Development Of Rapid Immunoassay For Imidacloprid,Acetamiprid,Carbofuran And Aldicarb

Nowadays,food safety has been closely watched,the government has invested a lot of energy to manage the issues.The food safety problems about pesticide residue are so common that the detection of pesticide residue is very important.The traditional chemical analysis method is not enough to meet the huge agricultural market,so it is greatly significant to develop a rapid and sensitive detection method of pesticide residue for the initial screening of agricultural products.The aim of this study was to develop an indirect competitive enzyme-linked immunoassay?ic-ELISA?and immunochromatographic strip assays for the detection of four common nicotine and carbamate pesticides including imidacloprid,acetamiprid,carbofuran and aldicarb in vegetables and fruits.As the four pesticides are small molecules,have no immunogenicity and no reactive groups for coupling with carrier proteins.According to the chemical structure of the four pesticides,the corresponding haptens were respectively designed and synthesized.The haptens possess reactive groups such as carboxyl,amino groups,etc.which could conjugate with carrier proteins.To obtain different immunogens and coating antigens,the conjugation between haptens and carrier proteins keyhole limpet hemocyanin?KLH?,bovine serum albumin?BSA?and chicken egg albumin?OVA?)was performed via 1-Ethyl-carbodiimide hydrochloride?EDC?and glutaraldehyde?GA?,respectively.BALB/c mice were immunized by conventional and rapid immunization.Mice sera were detected by ic-ELISA to screen out the mouse with high titer and inhibition,then for cell fusion.The supernatant of hybridoma was detected by ic-ELISA,and the hybridoma cells with good specificity and high sensitivity were subcloned by limiting dilution method.The monoclonal hybridoma cell lines were expansively cultured to prepare ascites.Monoclonal antibodies imidacloprid 3F2,acetamiprid 1B3,carbofuran 3H2 and aldicarb 4D10 were purified by caprylic acid and ammonium sulfate precipitation method.The affinity constants were 2.04×109,9.22×108,4.32×108 and 1.96×108 L/mol,respectively.The IC₅₀ values of the monoclonal antibodies against imidacloprid,acetamiprid,carbofuran and aldicarb?0.274,0.396,0.284,18.505 ng/m L?were determined by optimizing pH,ionic strength?NaCl content?and acetonitrile content of standard diluent.And the working ranges?IC20–IC80?were 0.072–1.037,0.096–1.629,0.078–1.033,4.602–74.411 ng/m L.The monoclonal antibody against acetamiprid also has a high cross-reactivity with thiacloprid.The IC₅₀ value of thiacloprid was 0.093 ng/m L and the working range was 0.036–0.241 ng/m L.This indicated that simultaneous detection of acetamiprid and thiacloprid residues could be achieved.The cross-reactivities of the other three antibodies to corresponding similar drugs were less than 1%,which indicated the high specificity of antibodies.Furthermore,cucumber and apple samples spiked with analysis?imidacloprid,acetamiprid,thiacloprid,carbofuran and aldicarb?were detected.The recoveries were between 80%–120%,which indicated the feasibility of ic-ELISA detection method for the actual samples of fruits and vegetables.Immunochromatographic strip assays were also established to detect cucumber and apple juice.And cucumber juice was not diluted,while apple juice was diluted three times after adjusted the pH to neutral with 1 M of NaOH.The cut-off values of strip assay for imidacloprid,acetamiprid,thiacloprid,carbofuran and aldicarb in cucumber and apple were 10 ng/g and 20 ng/g;5 ng/g and 10 ng/g;2.5 ng/g and 5 ng/g;5 ng/g and 10 ng/g;100 ng/g and 200 ng/g,respectively.The result can satisfy the market demand.Therefore,these two methods can be used in the initial screen of agricultural products for imidacloprid,acetamiprid,thiacloprid,carbofuran and aldicarb residues.