Determination of Total Aflatoxin Levels in Peanut Butter by Enzyme-Linked Immunosorbent Assay: Collaborative Study

Abstract Laboratories in Australia, Japan, Spain, the United Kingdom, and the United States participated in a collaborative study to evaluate a commercial enzyme- linked immunosorbent assay for the determination of total aflatoxin. Collaborators were sent 10 randomly numbered samples (5 blind duplicates) of roasted peanut butter. Two pairs were "blank" peanut butters to which aflatoxin B1, B2, G1, and G2 standards had been added. The other 3 pairs of peanut butters were 1 low aflatoxin level sample and 2 naturally contaminated samples. The assay is based on indirect competition. Test samples containing (free) aflatoxin, added to aflatoxin-coated microwells, compete for specific monoclonal rat anti-aflatoxin. As the concentration of aflatoxin in the test samples increases, the amount of rat antiaflatoxin binding to the aflatoxin attached to the well decreases. After a wash step to remove unbound material, the amount of rat anti-aflatoxin bound to the well is determined by its reaction with peroxidase conjugated rabbit anti-rat globulin. Bound peroxidase activity is then determined by the addition of a substrate, whose color development is inversely proportional to the aflatoxin concentration and is measured by absorbance. Coefficients of variation (CV) for total aflatoxin concentrations, for mean levels of 9,30, and 89µg/kg, were between 28 and 37% for the low level and 2 naturally contaminated samples, which contained mainly aflatoxin B1. CVs for the spiked samples were lower (24-25%) for mean levels of 11 and 20 µg/kg; recoveries were 84 and 89%, respectively. Ranges for relative standard deviations for repeatabilty and reproducibility were 9-30% and 25-37%, respectively. The method has been adopted first action by AOAC International.