Antigen synthetic strategy and immunoassay development for detection of acrylamide in foods

Abstract

Acrylamide, a toxic and carcinogenic compound, has been found to be present in a range of processed starchy foods. To prepare an effective immunogen compound for acrylamide, N-acryloxysuccinimide (NAS) was conjugated to bovine serum albumin (BSA) at a high molar ratio of 21.2:1. Antisera were obtained by immunization of rabbits with additional booster injections of the NAS-BSA conjugate after the regular process. The IgGs purified by an ammonium sulfate precipitation method were further fractionated with a BSA-immobilized immunoaffinity column. The affinity constant between the collected antibody and coated antigen (NAS-ovalbumin) is found to be 6.7 x 10(7) L mol(-1). Asparagine, the key precursor of acrylamide formation in foods, showed negligible cross-reactivity to the antibody. A biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) was developed and the optimum assay medium was found to be 0.1 mol L(-1) NaHCO(3) (pH 8.3, containing 0.5 mol L(-1) NaCl). The BA-ELISA afforded a practical sensitivity with a working range of 10-100,000 ng mL(-1) and a detection limit of 6 ng mL(-1). The assay was applied to detect acrylamide in potato fries and biscuits and the quantitative results were in good agreement with those obtained by the high-performance liquid chromatography method. This immunoassay will be very useful for monitoring acrylamide in food samples.