The Assay System for Dehydrogenases by Chemiluminescent Reaction and Its Application

化学发光反应脱氢酶检测系统及其应用

• 批准号: 01571185
• 负责人: MAEDA Masako
• 金额: $ 1.41万
• 依托单位: School of Pharmaceutical Sciences, Showa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01571185/
• 关键词: chemiluminescence; 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

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.

我们开发了一种使用 1-甲氧基 5-甲基吩嗪鎓甲基硫酸盐 (l-MPMS)/异鲁米诺 (IL)/micro-POD(m-POD) 系统测定 NAD(P)H 的化学发光方法。化学发光强度与 NAD 之间呈线性关系（ P)H浓度(log/log)为10^-9至10^-12mol/l。该化学发光检测已与葡萄糖-6-磷酸脱氢酶 (G6PDH)、β-D 半乳糖苷酶 (β-Gal) 和碱性磷酸酶 (ALP) 的检测联用。 G-6-PDH、β-Gal和ALP的检测限分别为10^<-18>、10^<-20>和10^<-18>mol/次。采用化学发光法对这些酶进行检测。用于 17ck 羟基孕酮化学发光酶免疫分析和 DNA 杂交分析。为了增加 NADH 化学发光反应的灵敏度，酶耦合循环系统，获得3×10^-14-5×10^-12mol/l范围内的标准曲线。 NADH的检测限为30 fmol/检测，与使用细菌荧光素酶的生物发光法的检测限相当。还使用己糖激酶/G6PDH和1-MPMS/IL/m-POD系统开发了ATP的化学发光检测。利用ATP与己糖激酶/丙酮酸激酶的酶循环反应开发了ATP的增强化学发光测定法。该方法比以前的方法灵敏1000倍。 ATP 的检测限为 10 fmol/测定。我们还将NADH化学发光反应应用于化学发光HPLC检测胆汁酸。

项目成果

Masako Maeda: "Chemiluminescent Assay of VariousEnzyme Activities and its Application to Enzyme Immunoassays." J. Biolum & Chemilum. 4. 140-148 (1989)

Masako Maeda：“各种酶活性的化学发光测定及其在酶免疫测定中的应用。”

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)

Masako MAEDA：“采用 3-羟基类固醇脱氢酶柱反应器和异鲁米诺微过氧化物酶化学发光检测的高效液相色谱”J.Chromatography 515. 329-335 (1990)

Hidetoshi Arakawa: "Highly Sensitive Biotin-Labelled Hybridization Probe." Chem. Pharm. Bull. 37. 1831-1833 (1989)

Hidetoshi Arakawa：“高灵敏生物素标记杂交探针。”

Y. Uji: "Chemiluminescent Determination of Nonesterified Fatty Acids in Serum" Clin. Chem.35. 325-326 (1989)

Y. Uji：“血清中非酯化脂肪酸的化学发光测定”临床。

Masako Maeda: "High-performance liquid chromatography with a 3 -hydroxysteroid dehydrogenase postcolumn reactor and isoluminol-microperoxidase chemiluminescencedetection." J. Chromatography. 515. 329-335 (1990)

Masako Maeda：“采用 3-羟基类固醇脱氢酶柱后反应器和异鲁米诺-微过氧化物酶化学发光检测的高效液相色谱。”