Production of Polyclonal Antibody and Development of a Competitive Enzyme-Linked Immunosorbent Assay for Benzoic Acid in Foods

摘要

A polyclonal antibody (PcAb)-based enzyme-linked immunosorbent assay (ELISA) for the determination of benzoic acid (BA) was developed. For this, 4-aminobenzoic acid was conjugated to keyhole limpet hemocyanin (KLH) by diazotization and used as an immunogen to produce a PcAb against BA. Two spectrophotometric formats, indirect and direct competitive ELISA (ci- and cd-ELISA, respectively), were developed, and the effects of heterologous coatings and assay conditions on assay performance were examined. Under optimal conditions, comparative sensitivities were obtained for a heterologous ci-ELISA and homologous cd-ELISA. The cd-ELISA showed a detection limit (LOD) of 1.25, 2.5 and 2.5 mu g mL(-1), linear range of 5.0-530.0, 10.0-1,060.0 and 10.0-1,060.0 mu g mL(-1), in carbonated drink, sausage and jelly, respectively. The assay time was 55 min (simple sum of incubations without time for the necessary intermediate manipulations). This assay exhibited good specificity for BA and its salt, sodium benzoate, and no cross-reactivity with structurally related food additives. Averaged recoveries from BA-spiked carbonated drink, sausage and jelly using this proposed dc-ELSIA ranged from 85.8 to 104.6 %. In addition, good correlation was observed between results from cd-ELISA and standard HPLC analyses (R (2) = 0.9484). Overall, these results indicated that this cd-ELISA could be used effectively for simple, rapid and low-cost screening of BA abuse in food samples.