Satoh Tetsuya, Kato Junichi, Takiguchi Noboru, Ohtake Hisao, Kuroda Akio
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University.
Biosci Biotechnol Biochem. 2004 Jun;68(6):1216-20. doi: 10.1271/bbb.68.1216.
We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.
我们开发了一种超灵敏的ATP生物发光测定法，该方法采用：(i) 腺苷酸激酶（ADK）将AMP + ATP转化为两分子ADP；(ii) 多聚磷酸（polyP）激酶（PPK）将ADP再转化回ATP（ATP扩增）；以及(iii) 一种市售的萤火虫荧光素酶。一种高度纯化的PPK-ADK融合蛋白能有效地扩增ATP，从而在萤火虫荧光素酶反应中产生高水平的生物发光。本方法对ATP的灵敏度比传统生物发光测定法高约10000倍，使我们能够在每次测定中检测到低至一个大肠杆菌菌落形成单位（CFU）的细菌污染。
