Study Of Rapid Immunoassay For Detecting Fipronil,Mebendazole And Endosulfan

The residues of pesticides and veterinary drugs in food have always the focus of attention,the ministry of agriculture also set corresponding rules and regulations to control the use of pesticides and veterinary drugs.However,the illegal use of outlaws has led to serious problem of pesticides and veterinary drugs residues in food.At present,the detection of pesticides and veterinary drugs mainly relies on traditional instrumental analysis methods.These methods are high sensitive and accurate;however,they are generally time-consuming and high costly,which cannot satisfy the demand for the detection of a large number of samples in the market.It is required to develop rapid and reliable method that suitable for on-site detection of a large of samples.The fipronil?FIP?,mebendazole?MBZ?and endosulfan?END?were used as the target in the paper,based on the prepared high-sensitivity monoclonal antibody,the corresponding ic-ELISA and colloidal gold immunochromatographic strip were developed and applied in real samples analysis.The main research contents and results are as follows:1.The complete antigens of FIP,MBZ and END were designed and synthesized successfully.The mice were immune with the prepared immunogen and mice sera were analyzed by the ic-ELISA method,the mouse that showed good affinity and inhibition for the targeted compound was selected for cell fusion and then obtained hybridoma cells.The ascites was prepared and obtained monoclonal antibodies.The affinity constants of FIP,MBZ and END antibodies were 4.12×10¹⁰,2.52×10¹⁰ and 3.80×10⁹L/mol respectively,the subtypes were IgG1,IgG2a and IgG1,the light chain were Kappa,Kappa and Lambda,respectively.2.The standard diluent of ic-ELISA were optimized,and the standard curves were established under optimal conditions.The IC₅₀ of monoclonal antibodies against FIP was 0.43 ng/mL and had good cross-reactivity with fipronil-desulfinyl?FIPCF₃?,fipronil-sulfone?FIPSO₂?and fipronil-sulfide?FIPSO?,the IC₅₀0 was 0.38,0.58 and 0.66ng/mL respectively,which can simultaneously detect fipronil and its three metabolites.The IC₅₀ of monoclonal antibodies against MBZ was 0.16 ng/mL,and also had cross-reactivity with albendazole?ABZ?,flubendazole?FLU?and fenbendazole?FBZ?,the IC₅₀ was 0.16,0.44,0.11 and 0.61 ng/mL respectively,which can detect 4benzimidazoles residues.The END antibody only specifically recognized END,the IC₅₀0 of END,?-endosulfan??-END?and?-endosulfan??-END?was 5.16,4.95 and 6.65ng/mL.The ic-ELISA method was developed based on the corresponding monoclonal antibody and applied in real samples analysis.The recovery rate was 83.46%-118.92%,which indicated the high accuracy and feasibility of the method.3.The corresponding colloidal gold immunochromatographic strip based on monoclonal antibodies was developed and applied in egg,carp and pear analysis.The detection limits of FIP,FIPSO₂,FIPSO and FIPCF₃ in egg were 10,10,10 and 5 ng/g,respectively.The detection limits of MBZ,ABZ,FLU and FBZ in carp were 2,4,1 and10 ng/g,respectively.The detection limits of END,?-END and?-END in pear were all40 ng/g.The above three kinds of colloidal gold immunochromatographic strips all satisfied the detection demand of the market.