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

A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the de termination of the fungicide benalaxyl (methyl N-phenylacetyl-N-2,6-xylyl a laninate) has been developed. A derivative of benalaxyl (4-[2-(N-phenylacet yl-N-2,6-xylylamino) propionamido] butyric acid) has been synthesized and l inked 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-reactiv ity to various structurally related pesticides. The calibration curves obta ined 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-xylyla mino) propionamido]butyric acid - ovoalbumin conjugate) show that it is pos sible to set up a specific assay for benalaxyl in aqueous samples. Detectio n limits of 0.35 mu g l(-1) (with mouse antibodies) and 0.20 mu g l(-1) (wi th chicken antibody) are obtained with a working range of about 0.5-50 mu g l(-1). The suitability of the ELISA for benalaxyl quantification in wine w as also studied. Therefore, samples were spiked with benalaxyl in the 0.5-2 00 mu g l(-1) range and directly analyzed without any pretreatment other th an a 1+3 dilution in PBS to minimize matrix effects. Good accuracy and prec ision were obtained with recoveries ranging from about 80% to 120% in a wor king range of 2-50 mu g l(-1). No false positive was found. (C) 1999 Elsevi er Science B.V. All rights reserved.