Development of an enzyme-linked immunosorbent assay for the detection and quantification of the insecticide tebufenozide in wine

A CD-ELISA assay has been developed for the quantification of the insecticide tebufenozide in red and white wine. Polyclonal antisera, raised against both KLH- and OVA-linked immunogens of the hapten 1-tert-butyl-2-[4-(carboxypropan-3-yl)benzoyl]-1-(3,5-dimethyl benzoyl)hydrazide NHS ester (Ib) were trialled for use in a quantitative immunoassay and both were found to be suitable for use, with IC50 values in the vicinity of 10 mug L-1 regardless of the carrier protein used and matrix (red versus white wine). The limits of detection were 0.7 mug L-1 and 3 mug L-1 for OVA- and KLH-linked polyclonal sera, respectively. In both cases, the IgG fraction isolated from the antisera was highly specific for tebufenozide and its hapten derivatives, showing no detectable cross-reactivity with eleven other potential pesticide contaminants used in the wine-making industry. Spiked red and white wine samples were successfully analyzed over the range 10-1000 mug L-1 using the immunoassay developed with IgG isolated from the antiserum rabbit anti-OVA-Ib as the immobilized capture antibody. Reported maximum residue limits for wine are 100 mug L-1 and thus would be readily detected using this technique. The immunoassay format represents a cost-effective, relatively simple means of screening large numbers of samples for the presence of this insecticide.