Chemiluminescent assay of various enzymes using indoxyl derivatives as substrate and its applications to enzyme immunoassay and DNA probe assay

Abstract

Chemiluminescent assays of various enzymes have been developed using indoxyl derivatives as substrates. The principle of the method is as follows: an enzyme causes hydrolysis of an indoxyl derivative to an intermediate indoxyl that is readily oxidized to indigo dye and simultaneously produces hydrogen peroxide (H2O2). Hydrogen peroxide is detected chemiluminescently using isoluminol-microperoxidase. Alkaline phosphatase (ALP), beta-D-galactosidase (beta-gal), and beta-glucosidase were assayed by this method using 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), and 5-bromo-4-chloro-3-indolyl-beta-D-glucoside, respectively, as substrates. Using BCIP and X-Gal substrates, we have been able to detect 10(-19) mol of ALP and beta-gal, respectively. This assay system can be applied to enzyme immunoassay and DNA probe assay.