Rapid Immunoassay For The Detection Of Four Pesticide Residues In Agricultural Products

The development of modern agriculture is inseparable from the use of pesticides,but pesticide residues bring not only environmental pollution and food safety problems,but also problems that affect the development of the national economy.At present,the detection of pesticide residues mainly relies on instruments,because of the high cost of instruments,it is difficult to be popularized.Therefore,in order to increase the monitoring of pesticide residues,meet market demand,and ensure food safety,it is significant to build a high-sensitivity,high-throughput,convenient,and rapid method that can be applied in more case.In this study,four pesticides,Procymidone,Trifluralin,Isoprocarb and Metalaxyl,were prepared to prepare monoclonal antibodies with strong specificity and high sensitivity.On this basis,an indirect competitive enzyme-linked immunosorbent assay（ic-ELISA）method and a colloidal gold immune-chromatographic assay（GICA）method were established and used to detect actual samples.First,the hapten was designed and synthesized based on the structure of Procymidone,Trifluralin,Isoprocarb,Metalaxyl,and the hapten was coupled to the carrier protein by the carbodiimide（EDC/NHS）method.Complete antigen,identified by UV-Vis spectrophotometer after successful coupling,immunized BALB/c mice with it,and screened out mice with positive spleen cells for cell fusion,after 3 subclonal screening and ascites preparation.After the steps of antibody purification and other steps,the monoclonal antibodies of four drugs were finally obtained:Procymidone 1E6,Trifluralin 2D7,Isoprocarb 1E1,Metalaxyl 2E2,and the affinity constants were:6.9×10⁸ L/mol,9.4×10⁸ L/mol,6.2×10⁸ L/mol,7.5×10⁸ L/mol,antibody subtypes are:IgG1,IgG2b,IgG1,IgG3,respectively.Based on the above four monoclonal antibodies,an ic-ELISA detection method for Procymidone,Trifluralin,Isoprocarb,and Metalaxyl was established,and the standard dilutions in the method were optimized.The IC₅₀ of the four monoclonal antibodies measured under the optimal conditions were 2.13 ng/m L,4.06 ng/m L,1.04 ng/m L,0.54 ng/m L,and the detection linear range was 1.79～6.676 ng/m L,1.47～11.21 ng/m L,0.381～3.77 ng/m L,0.151～1.82 ng/m L;similar to the corresponding drug structure,the cross-reaction rate of the substances is less than1%.The recovery rates of the four drugs in different actual samples are all in the range of90%～120%,and the coefficient of variation is less than 10%,which can be used for actual sample detection.Finally,the corresponding GICA detection methods were established for the four monoclonal antibodies,and the actual samples were tested.The delineation values of Procymidone test strips in leek,corn,green pepper,and cucumber samples were 1000 ng/g,1000 ng/g,200 ng/g and 200 ng/g;The line elimination values of the samples of Trifluralin test strips in cucumber,grreen pepper,and rice are 200 ng/g,500 ng/g,and 500 ng/g respectively;the line elimination values of the isopropanol test strip in the samples of corn,green pepper,and rice are 20 ng/g,50 ng/g,200 ng/g;the elimination values of metalaxyl test strips in rice,green pepper,and tobacco samples are 20 ng/g,50 ng/g,and 200 ng/g,respectively.The four test strips have good sensitivity,can meet market demand,and can be used for on-site rapid detection of pesticide residues in agricultural products.