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A novel bioluminescent assay for pyruvate phosphate dikinase (PPDK) Katsutoshi Ito*1; K Nishimura1; S Murakami2; H Arakawa1; M Maeda1 (1) School of Pharm. Sci., Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, JAPAN; (2) Research & Development Division, Kikkoman Corporation, 399 Noda, Chiba, 278-0037, JAPAN      *(itok@pharm.showa-u.ac.jp) A novel bioluminescent (BL) assay of PPDK has been developed using luciferin-luciferase reaction. PPDK catalyzes the formation of ATP from AMP, PPi and phosphoenolpyruvate (PEP). We presented that the BL assay for PPDK was achieved highly sensitive by a low background, because the BL assay was not affected by adenylate kinase which generates ATP from 2 mol of ADP and exists in various microorganisms. The proposed assay was performed as follows: 10µL of standard PPDK solution was added to a microtiter plate, followed by addition of 100µL of bioluminescent solution (containing AMP, PPi, PEP, Mg2+, luciferin and luciferase). The BL intensity was integrated for 5 s after incubation for 15 min. In this method, the range of calibration linearity and detection limit of PPDK were from 3.5 x 10-20 to 4.5 x 10-16 mol/assay and 1.36 x 10-20 mol/assay, respectively. The within-assay precision profiles of PPDK were 1.2-2.8% (n=8). The BL intensity was sable for at least 120 min at 37 °C. We applied the proposed assay to BL-EIA for alpha-fetoprotein (AFP) using PPDK as a labeled enzyme. The detection limit of AFP was 2.79 x 10-18 mol/assay and the serum AFP concentration could be measured using the BL-EIA. [Poster: ito.katsut.12322]

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