Chemiluminescent assay of various enzyme activities and its application to enzyme immunoassays

A highly sensitive chemiluminescent assay for NAD(P)H have been developed. The principle of the method is as follows; NAD(P)H reduces molecular oxygen to superoxide anion (O2-) and hydrogen peroxide (H2O2) in the presence of 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS) as electron mediator. The produced O2- and H2O2 can be measured by chemiluminescent reaction using isoluminol (IL) and microperoxidase (m-POD). A linear relationship between chemiluminescence intensity and NAD(P)H concentration (log/log) was obtained ranged from 10(-9) mol/l to 10(-5) mol/l. This chemiluminescent reaction has been coupled to the assay of glucose-6-phosphate dehydrogenase (G6PDH), beta-D-galactosidase (beta-Gal) and alkaline phosphatase (ALP). The detection limits of G6PDH, beta-Gal and ALP were 10(-18) mol, 10(-20) mol and 10(-18) mol per assay, respectively. The chemiluminescent assay of these enzymes applied to chemiluminescent enzyme immunoassay for 17 alpha-hydroxy-progesterone and DNA hybridization assay using these enzymes as label.