Development of an enzyme-linked immunosorbent assay for benalaxyl and its application to the analysis of water and wine Original Research Article

A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the fungicide benalaxyl (methyl N-phenylacetyl-N-2,6-xylyl alaninate) has been developed. A derivative of benalaxyl (4-[2-(N-phenylacetyl-N-2,6-xylylamino) propionamido] butyric acid) has been synthesized and linked to bovine serum albumin using the N-hydroxysuccinimide reaction. The immunogen was used to immunize three mice and one chicken, which responded by producing specific antibodies to benalaxyl with negligible cross-reactivity to various structurally related pesticides. The calibration curves obtained on two different types of haptens on solid phase (N-phenylacetyl-N-2,6-xylyl alanine – ovoalbumin conjugate and 4-[2-(N-phenylacetyl-N-2,6-xylylamino) propionamido]butyric acid – ovoalbumin conjugate) show that it is possible to set up a specific assay for benalaxyl in aqueous samples. Detection limits of 0.35 μg l−1 (with mouse antibodies) and 0.20 μg l−1 (with chicken antibody) are obtained with a working range of about 0.5–50 μg l−1. The suitability of the ELISA for benalaxyl quantification in wine was also studied. Therefore, samples were spiked with benalaxyl in the 0.5–200 μg l−1 range and directly analyzed without any pretreatment other than a 1+3 dilution in PBS to minimize matrix effects. Good accuracy and precision were obtained with recoveries ranging from about 80% to 120% in a working range of 2–50 μg l−1. No false positive was found.