Establishment Of Immunoassays For Four Pesticides In Cucumber

Pesticides are generally divided into insecticides,fungicides,herbicides,plant growth regulators and so on.Pyrimethanil and picoxystrobin are fungicides,isoproteron belongs to herbicides,and gibberellin acid belongs to plant growth regulators.They are often applied to crops to prevent diseases and insect pests or regulate plant growth.In actual agricultural production,failure to observe the drug withdrawal period or excessive use can easily lead to pesticide residues in food and agricultural products,which will bring great harm to people's health.Therefore,developing on-site and rapid detection methods is an important way to guide the use of pesticides.Indirect competitive enzyme-linked immunoassay?ic-ELISA?and immunochromatographic test strip analysis?ICS?methods have the advantages of simple operation,low cost,high sensitivity,and short time.The four pesticides of pyrimethanil,picoxystrobin,isoproturon and gibberellin acid were the targets,and two kinds of rapid immunoassay method were successfully developed and applied in actual determination.First,according to the structural characteristics of pyrimethanil,picoxystrobin,isoproturon and gibberellin acid,the structural transformations were carried out to prepare haptens,and then coupled with carrier proteins to synthesize their respective complete antigens,immunized mice,using hybridoma cell technology,specific monoclonal antibodies were obtained by screening.The affinity constants of the four monoclonal antibodies of pyrimethanil antibody 1E7,picoxystrobin antibody 3C2,isoproteron antibody 3G9,gibberellin acid antibody 4H11 were5.54×10⁹ L/mol,2.42×10¹⁰ L/mol,4.49×10⁹ L/mol,1.90×10⁹ L/mol.Optimized the organic solvent?methanol content?,pH and ionic strength?NaCl content?of the test solution,under the optimal conditions,for four pesticides:pyrimethanil,picoxystrobin,isoproturon and gibberellin acid,the half-inhibitory concentrations(IC₅₀)of monoclonal antibodies were4.00 ng/mL,0.10ng/mL,1.94 ng/mL,71.48 ng/mL;working range(IC₂₀?IC₈₀)were 1.20?15.53 ng/mL,0.04?0.25 ng/mL,0.46?8.19 ng/mL,21.76?234.87 ng/mL,respectively.The cross-reactivities of pyrimethanil 1E7,picoxystrobin 3C2,isoproteron 3G9 was less than 5%.the cross-reactivities of gibberellin acid 4H11 with structural analogs gibberellin A1,gibberellin A3,gibberellin A4,gibberellin A7 were:6.8%,5.8%,4.5%,3.4%,respectively.These showed that the cross-reactivities of the four monoclonal antibodies were less than 10%,and had good specificity.Based on the prepared monoclonal antibody and complete antigen,using cucumber as the detection matrix,the ic-ELISA detection method was evaluated by adding recovery experiment.At the addition levels of 20.0 ng/g,40.0 ng/g,and 80.0 ng/g,the recovery of pyrimethanil was96.6%?99.7%,and the coefficient of variation was 1.1%?1.8%;at the addition levels of 0.5ng/g,1.0 ng/g and 2.0 ng/g,the recovery of the picoxystrobin was 95.2%?98.5%,and the coefficient of variation was 1.5%?6.5%;at the addition level of 10.0 ng/g,20.0 ng/g and 40.0ng/g,the recovery rate of isoproteron was 84.9%?97.4%,and the coefficient of variation was1.5%?5.0%;at the addition level of 50.0 ng/g,100.0 ng/g,and 200.0 ng/g,the recovery rate of gibberellin acid was 92.7%?99.9%,and the coefficient of variation was 3.3%?4.5%.Further,by optimizing the concentration of gold-labeled antibody,the concentration of sprayed membrane antigen,etc,the immunochromatographic test strips of pyrimethanil,picoxystrobin,isoproturon and gibberellin acid were developed.The elimination values of pyrimethanil,picoxystrobin,isoproturon and gibberellin acid were 50 ng/g,100 ng/g,25 ng/g and 200 ng/g,respectively.In summary,this project successfully established the enzyme-linked immunoassay method and immunochromatographic test strip analysis method of pyrimethanil,picoxystrobin,isoproturon and gibberellin acid,and achieved the rapid detection needs of four pesticide residues in agricultural products.