Determination Of Organophosphorus Pesticides Multi-residues By Immunoassay Methods

The aim of the study was to develope a multi-residue immuoassay for the determination of organophosphorus pesticides. First of all, the hapten (O, O-dimethyl-O - (4 - propionyloxy) phosphate) of methyl-paraoxon was synthesized through three steps using 3-(4-Hydroxyphenyl) propionic acid and dimethyl chlorophosphate as the raw materials. NH2-Parathion was choosen for the hapten of Parathion. Then, the two haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA) to synthesize immunogen and coating antigen respectively. According to the immune protocol, four New Zealand white rabbits were immunized for six times by two immunogens during three months, and the antiserum with high titres were choosen for the experiment.Using the parathion as the inhibitor, an indirect competitive ELISA was developed. For application to actual samples, the in?uence of several factors such as organic solvent, ionic strength and pH were studied. Under optimized conditions, for parathion, the quantitative working range was 0.014～8.24μg/mL with a limit of detection (LOD) of 0.005μg/mL and the IC50 was 0.343μg/mL; for methyl-paraoxon, the quantitative working range was 0.015～12.02μg/mL with a limit of detection (LOD) of 0.004μg/mL and the IC50 was 0.368μg/mL. There was negligible cross reactivity (CR) with other OP pesticides. The fortified recovery test of parathion and methyl-paraoxon was performed with the ic-ELISA method by spiking three level concentrations (0.1, 0.5, 1.0μg/mL) in cabbage. The recoveries were 79.00%～88.40% and 74.00%～81.40%; the C.V% were 3.48%～8.53% and 3.53%～7.89%, respectively.Furthermore, the research of multi-residue immuoassy for diethoxy organophosphorus pesticides was also performed. First of all, the structures of some diethoxy organophosphorus pesticides such as chlorpyrifos, diazinon, parathion, paraoxon and phoxim were analyzed, and it was found that the common structure was (C2H5O)2P(O) or (C2H5O)2P(S), then diethylphosphonoacetic acid(DPA) was selected as the general-hapten.Then the hapten was conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) using active ester method. New Zealand white rabbits were immunized with the hapten-BSA conjugates, and the polyclonal antibodies against the DPA-BSA were raised. Using the hapten (DPA) and paraoxon as the inhibitors, the indirect competitive EL1SA was developed and optimized, respectively. Under the optimal conditions, other similar organophosphorus pesticides were used as inhibitors to test for the cross-reactivity. The results of these assays showed that the pesticides which cause relative high inhibition to the antiserum obtained from DPA-BSA were paraoxon and pirimiphos.The IC50 were 1.69 and 2.33μg/mL; the cross-reaction rate were 13.3% and 9.6%. All the other Ops (parathion, chlorpyrifos, diazinon and malathion) tested showed no cross reactivity. The fortified recovery test of paraoxon and pirimiphos was performed with the ic-ELISA method by spiking three level concentrations (0.5, 1.0, 5.0μg/mL) in cabbage. The recoveries were 75.02%～78.37% and 75.02%～78.37%; the C.V% were 3.04%～3.79% and 2.82%～3.36%, respectively.