| 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)
武田 素子 昭和大学, 薬学部, 助手 (70053973)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1988: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | chemiluminescence / enzyme immunoassay / chemiluminescent enzyme immunoassay / NADH / NADPH / enzyme cycling method / bile acid / 17-hydroxyprogesterone / 化学発光分析法 / 1ーメトキシフェナジンメトサルフェ-ト / イソルミノ-ル / 胆汁酸 / アルカリホスファタ-ゼ / 酵素サイクリング / 化学発光 / NAD(P)H / 酵素 / 液体クロマトグラフィ- / ルミノ-ル / 固定化酵素 / グルコ-ス / 17αーヒドロキシプロゲステロン / 酵素イムノアッセイ / イソルミノール |
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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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