Study On Enzyme-linked Immunosorbent Assay For Pesticide Carbendazim

It is of concern that in China pesticide residues in vegetable and fruits are found frequently excess to MLs, particular in organophospherous and carbamate category due to improper use of pesticides. In order to meet the need from rapid detection of pesticide residues in agricultural products and in-situ testing, immunoassay technology was applied to develop a quantitative detection method, indirect competition ELISA for carbendazim, a wide-spectrum and widely-used carbamate insecticide in China, to provide technical support for establishment of ELISA kit, with a aim to provide an technical supporting blocks for rapid in-situ detection and surveillance of pesticide residues in vegetables and fruits.Carbendazim(MBC), C₉H₉N₃O₂, methyl 3H-benzoimidazol-2-ylaminoformate), shared similar affinitive group, benzoimidazol ring, with benomyl and the same as the metabolites of thiophanate and thiophanate methyl. Two procedures were designed for synthesis of hapten of carben dazol as follows: (1) To activate the imine group on benzoimidazol ring and insert activated hydroxyl group to produce one of haptens, 1-oxycarbonylethyl propionate-2- benzoimidazol-2-ylaminoformate, the affinitive specific groups, benzoimidazol ring and aminoformate were maintained;(2) To conduct hydrosis of Carbendazol under alkine condition and produce 2-amino-2- benzoimidazol-2-ylaminoformate and acylize with succinic acid to got the final hapten, 2-succinimide benzoimidazol. The results of HNM and IR analysis indicated the synthesis success of both haptens. Then MBC-BSA for preparation of antiserums and OVA-MBC for coating were also obtained with couping mole ratios of ranging from 3-35:1, suggesting meeting the requirement of immuning animals of small haptens.Multicolonial antiserums with high-titer over 64000 were obtained and puritified by acid-ammonium sulfate methods. It affiniively reacts to benomyl(affinitive 100%), but less cross reactive to other catolags such as thiophanate and thiophanate methyl. It applies to develop ELISA technique.Working parameters of indirect ELISA technique for detection of carbendazol was optimized, including selection of coating method, optimum dilution ratio of antiserum and medium mediation. A Indirect ELISA detection method for carbendazol was developed, in which, inhibition ratio closely related to LN value of carbendazol concentration and fitted into equation, Y=11.832Ln(x)+57.366, where Y is inhibition ratio and x represents the concentration of carbendazol, R²=0.9989**, detection concentration range from 0.05ng/L-15ng/L, and recovery of added standard reach 109% with C.V. of 6.2-16.The cross-reaction of anti-MBC-BSA to their corresponding analogs were tested. The maximal concentration for half inhibition of antibody-antigen reaction were 4.0 ng/g for Benomyl, 30 ng/g for thiabendazol, cross reactivity 8%, above 200 ng/g for thiophanate-methyl cross reactivity below 2%, above 500 ng/g for 2-aminobenzimidazole.lt is indicated this method can apply to detect Benomyl andMBC.At spiked levels of 0.05, 0.1, and l.Oug/mL, recoveries of MBC in soil samples ranged from 109 %to 88.5% with C.V.% of 4.06- 8.65, in tap water ranged from 99.4% to 76.5% with C.V. % of 5.85-3.21.Above results showed that the indirect ELISA detection method of caibendazol residue was satisfied for pesticide residues analysis > and to provide technical basis for establishment of ELISA kit.