| Project/Area Number |
13672431
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | Showa University |
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Principal Investigator |
ITO Katsutoshi Showa University, Sch. of Pharm.Sci., Lecture, 薬学部, 講師 (20223141)
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| Co-Investigator(Kenkyū-buntansha) |
MAEDA Masako Showa University, Sch. of Pharm.Sci., Professor, 薬学部, 教授 (00053869)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2003: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | acetate kinase / pyruvate phosphate dikinase / firefly luciferase / luciferin / ADP-dependent hexokinase / simultaneous assay / tandem immunoassay / bioluminescent enzyme immunoassay / ADP依存牲ヘキソキナーゼ |
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| Research Abstract |
We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetyiphosphate, and the ATP is detennined by the firefly luciferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03x 10^<-20> and 2.05 x 10^<-20> mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample
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