Chemiluminescent Assay of NAD (P)H and its Application to Clinical Chemistry

NAD(P)H的化学发光测定及其在临床化学中的应用

• 批准号: 63571112
• 负责人: TSUJI Akio
• 金额: $ 1.34万
• 依托单位: 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: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63571112/
• 关键词: 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

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.

我们开发了一种化学发光方法，使用 1-甲氧基-5-甲基吩嗪鎓甲基硫酸盐 (1-MPMS)/异鲁米诺 (IL)/微过氧化物酶 (m-POD) 系统来测定 NAD (P)H。获得化学发光强度和NAD(P)H浓度(log/log)之间的线性关系，其范围为10^-9mol/l至10^-12mol/l。该化学发光测定已与葡萄糖-6-磷酸脱氢酶 (G6PDH)、β-D-乳糖苷酶 (β-Gal) 和碱性磷酸酶 (ALP) 的测定联用。 G6PDH、β-D-Gal和ALP的检测限分别为10^ -18 、10^ -20 和10^ -18 molper测定。将这些酶的化学发光检测应用于17α-羟基孕酮的化学发光酶免疫分析和DNA杂交分析。为了提高该方法的灵敏度，将酶循环系统与NADH的化学发光分析相结合。在3×10^-14至5×10^-12mol/l范围内获得标准曲线。 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i-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。该方法的灵敏度比前一种方法高 1000 倍。 ATP 的检测限为 10 fmol/测定。

项目成果

A. Tsuji: "Chemiluminescent Assay of Co-factors" J. Biolumi. Chemilumi.4. 454 (1989)

A. Tsuji：“辅因子的化学发光测定”J. Biolumi。

M. Takeda: "Chemiluminescence High Performance Liquid Chromatography of Corticosteroids Using Lucigenin as Post-column Reagent." Biomedical chromatogr.4. 119 (1990)

M. Takeda：“使用光泽精作为柱后试剂的皮质类固醇化学发光高效液相色谱法。”

Akio Tsuji: "Chemiluminescent Enzyme Immunoassay" Analytical Sciences. 5. 497-506 (1989)

Akio Tsuji：“化学发光酶免疫分析”分析科学。

A. Tsuji: "Chemiluminescent Enzyme Immunoassay." Analytical Sciences. 5. 497 (1989)

A. Tsuji：“化学发光酶免疫分析。”

M. Maeda: "High-performance liquid chromatography with a 3alpha-hydroxysteroid dehydrogenase postcolumn reactor and isoluminol-microperoxidase chemiluminescence detection." J. Chromatogr.515. 329 (1990)

M. Maeda：“采用 3α-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。”