Bioluminescent enzyme immunoassay using thermostable mutant luciferase and acetate kinase as a labelled enzyme

A bioluminescent assay of acetate kinase (AK) have been developed using thermostable mutant firefly luciferase. The principle of the method is as follows: AK catalyzes the formation of ATP from acetylphosphate and ADP, which is readily consumed by the luciferin–luciferase reaction resulting in bioluminescence. AK activity was determined by measuring the light generated by the luciferase reaction. In this method, we have been able to detect 8.5×10−21 mol of AK. The bioluminescence was stable for at least 100 min at 37°C. This bioluminometric assay of AK was applied to the enzyme immunoassay of human growth hormone (hGH) and human chorionic gonadotropin (hCG), using biotinylated AK and streptavidin. In these enzyme immunoassays, the measurable ranges of hGH and hCG were 78–20,000 pg ml−1 and 39–20,000 μIU ml−1, respectively. This assay system can be applied to various enzyme immunoassays with high sensitivity.