Analytical Sciences

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 ITO,* Kazuto NAKAGAWA,* Seiji MURAKAMI,*^,** Hidetoshi ARAKAWA,* and Masako MAEDA* 

*School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan
**Research & Development Division, Kikkoman Corporation, 399 Noda, Chiba 278-0037, Japan

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 luciferase-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 x 10^-20 and 2.05 x 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.

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