Homogeneous bioluminescence competitive binding assay for folate based on a coupled glucose-6-phosphate dehydrogenase-bacterial luciferase enzyme system

被引：7

作者：

Huang, W ^{[1
]}

Feltus, A ^{[1
]}

Witkowski, A ^{[1
]}

Daunert, S ^{[1
]}

机构：

[1] UNIV KENTUCKY,DEPT CHEM,LEXINGTON,KY 40506

来源：

ANALYTICAL CHEMISTRY | 1996年 / 68卷 / 09期

关键词：

D O I：

10.1021/ac950757m

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

A homogeneous bioluminescence competitive binding assay for folate was developed by using a coupled enzyme system of glucose-6-phosphate dehydrogenase (G6PDH) and bacterial luciferase. A highly substituted G6PDH-folate conjugate was prepared by employing an N-hydroxysuccinimide/carbodiimide method. Folate binding protein inhibits the activity of the conjugate. In the presence of folate, there is a competition between folate and the G6PDH-folate conjugate for the binding site of the folate binding protein, and the activity of the conjugate is recovered. Thus, the concentration of folate can be related to the activity of the G6PDH-folate conjugate, which is directly related to the bioluminescence produced by the coupled enzyme reaction. Using this assay, dose-response curves with a detection limit of 2.5 x 10(-8) M folate were obtained, which is an improvement of an order of magnitude with respect to an assay that monitors G6PDH activity spectrophotometrically. The assay was validated using vitamin tablets and a cell culture medium.

引用

页码：1646 / 1650

页数：5

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