Chemiluminescent assay of alkaline phosphatase using phenacyl phosphate

Lucigenin is popular as a chemiluminescent reagent. It produces strong chemiluminescence by reaction with hydrogen peroxide or some organic reductants. The application of this intense chemiluminescence has been studied for high-sensitive enzymatic activity assay. Phenacyl phosphate was synthesized as a new substrate for alkaline phosphatase (ALP); the phenacyl alcohol formed in the enzyme-catalyzed reaction reacted to lucigenin giving luminescence. The detection limit for ALP was 3.4 × 10-1 mol per assay tube after overnight incubation. ALP is used for enzyme immunoassays as a labeling enzyme. The assay in the present study should be applicable to high-sensitivity enzyme immunoassays.