Rapid Immunoassays For Six Chemical Residues In Food Samples

The residues of chemical contaminants in food seriously affect the health of consumers.Due to its simplicity,high efficiency,portability,cheapness and suitable for batch detection,immunoassay has been widely used in food detection.Six chemical drugs,namely,diafenthiuron,thiophanate-methyl,fenthion,carbofuran,carbosulfan and piperacillin,were used as research objects.Six monoclonal antibodies against the drugs were prepared.Based on the antibodies,indirect competitive enzyme-linked immunosorbent assay and colloidal gold immunochromatography assay were established for the detection of six chemical residues in vegetables and dairy products.（1）After analyzing the synthesis process and structural characteristics of the target chemical drugs,screening the suitable analogues,the specific haptens were obtained by chemical modification of the suitable analogues.The first is to use the amino group of analogues or the amino group introduced by nitro reduction to acylation reaction with succinic anhydride,and to introduce 4C spacer arm,diafenthiuron hapten,fenthion hapten and thiophanate-methyl hapten 1 were synthesized.The second is to synthesize thiophanate-methyl hapten 2 by nucleophilic addition reaction of the amino group of the analogue with 3-carboxyphenyl isothiocyanate,introducing the spacer arm with a benzene ring.And the third is to utilize the hydroxyl group of the analogue,two kinds of haptens shared by carbofuran and carbosulfan were synthesized by three steps reaction（esterification,aminolysis and N-Boc protection,deprotection;esterification,aminolysis and ester hydrolysis）with spacer arm.The haptens were conjugated with carrier proteins by active ester method to synthesize complete antigens,which were identified and characterized by UV-Vis photometer and SDS-PAGE.Using hybridoma cell technology,the mice were immunized for cell fusion experiments and monoclonal antibodies were obtained by the ascites purification,they are 5B3（for diafenthiuron）,2E4（for thiophanate-methyl）,1H2（for fenthion）,1B2（for piperacillin）and 5E6（for carbofuran and carbosulfan）,and the subtypes were Ig G1,Ig G1,Ig G2a,Ig G2b and Ig G1,respectively.The affinity constants were 3.67×10⁷ L/mol,9.92×10⁸ L/mol,2.12×10⁹ L/mol,2.15×10⁹ L/mol and 6.35×10⁹ L/mol,respectively.（2）Six ic-ELISA methods were developed by optimizing the concentration of antigens and antibodies and the pH,ionic strength and organic solvent content of the working solution.After the vegetables were extracted with acetonitrile and centrifuged,the supernatants were dried with nitrogen,and then redissolved with 0.01 mol/L PBS（pH 7.4）containing methanol or DMF for testing.The milk sample was diluted tenfold for testing.The IC₅₀ values of diafenthiuron,thiophanate-methyl,fenthion,carbofuran,carbosulfan and piperacillin by ic-ELISA were 20.96 ng/m L,173.6 ng/m L,11.70 ng/m L,0.187 ng/m L,13.71 ng/m L and 0.136 ng/m L,respectively,and the linear detection ranges(IC₂₀～IC₈₀)were 3.84～114.48 ng/m L,45.76～658.32 ng/m L,2.27～60.38 ng/m L,0.047～0.749ng/m L,2.31～81.42 ng/m L and 0.036～0.518 ng/m L,respectively.According to the results of recovery of piperacillin in milk and five pesticides in vegetable matrix,the recovery rates of six chemical drugs were in range of 82.1%～113.0%,and the coefficient of variation was less than 11.4%.（3）The detection probes were prepared by labelling antibodies with colloidal gold nanoparticles.The colloidal gold immunochromatography strips were developed by optimizing the concentration of gold-labelled antibodies,coating antigens and the types of gold-labelled antibody suspension buffer.For vegetable samples,0.01 mol/L PBS containing 10%and 20%methanol（pH7.4）were used as extracting solutions to detect carbofuran and thiophanate-methyl,respectively;to detect fenthion and carbosulfan,the samples were extracted with methanol,centrifuged,and the supernatants were dried with nitrogen,and then redissolved with 0.01 mol/L PBS（pH 7.4,1%ON-870）containing 5%methanol and 10%methanol for testing,respectively;to detect diafenthiuron,the samples were extracted with acetonitrile,centrifuged,and the supernatants were dried with nitrogen,then redissolved with 0.01 mol/L PBS（pH 7.4,1%ON-870）containing 5%DMF.Milk samples were directly tested,and milk powder samples were diluted fourfold with 0.01 mol/L PBS（pH 7.4,1%ON-870）.When testing vegetable samples,the visual limit of detection（v LOD）and cut-off value of diafenthiuron were 0.1μg/g and 10μg/g;the v LOD and cut-off value of thiophanate-methyl were 1μg/g and 5μg/g;the v LOD and cut-off value of fenthion were 50 ng/g and 200 ng/g;the v LOD and cut-off value of carbofuran were 0.2 ng/g and 2 ng/g;the v LOD and cut-off value of carbosulfan were10 ng/g and 50 ng/g.When testing milk samples,the v LOD and cut-off value of piperacillin were 2ng/m L and 5 ng/m L.When testing milk powder samples,the v LOD and cut-off value of piperacillin were 5 ng/g and 20 ng/g.The results indicated that the colloidal gold immunochromatographic strips could be used for rapid screening of diafenthiuron,thiophanate-methyl,fenthion,carbofuran,carbosulfan and piperacillin in food samples.In conclusion,five ic-ELISA and GICA methods were developed,which met the detection requirements of six chemical contaminants in vegetables or dairy products and provide rapid analytical methods for the detection of food safety at the grass-roots level.