Novel bioluminescent assay of pyruvate phosphate dikinase using firefly luciferase-luciferin reaction and its application to bioluminescent enzyme immunoassay

A novel bioluminescence (BL) assay of pyruvate phosphate dikinase (PPDK) has been developed using the firefly luciferin-luciferase reaction. PPDK catalyzes the formation of ATP from AMP, PPi (diphosphate) and phosphoenolpyruvate (PEP). The BL assay for PPDK was highly sensitive with a low background because it was not affected by adenylate kinase, which generates ATP from 2 mol of ADP and is present in various microorganisms. The proposed assay was performed as follows: 10 mu l of standard PPDK solution was added to a microtiter plate, followed by addition of 100 mu l of bioluminescent solution (containing AMP, PPi, PEP, Mg2+, luciferin and luciferase). The BL intensity was integrated for 5 s after incubation for 15 min. The range of log-log calibration linearity and 3S.D. detection limit of PPDK ranged from 3.5 x 10(-20) to 4.5 x 10(-16) and 1.36 x 10(-20) mol per assay, respectively. The within-assay precision for PPDK was 1.2-2.8% (n = 8). The BL intensity was stable for at least 120 min at 37 degrees C. The proposed assay to was applied to a BL enzyme immunoassay (ELA) for alpha-fetoprotein (AFP) and insulin using PPDK as the enzyme label. The detection limits for AFP and insulin were 2.79 x 10(-18) and 9.26 x 10(-17) mol per assay, respectively. Serum samples could accurately be analyzed by the BL-EIA. A high degree of correlation was observed between the values obtained using the proposed BL-EIA and conventional methods. (C) 2000 Elsevier Science B.V. All rights reserved.