| Pesticides are widely used in the growth,protection,storage and transportation of agricultural products.While maintaining the rapid development of agriculture,it also brings health risks.The framework of pesticide residue detection systems in many countries were established by and large.However,it is necessary to establish fast and sensitive detection methods to meet the actual detection requirements.In this study,monoclonal antibodies were prepared with five pesticides,including 2,4-D,butralin,trifluralin,flubendiamde and isocarbophos.And two immunoassay methods,indirect competitive enzyme-linked immunosorbent assay（ic-ELISA）and colloidal gold immunochromatography（GICA）,were established.Considering the chemical structure,acquisition of raw materials,difficulty of derivatization and other factors,the haptens were designed,modified and synthesized.The complete antigens were synthesized by the carbodiimide method and applied to immunizations.The antiserum was detected by ic-ELISA for selecting the qualified positive splenic cell donors for cell fusion.Hybridoma cells have been acquired by selective culture and screening.Finally,the ascites was prepared by inducing method.After purification of ascites,we obtained 2,4-D antibody 3C9,butralin antibody 5G6,trifluralin antibody 4H6,flubendiamde antibody 2B1 and isocarbophos antibody 3G11.The subtypes of antibodies were Ig G1,Ig G1,Ig G2b,Ig G2a and Ig G2b,respectively.The affinity constants were6.773×109 L/mol,6.876×109 L/mol,3.871×109 L/mol,5.279×109 L/mol and 4.068×109 L/mol,respectively.The cross-reaction rates of the antibodies were all less than 5%,indicating high specificity.By determining the working point（concentration of antigen and antibody）and optimizing the buffer liquid system（p H,ionic strength,methanol content）,the corresponding ic-ELISA method was established.The detection linear ranges(IC20～IC80)of 2,4-D,butralin,trifluralin,flubendiamde and isocarbophos were 0.64～9.80 ng/m L,0.77～15.11 ng/m L,0.41～8.79 ng/m L,0.46～5.22 ng/m L and 0.86～23.50 ng/m L,respectively,and the half inhibition rates(IC50)were 2.50 ng/m L,3.42 ng/m L,1.89 ng/m L,1.55 ng/m L and 4.48 ng/m L,respectively.Combined with the requirement of national standard and the actual situation,the vegetable samples such as cabbage and green pepper were selected for recovery experiment.The ic-ELISA of five pesticides achieved good recoveries ranging from 80%to 120%with a coefficient of variation（CV）less than 10%.By synthesizing GNP-labeled m Abs and optimizing the scribing concentration,the corresponding GICA method was established,which can be used for qualitative and quantitative analysis.Qualitative results showed the cut-off values of 2,4-D strip in cabbage and bean sprouts both were 200 ng/g,the cut-off values of butralin strip in cabbage and green pepper both were 200 ng/g,the cut-off values of trifluralin strip in cabbage and green pepper both were 100 ng/g,the cut-off values of flubendiamde strip in cabbage and cucumber both were 100 ng/g,and the cut-off values of the isocarbophos strip in cabbage and radish both was100 ng/g.The quantitative detection results showed the limit of detection（LOD）of 2,4-D in cabbage and bean sprout were 1.2 ng/g and 3.2 ng/g.The LOD of butralin in cabbage and green pepper were 2.21 ng/g and 0.64 ng/g.The LOD of trifluralin in cabbage and green pepper were 3.83 ng/g and 2.92 ng/g.The LOD of flubendiamde in cabbage and cucumber were 3.87 ng/g and 1.17 ng/g,and the LOD of isocarbophos in cabbage and radish were 1.76ng/g and 1.23 ng/g.The GICA was successfully applied to detect vegetables and achieved good recoveries ranging from 80%～120%with a coefficient of variation less than 15%.Confirmed by LC-MS/MS method,the GICA provides a sensitive,efficient and reliable rapid detection method,which could be used for rapid screening of pesticide residues in vegetables. |
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