Chemiluminescent assay of beta-D-galactosidase based on indole luminescence

We developed a novel chemiluminescent assay of beta-D-galactosidase (beta-gal) based on the chemiluminescence of indole. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) was used as a substrate for beta-gal and also as a light emitter. X-gal was hydrolysed by beta-gal to liberate free indoxyl, followed by oxidation to indigo dye, and simultaneously produces hydrogen peroxide (H2O2). H2O2 reacts with the residual X-gal in the presence of horseradish peroxidase (HRP) to emit light. The measurable range of beta-gal obtained by this method was 6 x 10(-14) mol/L to 6 x 10(-11) mol/L; the detection limit was 3 amol/assay. This chemiluminescent assay could be applied to an enzyme immunoassay of thyroxine using beta-gal as the enzyme label.