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Antibodies directed against Aflatoxin B1 (AFB1) were produced in laying hens, isolated from egg yolk and applied in an enzyme-linked immunosorbent assay (ELISA) for AFB1 in ground peanuts. The ELISA sensitivity was improved by reduction of the amount of the coating antigen absorbed onto the microplate well surfaces. The main aflatoxins B1, B2, G1, G2, M1, aflatoxicol, sterigmatocystin were found to cross-react in the competitive ELISA, 100, 25, 23, 4, 0, 0, 0%, respectively. Aflatoxin B1 could be reproducibly detected and quantified in spiked ground peanuts at levels greater than 5 ppb using a hexane and methanol solvent based peanut extraction protocol.