1990 Fiscal Year Final Research Report Summary
Chemiluminescent Assay of NAD (P)H and its Application to Clinical Chemistry
| Project/Area Number |
63571112
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory medicine
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| Research Institution | Showa University |
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Principal Investigator |
TSUJI Akio Showa University, Pharmaceutical Sciences, Professor, 薬学部, 教授 (80053784)
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| Co-Investigator(Kenkyū-buntansha) |
ARAKAWA Hidetoshi Showa University, Pharmaceutical Sciences, Lecture, 薬学部, 講師 (70129807)
MAEDA Masako Showa University, Pharmaceutical Sciences, Assistant Prof, 薬学部, 助教授 (00053869)
ARAKAWA Hidetoshi Showa University, Pharmaceutical Sciences, Lecture (70129807)
ARAKAWA Hidetoshi Showa University, Pharmaceutical Sciences, Lecture (70129807)
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| Project Period (FY) |
1988 – 1990
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| Keywords | chemiluminescence / enzyme immunoassay / chemiluminescent enzyme immunoassay / NADH / NADPH / enzyme cycling method / bile acid / 17-hydroxyprogesterone |
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| Research Abstract |
We have developed a chemiluminescent method for the assay of NAD (P)H using the 1-methoxy- 5-methylphenazinium methylsulphate (1-MPMS) / isoluminol (IL) / microperoxidase (m-POD) system. A linear relationship between chemiluminescence intensity and NAD (P)H concentration (log/log) was obtained ranged from 10^<-9> mol/l to 10^<-12> mol/l. This chemiluminescent assay has been coupled to the assay of glucose-6-phosphate dehydrogenase (G6PDH), beta-D-glactosidase (beta-Gal) and alkaline phosphatase (ALP). The detection limits of G6PDH, beta-D-Gal and ALP were 10^<-18>, 10^<-20> and 10^<-18> molper assay, respectively. The chemiluminescent assay of these enzymes were appplied to chemiluminescent enzyme immunoassay for 17alpha-hydroxyprogesterone and DNA hybridization assay.
In order to increase the sensitivity of this method, enzyme cycling system was coupled to the chemiluminescent assay of NADH. The standard curve was obtained in the range from 3 x 10^<-14> to 5 x 10^<-12> mol/l. The detection limit of NADH was 30 fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase.
The chemiluminescent assay of ATP was also developed using hexokinase/G6PDH and i-MPMS/ IL/m-POD system. The enhanced chemiluminescent assay of ATP was developed by using enzyme cycling reaction of ATP with hexokinase/pyruvate kinase. This method is 1000 fold more sensitive than the former method. The detection limit of ATP was 10 fmol/assay.
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