1991 Fiscal Year Final Research Report Summary
The Assay System for Dehydrogenases by Chemiluminescent Reaction and Its Application
| Project/Area Number |
01571185
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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 |
Physical pharmacy
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| Research Institution | School of Pharmaceutical Sciences, Showa University |
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Principal Investigator |
MAEDA Masako Showa University, Pharmaceutical Science, Asociated Professor, 薬学部, 助教授 (00053869)
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| Co-Investigator(Kenkyū-buntansha) |
ARAKAWA Hidetoshi Showa University, Pharmaceutical Science, Lectureler, 薬学部, 専任講師 (70129807)
TSUJI Akio Showa University, Pharmaceutical Science, Professor, 薬学部, 教授 (80053784)
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| Project Period (FY) |
1989 – 1991
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| Keywords | chemiluminescence / NAD (P) H / enzyme cycling method / enzyme immunoassay / chemiliuminescent HPLC / chemiluminescent enzyme immunoassay / bile acid / ATP |
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| Research Abstract |
We have developed a chemiluminescet assay method for NAD(P)H using 1-methoxy5-methylphenazinium methylsulfate(l-MPMS)/isoluminol(IL)/micro-POD(m-POD)system.
A linear relationship between chemiluminescence intensity and NAD(P)H concentration(log/log)was obtained from 10^<-9> to 10^<-12> mol/l. This chemiluminescent assay has been coupled to the assay of glucose-6-phosphate dehydrogenase(G6PDH), beta-Dgalactosidase(beta-Gal)and alkaline phosphatase(ALP). The detection limits of G-6-PDH, beta-Gal and ALP were 10^<-18>, 10^<-20> and 10^<-18> mol/assay, respectively.
The chemiluminescent assay of these enzymes were applied to chemiluminescent enzyme immunoassay for 17ckhydroxyprogesterone and DNA habridization assay.
In order to increase the sensitivity of this chemiluminescent reaction of NADH, enzyme cycling system was coupled, the standard curve obtained in the range of 3 x 10^<-14> - 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 bacteria luciferase.
The chemiluminescent assay of ATP was also developed using hexokinase/G6PDH and 1-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 former method. The detection limit of ATP was 10 fmol/assay. We also applied to chemiluminescent HPLC for bile acid detected by using NADH chemiluminescent reaction.
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