Abstract
We developed a chemiluminescent method for determining alkaline phosphatase (ALP) based on the following reaction ; hydrolysis of NADP (ALP substrate), NAD/NADH cycling reaction with alchohol dehydrogenase and 1-methoxy-5-methylphenaziniummethylsulfate (M-PMS) in the presence of superoxide dismutase (SOD), and subsequent luminol reaction with peroxidase. SOD resulted in about a 4 times higher yield of H2O2 and its accumulation in the cycling reaction by immediate conversion of O2- to H2O2.
The determination limit of ALP activity assayed in a ten-minute cycling reaction system was similar to that estimated with 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD) reagent. This technique was applied to ELISA for sensitive determination of urinary FDP.
