Study On Methods Of Enzyme Linked Immunoassay And Chemiluminescence Enzyme Immunoassay For Prothioconazole Residues

Prothioconazole is a new type of triadimefon fungicide,which can be used to control a variety of cereal crop diseases and has an excellent control effect on almost all wheat diseases.However,due to the unscientific use of prothioconazole,the residue of prothioconazole and its metabolite prothioconazole-desthio do harm to the environmental safety,the quality and safety of agricultural products,and the exposure risk to the application personnel is significant.At present,the main method for residue analysis of prothioconazole and its metabolites is instrumental analysis.This method has the characteristics of high sensitivity,good accuracy and high reliability of results,but it has some shortcomings such as complex pretreatment,expensive equipment and not suitable for field rapid detection and so on.Therefore,in this study,anti-prothioconazole polyclonal antibody was obtained by synthesizing prothioconazole artificial antigen,immunizing rabbits and establishing enzyme linked immunosorbent assay（ELISA）and chemiluminescence enzyme immunoassay（CLEIA）for agricultural products and environmental samples.The research results provide good technical support for ensuring environmental safety and the quality and safety of agricultural products.The main research contents are as follows:1.Screening and identification of polyclonal antibodies against prothioconazoleProthioconazole hapten was synthesized by reaction of prothioconazole with methyl3-bromopropionate,and was identified by nuclear magnetic resonance as the target hapten.Coated antigens and immune antigens were prepared by coupling prothioconazole hapten with chicken egg albumin（OVA）and bovine serum protein（BSA）.The UV spectrum analysis proved that the coupling was successful.The coupling ratios of coated antigen and immune antigen were 31:1 and 34:1,respectively.New Zealand white rabbits were immunized with the successfully conjugated immunogen,and the serum titer of the New Zealand white rabbits reached 1:512000 through five immunizations.A New Zealand white rabbit with good competition results was selected to collect heart blood,purify serum with capra-ammonium sulfate method,and obtain polyclonal antibody against prothioconazole.The binding of polyclonal antibody to prothioconazole was determined by indirect competitive ELISA（ic-ELISA）method with IC₅₀of 0.27μg/m L.The cross-reaction rate of the anti-prothioconazole polyclonal antibody against six prothioconazole analogues except prothioconazole-desthio was not less than or equal to 0.1%.2.Establishment of ELISA method for prothioconazole residueThe obtained prothioconazole artificial antigen and anti-prothioconazole polyclonal antibody were used to establish the ELISA method of prothioconazole residue.By optimizing the working concentration of antigen and antibody,the content of methanol,the concentration of sodium ion and the p H value in the working buffe,the ELISA method was established for the analysis of agricultural products and environmental samples.The results showed that when the concentration of antigen was 0.8625 mg/L and the concentration of antibody was 0.1606 mg/L,the IC₅₀of the ELISA method was 102.3 ng/m L,and the detection limit was 10.7 ng/m L.The established method had 68.0%cross-reaction against prothioconazole-desthio.There was no significant cross-reaction（≤0.1%）with the other six prothioconazole analogues.At the concentration of 7.5μg/g-30μg/g,the recoveries of prothioconazole in wheat grains and water ranged from 81.9%to 104.7%,and the relative standard deviation（RSD）was from 1.5%to 5.6%.Compared with the results of UPLC-MS/MS detection,the linear regression equation was y=1.158x-4.294,and the coefficient of determination was 0.99,indicating that the established ELISA method was in good agreement with the results of UPLC.3.Establishment of CLEIA method for prothioconazole residueBased on the optimal ratio of chemiluminescence substrate,the CLEIA method was established by optimizing the working concentration of antigen and antibody,the content of methanol,sodium ion and p H value in the working buffer solution.The results showed that when the antigen concentration was 0.22 mg/L and the antibody concentration was0.32 mg/L,the IC₅₀of the CLEIA method was 38.0 ng/m L,and the detection limit was 1.2ng/m L.In addition,the established method had 40.4%cross-reaction to prothioconazole-desthio.There was no significant cross-reaction（<0.1%）with the other six prothioconazole analogues.The addition and recovery experiments were carried out on soybean and water.At 100 ng/g-500 ng/g concentration,the recovery ranges from 89.5%to118.4%,and RSD ranges from 2.0%to 11.9%.Compared with the UPLC-MS/MS detection junction,the linear regression equation is y=0.855x+44.545,and the coefficient of determination is 0.938,which indicates that the CLEIA method is in good agreement with the UPLC results.Compared with ELISA method,the detection sensitivity of this method is nearly 3 times higher.In this study,the artificial antigen of prothioconazole was successfully synthesized,and after immunizing New Zealand white rabbits with it,polyclonal antibodies against prothioconazole were screened and obtained.The obtained polyclonal antibodies were used to establish ELISA method and CLEIA method for residues of prothioconazole in agricultural products and environmental samples,and the obtained antibodies had good specificity.The two immunoassay methods for prothioconazole residue established by using the antibody have the advantages of simple treatment,low cost,high sensitivity and environmental friendliness,and can meet the requirements of the current maximum residue limit of prothioconazole residue in agricultural products and environmental samples.