Screening Of Anti-Thiamethoxam Monoclonal Antibody And Developemnt Of Immunoassay For The Detection Of Acetamiprid

Thiamethoxam and acetamiprid are neonicotinoid insecticides,which act by inhibiting acetylcholinesterase activity,thereby interfering with insect nerve signaling and achieving lethal purposes.Because of its high speed,long-lasting effect,wide insecticidal spectrum and low toxicity to mammals,it is widely used in agricultural production,which may cause thiamethoxam and acetamiprid residues in agricultural products.Therefore,there is an urgent need to establish a rapid and sensitive immunoassay method to achieve screening of thiamethoxam and acetamiprid residues in agricultural products.In this paper,the anti-thiamethoxam monoclonal antibody was successfully prepared by fusion technology.Based on this,an immunoassay method for the residues of acetamiprid pesticide was established.The results are as follows:In this study,thiamethoxazine hapten was synthesized using thiamethoxazine as research object.Thiamethoxazine hapten was conjugated with bovine serum albumin(BSA)and ovalbumin(OVA)to form immunogen and coated antigen by active ester method to immunize BALB/c mice.A stable monoclonal antibody against thiamethoxazine was successfully prepared by cell fusion technique,which was 3F7 and identified as IgG1.Two kinds of coated antigens H1-OVA and H2-OVA were prepared by the active ester method using the synthetic acetamiprid hapten(HI)and thiamethoxam hapten(H2),based on titer and competition determination.The H2-OVA with better sensitivity was analyzed for subsequent experiments..By optimizing the experimental conditions such as antigen-antibody concentration,ionic strength and pH value,a standard curve of biotinylated indirect competitive enzyme-linked immunosorbent assay(Bic-ELISA)for the determination of acetamiprid residues was established.The regression equation is y.=0.4102x+0.5978,R2=0.9908,and the minimum detection limit(LOD)was 0.17 ng/mL.The method has good specificity and can be used for qualitative and quantitative detection of acetamiprid.Based on the pre-laboratory study,we successfully detected the residues of acetamiprid in three pollen(water pollen,camellia pollen and rape pollen)by Bic-ELISA,and the recovery rate was 81.1%-108.0%.The relative standard deviations were 4.8%-10.9%,6.1%-11.7%,consistent with HPLC-MSMS results.It shows that the method we have established is accurate and reliable,and the experimental results will provide certain technical support for the market regulation of acetamiprid residue in pollen samples.