Detection of Aflatoxin B1 by Enyzme - Linked Immunosorbent Assay (ELISA)

Abstract

A specific microtest plate enzyme - linked inmunosorbent sassay has been developed for the rapid quantiation of Aflatoxin B1 as low as 0.1 ug per assay, Multiple site injection of rabbit with the commercially available haptens, Aflatoxin B1 - oxime - brovine serum albumin conjugate, was used for the production of antiserum. ELISA titration of Aflatoxin B1 antibody was obtained from four rabbits as high as 1: 15,000. It was the first production of antiserum against Aflatoxin B1 in Thailand which will further be used for the Aflatoxin detection by ELISA in agricultural commodities.
Direct competitive ELISA was used for detection of Aflatoxin B1. Aflatoxin B1 was extracted in methanol from naturally contaminated corn and feeds. Wells of a polystrene microplate were coated with diluted antibody. The plates were washed in PBS - Tween. Thereafter aflatoxin B1 standards or sample extracts were added to each well, followed by equal amount of Aflatoxin B1 - peroxidase conjugate, then plates were incubated. The plates were again washed, and the amount of conjugate perosidase conjugate bound to the antibody was determined after addition of the substrate solution consistion of O - phenylene diamine and hydrogen peroxide. The change in absorbance was monitored at a wavelenght of 490 nanometer. ELISA method is more rapid, less expensive, and saver than the physio - chemical method of aflatoxin analysis.