Study On Immunoassay Of Quinclorac And Bensulfuron Methyl

Immunoassay has the characteristics of high sensitivity,strong specificity,and can detect a large number of samples in a short time,so it has an important application value in the detection of pesticide residues.In this study,quinclorac and bensulfuron methyl were used as research objects to prepare monoclonal antibodies against quinclorac and bensulfuron methyl.On the basis of optimizing the immunoassay factors and evaluating the sensitivity,specificity,accuracy and precision of the method,an enzyme-linked immunosorbent assay for quinclorac and bensulfuron methyl was established,and the method was applied to the residue analysis of quinclorac and bensulfuron methyl in soil,water and agricultural products.The established immunoassay methods of quinclorac and bensulfuron methyl can provide technical support for monitoring and controlling herbicide residues,ensuring the quality of agricultural products and environmental safety.1.The hapten was prepared by chemical modification of the structures of quinclorac and bensulfuron methyl.The hapten was verified by ¹H-NMR.The hapten was coupled with BSA and OVA carrier protein by NHS/DCC method to prepare immune antigen and coating antigen.The synthetic artificial antigen was verified by UV spectrophotometry,which showed that the artificial antigen was successfully coupled.2.Mice were immunized with quinclorac and bensulfuron methyl immune antigens to obtain cell lines that could stably secrete monoclonal antibodies against quinclorac and bensulfuron methyl.When the concentration of coating antigen was 1 mg/L,the titers of monoclonal antibodies secreted by hybridoma cells of quinclorac and bensulfuron methyl were 2×10⁵and 2.4×10⁶,respectively.The ascites of mice were induced by intraperitoneal injection of single positive cell strain.The ascites was purified by octanoic acid ammonium sulfate method.Four monoclonal antibodies against quinclorac and two monoclonal antibodies against bensulfuron methyl were prepared.The optimal titer and inhibition of monoclonal antibodies against quinclorac D12 and bensulfuron methyl G11 were selected for subsequent experiments.3.The optimal reaction conditions of ic-ELISA were determined by optimizing the immune analysis factors:the concentration of quinclorac coating antigen was 0.25 mg/L,the dilution ratio of antibody was 20000 times,the concentration of antibody was 2.52 mg/L,the methanol content was 10%,the p H was 7.5 and the Na⁺concentration was 0.4 mol/L.Under the optimal immune reaction conditions,the standard curve of ic-ELISA was established.The concentration of IC₅₀in inhibition was 0.138 mg/L,the LOD(IC₁₀)was 0.007 mg/L,and the detection range(IC₁₀-IC₉₀)was 0.007-1.288 mg/L.The cross reaction rate of dichloroquinoline acid and its structural analogues was lower than 2%,and there was no obvious cross reaction.The coefficient of variation of dichloroquinoline acid in the plate was 2.79-7.77%,and the coefficient of variation between plates was 2.74-7.28%.The average recovery of dichloroquinoline in river water,soil and rice is 90.36-107.21%,and the coefficient of variation is 0.27-6.51%.4.The optimal reaction conditions of bensulfuron methyl ic-ELISA were obtained by optimizing the immune conditions:the concentration of bensulfuron methyl coating antigen was 0.5 mg/L,the dilution ratio of antibody was 27000 times,the concentration of antibody was 1.37 mg/L,the methanol content was 5%,p H was 8.5 and Na⁺concentration was 0.5 mol/L.The standard competition curve of bensulfuron methyl ic-ELISA was established under the optimal immune reaction conditions.The IC₅₀of bensulfuron methyl ic-ELISA was 1.377 mg/L,LOD(IC₁₀)was 0.0249 mg/L,and the detection range(IC₁₀-IC₉₀)was 0.0249-50.132 mg/L.The cross reaction rate of bensulfuron methyl and its structural analogues was lower than 4%,and there was no obvious cross reaction.The coefficient of variation of bensulfuron methyl in the plate was 1.98-7.29%,and the coefficient of variation between plates was2.30-7.28%.The average recovery of bensulfuron methyl in water,soil and rice was 74.99-95.80%,and the coefficient of variation was 1.19-7.05%.