kinds of samples. In this work, polycolonal and monocolonal antibodies of some of the chemical pesticides widely used in our country were developed and enzyme-linked immunosorbent assay methods were standardized, which were approved in the residue detection of the pesticide in the fields and residue elimination determination of pesticides in the vegetables. The results were described as bellow.1. One hybridoma-cell line 3C₃ secreting monoclonal antibodies against insecticide fipronil were produced by fusing mouse myeloma cells (sp2/0) with spleen cells from Balb/c mice immunized by the hapten fipronil analygue conjugated to bovine serum albumin. The production and characterization of hybridoma cell line secreting positive antibodies permanently are described. The effect of several physicochemical factors on the monoclonal antibodies for fipronil was studied and the results were used to optimize the assay system, when incorporated into a competitive indirect ELISA, the antibody provided a detection limit of 0.074ngmL-1 and I50 5.99ng·mL-1 of fipronil. The specificity of the resultant fipronil-specific monoclonal antibodies was tested with several insecticides including Chlorfluazuron, Cyhalothin, Trifluralin and Flufenoxuron, the cross-reaction was less than 0.67%, indicating there no interfering comfounds was found.In different matrix, the fipronil recovery is in the range of 94.7-108% and the coefficient of variation 1.8-9.8%.2. The indirect competitive ELISA based on monoclonal antibody introduced previously was applied to moitor the degradation kinetics of fipronil in vegetable and soil. The results showed that the semi-life of fipronil in vegetable is 10.4 d., and 15.1 d. in soil. The immunological assay method was applied to evaluate the effect of PH value, ozone in aqueous solution, Co-60 irradiation and UV-light irradiation on thedegration of fipronil in the lab. The reduction of the amount of the pesticide indicated that dissipation rate of fipronil was significantly accelerated by PH value, ozone into aqueous solution and UV-light irradiation.3. Diethylphosphono acetic acid (DPA) was used as a generic hapten in order to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. In an indirect enzyme-linked immunosorbent assay (ELISA) format, the monoclonal-antibody has a high affinity to DPA , chlorpyrifos , diazinon , omethoate , parathion-ethyl and profenofos, showed preferential binding towards pesticides containing diethyl-phosphate and p=o functionalities rather than those containing dimethyl-structure and p=s group. The potential for using DPA was discussed.