Chemiluminescence assay of β- d-galactosidase and its application to competitive immunoassay for 17α-hydroxyprogesterone and thyroxine

Abstract

A highly sensitive chemiluminscence assay of β- d-galactosidase and its application to chemiluminescence enzyme immunoassay is described. In the first step, β- d-galactose was produced from o-nitrophenyl β- d-galactoside as a substrate by hydrolysis with β- d-galactosidase, and subsequently NADH generated by the action of d-galactose dehydrogenase and NAD +. The generated NADH was then measured separately by chemiluminescent reaction with 1-methoxy-5-methylphenazinium methyl sulphate and isoluminol-microperoxidase. The detection limits of β- d-galactosidase for 120- and 1000-min assays were 3.3 × 10 −10 and 3.3 × 10 −21 mol per assay, respectively. The relative standard deviation (R.S.D.) of β- d-galactosidase assay was from 4.1 to 8.1%. The chemiluminescence assay of β- d-galactosidase could be applied to the chemiluminescent enzyme immunoassay of 17α-hydroxyprogesterone (17-OHP) and thyroxine. The measurable ranges were from 0.033 to 100 pg per assay for 17-OHP and from 10 to 100 pg per assay for thyroxine. The detection limits were 33 fg (0.1 amol) per assay for 17-OHP and 10 pg (13 fmol) per assay for thyroxine. The R.S.D.s of these assays were in the range 1.0–11.7% for 17-OHP and 7.3–9.8% for thyroxine in the linear range of the calibration graph.