The Sigma-Aldrich® Low Matrix Total Aflatoxin ELISA (Enzyme-Linked Immunosorbent Assay) kit is a solid phase direct competitive enzyme immunoassay. An aflatoxin specific antibody optimized to cross react with all four subtypes of aflatoxin (see cross-reactivity information), is coated to a polystyrene microwell. Toxins are extracted from a ground sample with either 80% methanol or 80% acetonitrile and after dilution, added to the appropriate well. If aflatoxin is present it will bind to the coated antibody. Subsequently, aflatoxin bound to horse-radish peroxidase (HRP) is added and binds to the antibody not already occupied by aflatoxin present in the sample or standard. After this incubation period, the contents of the wells are decanted, washed and an HRP substrate is added which develops a blue color in the presence of enzyme. The intensity of the color is directly proportional to the amount of bound conjugate and inversely proportional to the amount of aflatoxin in the standard or sample. Therefore, as the concentration of aflatoxin in the sample or standard increases, the intensity of the blue color will decrease. A solution containing a red dye is added to stop the enzyme reaction and simultaneously causes a color change leaving the bluest wells deep blue/purple and the lightest wells a mauve/pink, thus facilitating a visual qualitative estimate of Aflatoxin in the sample by comparing to the color of the standards.
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