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.
Maeda
M
M
School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Arakawa
H
H
Tsuji
A
A
D000469:Alkaline Phosphatase / Q000032:analysis; D004247:DNA / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D009243:NAD / Q000032:analysis; D009249:NADP / Q000032:analysis; D009693:Nucleic Acid Hybridization; D001616:beta-Galactosidase / Q000032:analysis
D009243:NAD; D009249:NADP; D004247:DNA; D005954:Glucosephosphate Dehydrogenase; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase