Enzyme-Linked Immunosorbent Assay (ELISA) for Aflatoxin B1 Estimation in Groundnuts

The commercially available hapten, afla B1-oxime-bovine serum albumin, was used to produce an antiserum in rabbits. The same hapten was coupled with alkaline phosphatase (hapten-BSA-ALP) and used in the competitive direct enzyme-linked immunosorbent assay (ELISA) for the detection of aflatoxin B1. Aflatoxin B1 was extracted in methanol from naturally contaminated or 'spiked' groundnut seed samples. Wells of a polystyrene microtitre plate were coated with the antiserum, the plates were washed in PBS-Tween, aflatoxin B1 standards or groundnut sample extracts, and hapten-BSA-ALP conjugate were added and the plates incubated. The plates were again washed, and the amount of conjugate bound to the antibody was determined after addition of the substrate, p-nitro-phenylphosphate. The hapten-BSA-ALP conjugate has advantages in stability, simplicity of preparation, and high specificity, over the conventional toxin-enzyme conjugate in direct competitive ELISA. The assay method is more rapid and less expensive than the physico-chemical methods of aflatoxin analysis and it can detect levels of aflatoxin B1 as low as 50 picograms