Analytical Sciences
Online ISSN : 1348-2246
Print ISSN : 0910-6340
ISSN-L : 0910-6340
Original Papers
Highly Sensitive Simultaneous Bioluminescent Measurement of Acetate Kinase and Pyruvate Phosphate Dikinase Activities using a Firefly Luciferase-Luciferin Reaction and Its Application to a Tandem Bioluminescent Enzyme Immunoassay
Katsutoshi ITOKazuto NAKAGAWASeiji MURAKAMIHidetoshi ARAKAWAMasako MAEDA
Author information
JOURNAL FREE ACCESS

2003 Volume 19 Issue 1 Pages 105-109

Details
Abstract
We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luniferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03×10-20 and 2.05×10-20 mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample.
Content from these authors
© 2003 by The Japan Society for Analytical Chemistry
Previous article Next article
feedback
Top