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The Assay System for Dehydrogenases by Chemiluminescent Reaction and Its Application

Research Project

Project/Area Number 01571185
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Physical pharmacy
Research InstitutionSchool of Pharmaceutical Sciences, Showa University

Principal Investigator

MAEDA Masako  Showa University, Pharmaceutical Science, Asociated Professor, 薬学部, 助教授 (00053869)

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)
Project Period (FY) 1989 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1990: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywordschemiluminescence / NAD (P) H / enzyme cycling method / enzyme immunoassay / chemiliuminescent HPLC / chemiluminescent enzyme immunoassay / bile acid / ATP / 化学発光法 / ガラクトシダ-ゼ / アルカリホスファタ-ゼ / NADH / 固定化酵素 / 酵素イムノアツセイ / 酵素 / 酵素活性 / 酵素サイクリング / 酵素イムノアッセイ
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.

Report

(4 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • 1989 Annual Research Report
  • Research Products

    (39 results)

All Other

All Publications (39 results)

  • [Publications] 河本 美津子: "酵素サイクリング法を用いる還元型ニコチンアデニンジヌクレオチドの高感度化学発光測定法とその応用" 分析化学. 40. 537-542 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Masako MAEDA: "Chemiluminescent assay of alkaline phosphatase using ascorbic acid-2-phosphate as substrate and its application to CL-enzyme immunoassays" J.Bioluminescence & Chemiluminescence. 119-122 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Masako MAEDA: "Chemiluminescence assay ofβ-D-galactosidase and its application to enzyme immunoassay" Anal.Chim.Acta.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Masako MAEDA: "A flow injection analysis of glucose,bile acid and ATP using immobilized enzyme reactor and CL assay of NAD(P)H" J.Biloluminescence & Chemiluminescence.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] 前田 昌子,辻 章夫: "注目の臨床実験検査法" 中山書店, 320 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Akio Tsuji: "Chemiluminescent Assay of Co-factiors" J. Biolum & Chemilum. 4. 454-462 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Masako Maeda: "Chemiluminescent Assay of VariousEnzyme Activities and its Application to Enzyme Immunoassays." J. Biolum & Chemilum. 4. 140-148 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Hidetoshi Arakawa: "Highly Sensitive Biotin-Labelled Hybridization Probe." Chem. Pharm. Bull. 37. 1831-1833 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Akio Tsuji: "Chemiliuminescent Enzyme Immunoassay. (Review)" Analytical Sciences. 5. 497-506 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Y. Uji: "Chemiluminescent Determination of Nonesterified Fatty Acids in Serum" Clin. Chem.35. 325-326 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Hidetoshi Arakawa: "Fluorescent Enzyme Immunoassay of Free Thyroxine Using Glucose Oxidase as Enzyme Label." Analytical Sciences. 6. 33-37 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Masako Maeda: "High-performance liquid chromatography with a 3 -hydroxysteroid dehydrogenase postcolumn reactor and isoluminol-microperoxidase chemiluminescencedetection." J. Chromatography. 515. 329-335 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Mitsuko Hirukawa: "Separation of free amino acids by reversedphase ion-pair chromatography withcolumn swiching and isocratic elution." J. Chromatography. 507. 95-101 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Motoko Takeda: "Chemiluminescence High Performance Liquid Chromatography of Corticosteroids Ushin Lucigenin as Post-column Reagent." Biomedical Chromatography. 4. 119-122 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Masako Maeda: "Chemiluminescent Assay of Alkaline phosphatase using Ascorbic acid-2-phosphate as Substrate and Its Application to Chemilumi nescent Enzyme Immunoassay." J. Biolumin & Chemilum. 119-122 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Sekiya: "Fully-automated Analyzer for Chemiluminescent Enzyme Immunoassay." J. Biolumin & Chemilum. 123-126 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] S. Aoyagi: "The reduction of nonspecific binding in chemiluminescent sandwich enzyme immunoassay" J. Immunological Methods. 137. 73-78 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] H. Arakawa: "Chemiluminescent Assay of Various Enzymes Using Indoxyl Derivatives as Substrate and Its Application to Enzyme Immunoassay and DNA Probe Assay" Anal. Biochem.199. 238-242 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] 河本 美津子: "酵素サイクリング法を用いる還元型ニコチンアデニシンヌクレオチドの高感度化学発光測定法とその応用" 分析化学. 40. 537-542 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Masako MAEDA: "Chemiluminescence assay of alkaline phosphatase using ascorbic acidー2ーphosphate as substrate and its application to CLーenzyme immunoassays" J.Bioluminescence & Chemiluminescence. 119-122 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Masako MAEDA: "Chemiluminescent assay of βーDーgalactosidase and its application to enzyme immunoassay" Anal.Chim.Acta.

    • Related Report
      1991 Annual Research Report
  • [Publications] Masako MAEDA: "A flow injection analysis of glucose,bile acid and ATP using immobilized enzyme reactor and CL assay of NAD(P)H" J.Bioluminescence & Chemiluminescence.

    • Related Report
      1991 Annual Research Report
  • [Publications] 前田 昌子,辻 章夫: "注目の臨床実験検査法" 中山書店, 320 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Masako MAEDA: "High Performance Liquid Chromatogrphy with 3 ーHydroxysteroid Dehydrogenase Post Column Reactor and Isoluminolーmicroperoxidase Chemiluminscence Detection" J.Chromatography. 515. 329-335 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Motoko TAKEDA: "Chemiluminescence High Performance Liquid Chromatography of Corticostroide Using Lucigenin as Postーcolum Reagent" Biomedical Chromatogrphy. 4. 119-122 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Hidetoshi ARAKAWA: "Fluorescent Enzyme Immunoassay of Free Thyroxin Using Glucose Oxidase as Enzyme Label" Analytical Sciences,. 6. 33-37 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Akio,TSUJI: "Chemiluminescent Assay of Enzymes and Its Application to Flow Injection Analysis" J.Flow Injection Analysis. 7. 87-100 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Shingo SHIMADA: "A Sensitive Enzymatic Colorimetric Assay for Total Bile Acids Using Microtiter Plate" J.Analytical BioーScience. 13. 70-75 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Masako MAEDA: "Chemiluminescent Assay of Alkaline Phosphatase using Ascorbic Acid 2ーphosphate as substrate and Its Application to CLーEIA" J.Bioluminescence & Chemiluminescence.

    • Related Report
      1990 Annual Research Report
  • [Publications] Masako MAEDA: "Luminescence Immunoassay and Molecular Application (Ed.by K.Van Dyke)" CRC Press Inc., 341 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Masako MAEDA: "Advances in Clinical Chemistry" Academic Press INC., 248 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Masako MAEDA: "Ultrasensitive Chemiluminescence Assay for NADH using Enzyme Cycling and Its Application to Determination of Enzyme Activity" BUNSEKI KAGAKU. 38. 495-499 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Masako MAEDA: "Chemuluminescent Assay of Various Enzyme Activities and Its Application to Enzyme Immunoassays" J.Bioluminescence & Chemiluminescence. 4. 140-148 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Akio TSUJI: "Chemiluminescence Assay of Co-factors" J.Bioluminescence & Chemiluminescence. 4. 454-462 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Akio TSUJI: "Chemiluminescence Enzyme Immunoassay" Analytical Sciences. 5. 497-506 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Yoshinori UJI: "Chemiluminescent Determination of Nonesterified Fatty Acids in Serum" Clinical Chemistry. 35. 325-326 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Hidetoshi ARAKAWA: "Highly Sensitive Biotin-Labelled Hybridazation Probe" Chemistry and harmaceutical Bulletan. 37. 1831-1833 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 前田昌子(石川栄治編): "エンザイムイムノアッセイ" 東京化学同人, 497 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 前田昌子(今井一洋編): "生物発光と化学発光 基礎と実験" 広川書店, 284 (1989)

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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