A novel automated flow immunoassay system is developed for the detection of wheat protein as a food allergen. A polyclonal immunoglobulin G (IgG) antibody was isolated from serum of a rat with a wheat allergy, then reductively treated using dithiothreitol and covalently coupled to alkaline phosphatase. This conjugate was used in a non-competitive binding assay. The automated system was equipped with an immunoreaction column, an anion-exchange column, a luminescent reaction column and a photodetector. Antibody-antigen complexes were separated from their free conjugate on the basis of a difference in isoelectric point (pI) by an anion-exchange column. This simple technique permits the assay of wheat-protein allergen in 25min with a reliable detection limit of 5μg/ml. The anion-exchange column was regenerated by occasional elution with N-methylpiperazine buffer (pH 5.0) containing 0.5M NaCl, to remove free conjugate. Free conjugate recovered in this manner could be reused up to four times without significant decrease in sensitivity of the immunoassay. Copyright (C) 1998 Elsevier Science B.V.
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