Highly Sensitive Chemiluminescent Assays of Biological Substances using Enzyme Cycling and Cascade Reaction

使用酶循环和级联反应对生物物质进行高灵敏化学发光测定

• 批准号: 03452279
• 负责人: TSUJI Akio
• 金额: $ 2.62万
• 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 (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03452279/
• 关键词: 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

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.

利用NAD^+/NADH酶循环反应和光泽精化学发光反应，建立了高灵敏度的碱性磷酸酶（ALP）荧光分析法。这些方法已应用于使用ALP作为标记酶的酶免疫测定。(1)NAD^+/NADH酶循环化学发光法测定ALP：利用NAD^+/NADH的酶循环反应增强了对NADH的化学发光测定。NADH的检测限提高了10^3倍。该方法用于以NADP^+为底物测定ALP。ALP的检测限为4 × 10 ~（-6）<-20>mol/次。建立了17-羟基孕酮和hCG的化学发光酶免疫分析法。(2)基于光泽精与还原性化合物的化学发光反应的ALP的化学发光测定：在检测的还原性化合物中，二羟基丙酮与光泽精产生最强的化学发光。以NADP^+为底物，建立了一种新的碱性磷酸酶荧光分析法。ALP产生的NAD^+与甘油/甘油脱氢酶反应生成二羟基丙酮，用光泽精检测。该方法的检出限为1.25 × 10 ~（-1）<-19>mol/次，低于酶循环法，但测定过程简单、快速。用此方法建立了CCK-8和hCG的化学发光酶免疫分析法。

项目成果

A.Tsuji, M.Maeda, H.Arakawa: "Chemiluminescent assay of co-factors." J.Biolumi & Chemilumi.4. 454-462 (1991)

A.Tsuji、M.Maeda、H.Arakawa：“辅助因子的化学发光测定。”

M.Maeda,A.Tsuji,et al.: "Chemiluminescent assay of alkaline phosphatase asusing ascorbic acid-2-06phosphate as substrate and its application to chemiluminescent enzyme immunoassays" J.Biolumi & Chemilumi.4. 119-122 (1991)

M.Maeda,A.Tsuji,et al.：“以抗坏血酸-2-06磷酸为底物的碱性磷酸酶的化学发光测定及其在化学发光酶免疫测定中的应用”J.Biolumi

M.Kitamura,M.Maeda,A.Tshuji: "Sensitive chemiluminescence assay of NADH using enzyme cycling and its application." BUNSEKI KAGAKU. 40. 537-542 (1991)

M.Kitamura、M.Maeda、A.Tshuji：“使用酶循环的 NADH 灵敏化学发光测定及其应用。”

Masako MAEDA: "Chemiluminescence assay of β-D-galactosidase and its application to enzyme immunoassay" Anal.Chim.Acta.

Masako MAEDA：“β-D-半乳糖苷酶的化学发光测定及其在酶免疫测定中的应用”Anal.Chim.Acta。

H.Arakawa,M.Maeda,A.Tsuji: "Chemiluminescent assay of various enzymes using indoxyl derivatives as substrate and its applicationstto enzyme immunoassay and DNA probe assay." Anal.Biochem.199. 238-242 (1991)

H.Arakawa、M.Maeda、A.Tsuji：“使用吲哚酚衍生物作为底物的各种酶的化学发光测定及其在酶免疫测定和 DNA 探针测定中的应用。”