COMMERCIALLY AVAILABLE BENZIMIDAZOLE DERIVATIVES AS IMMUNIZING HAPTENS FOR THE DEVELOPMENT OF A POLYCLONAL ANTIBODY RECOGNIZING THE FUNGICIDE CARBENDAZIM

Carbendazim [methyl 2-benzimidazole carbamate] is a fungicide widely used for controlling a broad range of fungi affecting fruits, vegetables, field crops etc. Determination of carbendazim in water, soil and various crops -especially cereals- is frequently required to assure compliance with national/European laws and regulations. An anti-carbendazim polyclonal antibody was developed against a combination of three commercially available benzimidazole derivatives, namely 2-(2-aminoethyl) benzimidazole, 2-benzimidazole propionic acid and 2-mercaptobenzimidazole; this considerably accelerated the antibody development procedure and reduced cost. The above derivatives were conjugated to keyhole limpet hemocyanin, following well-established conjugation chemistry protocols, and a mixture of the conjugates was administered to New Zealand white rabbits; the IgG fraction of the antisera collected was isolated and its purity was verified with SDS-PAGE. Immunochemical functionality of the antisera and the isolated antibody was evaluated with in-house developed titer- and displacement ELISAs. In these ELISA systems, the commercially available chemical 2-mercaptobenzimidazole was coupled through 3-maleimidopropionic acid to an in-house synthesized lysine-dendrimer, prepared as previously described by our team (Papasarantos et al., 2010), and was finally used as the immobilized hapten. The antisera ELISA-titer values were in all cases higher than 1:10,000. ELISA-displacement curves were obtained in the presence of carbendazim at various concentrations (1 - 20 mu g/mL). As shown by ELISA-displacement experiments -and probably due to the variety of the immunizing haptens used for its development, the anti-carbendazim antibody developed was capable of recognizing not only carbendazim, but also the fungicide benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazole-carbamate] as well as the benzimidazole molecule and various benzimidazole derivatives. Considering its highly promising immunochemical profile, especially its broad specificity, the anti-carbendazim antibody reported here may be used in the development of immunoassay systems for analyzing samples of environmental interest. The above antibody is being currently tested as a specific biorecognition element in a cell biosensor platform for pesticide residue detection based on the Bioelectric Recognition Assay (BERA) technology, which has been developed in the framework of the EU Capacities Project FOODSCAN.