Preliminary Establishment Of Enzyme-linked Immunosorbent Assay And Immunochromatography For Thiamethoxam

Thiamethoxam is a new nicotine pesticide,which is widely used in agricultural production.Thiamethoxam residues may occur in agricultural products,which has a certain potential safety hazard to the health of consumers.Therefore,to strengthen the monitoring of new nicotine pesticides is a major food safety issue related to the national economy and people’s livelihood.The development of rapid detection technology has become the research trend of new nicotine pesticide residue detection.This study is mainly divided into two parts: Firstly,to prepare the monoclonal antibody against thiamethoxam,through the optimization of experimental parameters,the enzyme-linked immunosorbent assay(ELISA)method for the monoclonal antibody against thiamethoxam has been successfully established;Secondly,a colloidal gold immunochromatography method for the detection of thiamethoxam pesticide residues was developed by using the colloidal gold labeling technology and the optimization of experimental parameters,and to make the colloidal gold immunoassay Hin test strip.The main conclusions are as follows:The first,three coated antigens,were h1-ova,h2-ova and h3-ova,were prepared by active ester method using the synthesized acetamiprid hapten(H1),thiamethoxam hapten(H2)and imidacloprid hapten(H3).Based on the titer and competitive determination,h2-ova with better analytical sensitivity was selected for subsequent experiments.The indirect competitive enzyme-linked immunosorbent assay(BIC ELISA)for the determination of thiamethoxam residues was successfully established by optimizing the experimental conditions of antigen antibody concentration,ionic strength and p H value.The results show that the optimal working system is: when the coating agent and antibody are diluted 1000 times and 4000 times,there is 5% methanol in the buffer,the ion strength is 0.4 mol/L Na Cl,and the p H is 9.0.Under the optimized conditions,the performance of ELISA method was tested with thiamethoxam standard of different concentration gradients,and the standard curve was drawn.The linear range was 1.0-250.0 ng/ml,and the IC50 value was 28.6 ng/ml.Seven new nicotine pesticides were used for cross reaction,the highest cross reaction rate was 0.28%,and the cross reaction rate of other pesticides was lower than 0.2%,which proved that the method had good specificity and could be used for qualitative and quantitative detection of thiamethoxam.The second,based on the detection principle of double antibody sandwich,the colloidal gold was prepared by trisodium citrate reduction method.The prepared colloidal gold was used as a marker to mark the thiamethoxam monoclonal antibody.The artificial antigen of thiamethoxam was coated on the detection line,and the second antibody of Goat anti mouse was coated on the quality control line.The detection method of colloidal gold immunochromatography strip was successfully established.By optimizing the experimental parameters,the optimal working system was obtained,i.e.adding 20 μL 0.1 mol/l K2CO3 solution,labeling p H value,antibody labeling amount of 20 μL,and using antigen stock solution(undiluted)to draw the membrane.Finally,the sensitivity of the prepared colloidal gold strip was tested.In the experiment,the sensitivity of the strip was 20ng/ml.the strip was used to detect the thiamethoxam pesticide residues in agricultural products with high sensitivity,strong specificity,good stability and without special equipment.It was suitable for the rapid detection of thiamethoxam residues in agricultural products.