DEVELOPMENT OF A QUANTITATIVE AND SENSITIVE ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR OCHRATOXIN-A USING ANTIBODIES FROM THE YOLK OF THE LAYING HEN

Antibodies directed against ochratoxin A (OA) were obtained from hen egg yolk using an optimized purification procedure and applied in an enzyme-linked immunosorbent assay (ELISA) for OA in swine finisher diets. The egg yolk antibody could be recovered at levels as high as 70-80% with purities greater than 86-92% using a mixture of aqueous buffer and chloroform for lipid extraction and poly(ethylene glycol) for antibody precipitation. Ochratoxins C, B, and alpha and the structurally related mycotoxin citrinin were found to cross-react with the antibody 400, 100, 33.3, and 2%, respectively, in an indirect competitive ELISA. Ochratoxin A could be detected in swine finisher diets at levels greater than 50 ppb using a simplified sample preparation procedure and a indirect competitive ELISA. Recoveries of OA from the diets were validated by conventional HPLC analysis using a proven sample extraction protocol. ELISA-determined OA values correlated highly with those obtained using HPLC analysis (r = 0.98). Assay sensitivity was found to be dependent on background absorbance. The mixed anhydride (MA) coupling chemistry used to prepare the immunogens promoted high background absorbances in the quantitative ELISA. The background was overcome by using N-hydroxysuccinimide activated ester coupling chemistry for the preparation of plate coating antigen and or incubation of the antibody with bovine serum albumin that had been subjected to the MA reaction. This study demonstrates that antibodies from hen egg yolk can be readily obtained in good yield and purity and used to develop a highly sensitive ELISA for OA.