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

我们建立了一种同时测定醋酸激酶(AK)和丙酮酸磷酸二激酶(PPDK)活性的生物发光方法，并将其应用于串联酶免疫分析。建议的化验原理如下。在第一步中，AK由ADP和乙酰磷酸生成ATP，并通过萤火虫荧光素酶-荧光素反应来测定ATP。在第二步中，加入葡萄糖和依赖于ADP的己糖激酶来消除AK的生物发光强度，后者由ADP形成AMP。同时，PPDK催化AMP、二磷酸和磷酸烯醇式丙酮酸相互转化为ATP、磷酸和丙酮酸。PPDK形成的ATP也是由萤火虫荧光素酶-荧光素反应决定的。AK和PPDK的检出限(空白+3SD)分别为1.03×10~(-20)和2.05×10~(-20)mol/次。该方法适用于同一样品中胰岛素和C-肽的生物发光酶免疫测定

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 acetyiphosphate, and the ATP is detennined 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 1.03x 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