Develop Enzyme-linked Immunosorbentassay Kit Of Dimethoate

Pesticide residue is one of the important hazards to human health.At present,organophosphorus pesticide is the most popular pesticide used in agriculture, occupying about 70% to the total used. So detection of organophosphorus pesticide is of significance for food safety.Dimethoate is one of medium toxic organophosphorus pesticide, but its metabolite, O-Dimethoate is much more toxic. Currently, gas chromatography(GC) or high performance liquid chromatography(HPLC) are two common approaches for detection of Dimethoate, involved in long sample cleanup procedureand considered as relatively time-consuming, expensive and laborious. They are not feasible for detection of large numbers of samples and fast detection at spot.On the other hand, ELISA is a rapid,sensitive and selective detection at trace levels. It depends on the high specificity reaction between antibody and antigen, with low cost, safety, easily-handling and reliability.So development of Enzyme-linked immunoassay kit of dimethoate of importance in practice.This research aims to address the key technologies and scientific issues related to establishment of Enzyme-linked immunoassay kit for rapid detection of Dimethoate.According to the molecular structure and biology characteristic of dimethoate,the hapten was synthesized, which then was conjugated to bovine serum album and ovalbumin for prodcuing immunogen and coating antigen. A polyclonal antibody of dimethoate with a titer of 1:256000 and affinity constant of 3.84 ×10¹⁰ had been obtained by immunizing mice. The titer of HRP-labeled antibody is 1:4000. It has relatively low cross-reactivity with other major organophosphorus pesticide, except for O- dimethoate. By indirect and direct ELISA, the optimum coating medium of Dimethoate is 0.05mol/L carbonate buffer, with pH9.6; the optimum coating tempreture and time is 4℃ for 12h; the optimum working concentration of coating antigen, antibody and HRP-labeled goat anti-mouse IgG is 0.8ug/mL, 1:32000 and 1:4000,respectively; the detection limit is 0.01mg/L; the recovery of sample is 95.61%; a good correlation is observed between the ELISA and GC wirh R= 99.46%.The viation coefficient of among batches and within a batch are all lower than 7.72%, showing the stability of the setted kits.