Chemiluminescent assay of enzymes using proenhancers and pro‐anti‐enhancers

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Abstract
Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proehancer and pro‐anti‐enhancer substrates. Alkaline phosphatase is measured using disodium para‐iodophenyl phosphate (proenhancer) which is converted to para‐iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para‐nitrophenyl phosphate which is converted by alkaline phosphatase to para‐nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro‐anti‐enhancer assays was 100 attomoles and 1 picornole, respectively. The proenhancer strategy was effective in assays for beta‐D‐galactosidase, beta‐D‐glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha‐fetoprotein showed good agreement.