Spectrophotometric enzyme-amplified immunoassay for thyroid stimulating hormone

Abstract

Thyroid stimulating hormone (TSH) regulates the function of the thyroid gland. Its determination at low concentrations in serum is useful in the diagnosis of hyperthyroidism. In this paper, it is detected using a spectrophotometric enzyme-amplified immunoassay. The reporter enzyme is alkaline phosphatase and its substrate is flavin adenine dinucleotide phosphate (FADP). Reaction with alkaline phosphatase converts FADP into flavin adenine dinucleotide (FAD), which, unlike FADP, re-activates apo-D-amino acid oxidase (apo-AOD). Re-activation of apo-AOD allows the product of the reporter enzyme to be amplified. The lower limit of detection for TSH by this method is 0.06 µU cm^–3. This compares with 0.54 µU cm^–3 for an identical assay in which p-nitrophenyl phosphate was the substrate for alkaline phosphatase. Contaminating alkaline phosphatase was removed from the reagents by affinity chromatography.