Development Of Enzyme Linked Immunosorbent Assay (Elisa) For The Multi-Residue Detection Of O-(2-Isopropylcarbonylphenyl)Thiophosphoryl Amide Pesticides

According to the common structure of O-(2-isopropylcarbonylphenyl) thiophosphoryl amide organophosphorus pesticides, universal hapten was synthesized from O-methyl dichlorothiophosphate and isopropyl salicylate (2-hydroxy-benzoic acid1-methylethyl ester) and γ-aminobutanoic acid. Bovine serum albumin (BSA) and ovalbumin (OVA) were used as protein carriers respectively to couple with hapten by an active ester method. Complete antigens were prepared as immunogen and coating antigen. BALB/C mouse were immunized with immunogen. The cells were fused and screened with conventional method. At last, one cell strain of monoclonal antibody named6B5/E3was obtained. The subtype of immunoglobulin was IgGl and the isotype of light strains was κ. The affinity constant was4.64×10L/mol. The monoclonal antibody could only recognize isocarbophos, isofenphos-methyl and isofenphos, while could not recognize other pesticides. Based on the monoclonal antibody secreted by the hybridoma cell line6B5/E3, a multi-residue determinate method for the O-(2-isopropylcarbonylphenyl) thiophosphoryl amide pesticides was established by investigating the factors, such as blocking agents, ionic strength, pH and so on. Under the optimal detection system, the standard curves of three pesticides (isocarbophos, isofenphos-methyl and isofenphos) were established by indirect enzyme-linked immunsorbent assay respectively. Logit (B/Bo) was used as the lateral coordinates (y) and logarithm of concentration of these three pesticides was used as the longitudinal coordinates (x), three linear equations were obtained after inversion. The linear equation of isocarbophos was y=-2.1313x+2.9175with a coefficient of correlation0.9908, the I50was23.39ng/ml, the limit detection was12.19ng/ml, the average recoveries in soil, distiller water and rice were112.86%,103.54%and104.81%respectively. The linear equation of isofenphos-methyl was y=-1.9333x+3.5754with a coefficient of correlation0.9938, the I50was70.79ng/ml, the limit detection was34.51ng/ml, the average recoveries in soil, distiller water and rice were105.63%,107.84%and101.85%respectively. The linear equation of isofenphos was y=-1.2001x+2.1553with a coefficient of correlation0.9796, the I50was62.52ng/ml, the limit detection was19.68ng/ml, the average recoveries in soil, distiller water and rice were100.54%,91.63%and102.27%respectively.In our work, monoclonal antibody which could detect O-(2-isopropylcarbonylphenyl) thiophosphoryl amide pesticides was obtained. This paper had provided theoretical references for detecting these pesticides simultaneously. It also had provided a new method for quick detection of them and a condition for the development of pesticide detection kit.