The Preparation Of Monoclonal Antibodies And Development Of Immunoassays For Thiacloprid

Thiacloprid is a high-efficient,low-toxic,broad-spectrum neonicotinoid insecticide developed by Bayer,which is widely used to prevent piercing-sucking and chewing mouthparts pests.At present,high-performance liquid chromatography(HPLC)is still the mainly detect method for thiacloprid,however,there are some problems such as large cost,large dosage of organic solvents,complexed operation program and difficulties of realizing high-thoroughput detection by instrument detection.In this study,the high-level monoclonal antibody based on the thiacloprid was produced.And an indirect competition Enzyme-linked ImmunoSorbent Assay(ic-ELISA),a fluorescence polarization immuno-assay(FPIA)and an upconversion fluorescence immunochromatographic Assay(U-ICA)on basis of anti-thiacloprid monoclonal antibody has been developed.The study results showed that the three immunoassays would be the rapid detection for monitoring of thiacloprid in agricultural and environmental samples.In this study,the thiacloprid coating antigens was prepared with the haptens(produced in laboratory)by using active ester method and the molar ratio was 14:1.The BALB/c female mice were immunized by routine immunization and rapid adjuvant immunization using the thiacloprid immunogen prepared in the laboratory.The results showed that routine immunization was more effective,because one stability and highest sensitive cell(C9)which could secrete anti-thiacloprid monoclonal antibody,was chosen from the three hybridoma cell lines that the immunization produced.After the assay,the resulting of the monoclonal antibody subtype was IgG2b.The ic-ELISA method was established by using the prepared monoclonal antibody and the condition of concentrations of antibodies,coating antigen and buffer solution was optimized.Under the optimum conditions,the IC50 was 2.31 ng/mL and the linear range(IC10-IC90)was 0.44-16.97 ng/mL.The cross reactivities(CRs)with some analogues of thiacloprid were lower than 0.89%,except acetamiprid(CR=6.20%).Eight substrates(water,rice,soil,wheat,pear,apple,tomato and cucumber)were spiked with thiacloprid standards at 0.1-1 mg/kg.The average recoveries were 77.12%-109.81%and the relative standard deviations(RSDs)were 0.36%～19.50%.The immunoassay also valued by HPLC method.The line equation and correlation coefficient of two methods were y=0.9546x+0.0601(R2=0.9646),the results demonstrated that the ic-ELISA would be a great reliability and accuracy and was available method for the monitoring of thiacloprid in environmental samples and agricultural products.The Fluorescence polarization immunoassay(FPIA)was established by thiacloprid monoclonal antibody and optimized the fluorescent tracer,working concentration of antibodies and buffer solution.The most suitable buffer solution was 0.1 mol/L Na+ borate saline buffer including 5%methanol and pH 5.4.Under optimal conditions,The IC50 was 14.34 ng/mL and the linear range(IC10-IC90)was 2.43-103.19 ng/mL according to the standard curves that were obtained.The FPIA method was highly specific to thiacloprid,and the CR rate with analogues was lower than 3.31%(acetamiprid CR=3.31%,clothianidin CR=0.54%,imidaclothiz CR=0.33%,imidacloprid CR=0.11%).Four substrates(rice,soil,tomato and cucumber)were spiked with thiacloprid standards at 0.2-1 mg/kg.The average recoveries were 71.01%～109.94%and the relative standard deviations(RSDs)were 2.97%～15.37%.Detected the concentrations of thiacloprid in tomato samples with FPIA method,a good correlation was obtained between the FPIA and HPLC y=0.8567x+0.0716(R2=0.9658),the results showed that FPIA method was reliable and accurate,and could be used for the detection of thiacloprid in environmental samples and agricultural products.Based on the preparation of McAb,the upconversion fluorescence immuno-chromatographic assay(U-ICA)was developed.The up-conversion phosphor,working concentration of antibodies and buffer solution were optimized.The best condition of U-ICA is 0.2 mol/L Na+ phosphate buffer solution including 2.5%methanol by pH 7.4.Under optimal conditions,The IC50 was 70.09 ng/mL according to the standard curves that were obtained,the linear range is 17.86 ng/mL-461.71 ng/mL.The cross reactivities(CRs)with some analogues of thiacloprid were lower than 0.79%except with acetamiprid(CR=3.51%).The average recovery in soil and cucumber samples was 81.30%～101.72%,with the RSD was between 4.04%～13.63%.A good correlation was obtained by detected the concentrations of thiacloprid in tomato samples with U-ICA method.The line equation and correlation coefficient of U-ICA and HPLC methods were y=0.928x+0.028(R2=0.9809),which suggested the U-ICA method could be a rapid thiacloprid detection in environmental samples and agricultural products.