Monoclonal antibody generation and direct competitive enzyme-linked immunosorbent assay evaluation for the analysis of the fungicide fenhexamid in must and wine.

Fenhexamid is a common fungicide used to fight botrytis infections in a great variety of plant crops, particularly in vineyards. In this study, high-affinity and specific monoclonal antibodies against fenhexamid were produced for the first time, and an enzyme-linked immunosorbent assay (ELISA) in the antibody-coated direct competitive format was developed. The derivative 4-[2,3-dichloro-4-(1-methylcyclohexanecarboxamido)phenoxy]butanoic acid, with a carboxylic spacer arm attached to the phenolic hydroxyl group of fenhexamid, was synthesized for antibody production and ELISA development. To optimize this assay, the dependence of assay parameters on ionic strength and pH was assessed, and the influence of several organic solvents was determined. The IC(50) value of the optimized assay for fenhexamid and the calculated limit of detection in phosphate buffer were 0.52 +/- 0.06 and 0.13 +/- 0.03 microg/L, respectively. Preliminary studies using fortified must and wine samples indicated that the presence of fenhexamid below the maximum residue limits in grapes could be easily determined with the developed immunoassay by a simple dilution of the sample in water.