Monoclonal Antibody-Based Enzyme Linked Immunosorbent Assay of Aflatoxin B1, T-2 Toxin, and Ochratoxin A in Barley

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Abstract
Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1f 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile- 0.5% KCI-6% H2S04 (89 + 10 + 1 ) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonltrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCI buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1( 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, Inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were <12% for Bi and OA but as high as 17% forT2.