Optimization of indirect immunoassay for aflatoxin B1 detection


M.K.A. Kadir and I.E. Tothill


The development of indirect competitive immunoassay formats for the Enzyme Links Immunosorbent Assay (ELISA) construction was undertaken for aflatoxin B1 (AFB1)determination. An indirect assay was based on the competition between an immobilised aflatoxin B1 conjugated with bovine serum albumin (AFB1-BSA conjugate) and the free AFB1 for the binding site of monoclonal antibody against AFB1 (MAbAFB1). Then, the secondary anti-antibody IgG labelled with horseradish peroxidase (anti-IgG-HRP conjugate) was used as an enzyme label. A spectrophotometric assay using microtitre plate was used in optimizing the immuno-reagent used for competitive ELISA. The optimal conditions obtained for competitive ELISA were 1 μg/ml of BSA-AFB1, 10 μg/ ml of monoclonal antibody and 1 μg/ml of anti-antibody labelled HRP. The linear rage of standard curve (0.1 – 10 μg/litre) was achieved with a detection limit of 0.08 μg/litre. The achieved detection range for AFB1 was within the European (2 – 4 μg/litre) and Malaysian (5 – 15 μg/litre) required legislative limit of analyses.

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