| Project/Area Number |
03452279
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Physical pharmacy
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| Research Institution | SHOWA UNIVERSITY |
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Principal Investigator |
TSUJI Akio Showa University, School of Pharmaceutical Science, PhD, Professor, 薬学部, 教授 (80053784)
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| Co-Investigator(Kenkyū-buntansha) |
MAEDA Masako Showa University, School of Pharmaceutical Science, PhD, Assistant Professor, 薬学部, 助教授 (00053869)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1992: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1991: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | Chemiluminescence / Enzyme cycling / NADH / Alkaline phosphatase / Enzyme immunoassay / Lucigenin / hCG / CCK-8 / Dihydroxyacetone / NADP^+ / 酵素サイクリング反応 / 化学発光反応 / 生体成分 / 増幅化学発光法 / アルカリ性ホスファタ-ゼ |
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| Research Abstract |
We have developed highly sensitive chemiluminescent assays of alkaline phosphatase (ALP) enhanced by using NAD^+/NADH enzyme cycling reaction and lucigenin chemiluminescence reaction. These methods have been applied to enzyme immunoassays using ALP as label enzyme.
(1) Chemiluminescent assay of ALP using NAD^+/NADH enzyme cycling : The enzyme cycling reaction of NAD^+/NADH was applied to enhance the chemiluminescent assay of NADH. The detection limit of NADH is increased 10^3 times. This method was used for the assay of ALP using NADP^+ as substrate. The detecion limit of ALP was 4 x 10^<-20>mol/assay. Chemiluminescent enzyme immunoassays of 17-hydroxyprogesteron and hCG were developed.
(2) Chemiluminescent assay of ALP based on the chemiluminescent reaction of lucigenin with reducing compounds : Dihydroxyacetone in examined reducing compounds gives the most intensive chemiluminescence with lucigenin. The new chemiluminescent assay of ALP was developed using NADP^+ as substrate. NAD^+ generated by ALP reacted with glycerol/glycerol dehydrogenase to yield dihydroxyacetone which was detected by lucigenin. The detection limit of this method was 1.25 x 10^<-19> mol/assay which was lower than that of the enzyme cycling method but the assay procedure was simple and rapid. Chemiluminescent enzyme immunoassays of CCK-8 and hCG were developed by this method.
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