Facile chemiluminescent method for alkaline phosphatase determination Original Research Article

The determination of alkaline phosphatase activity is of wide applicability, both as a free enzyme or bound to antibodies (conjugates). Activity determinations employing chemiluminescent substrates have become increasingly important due to their high sensitivity, typically equivalent to or better than assays utilizing radioactive labels. We report here a new chemiluminescent methodology for the determination of alkaline phosphatase activity based on the hydrolysis of disodium 1-(2-methylpropenyl)phosphate, readily synthesized in three steps. The hydrolysis product, 2-methyl-1-propenol, is a known substrate of horseradish peroxidase, which catalytically oxidizes enols to a dioxetane intermediate, that in turn yields luminescence upon cleavage. The detection limit of this method was determined to be 1.5 femtomoles of free ALP per assay. This methodology was also tested with bound ALP conjugates showing excellent sensitivity. Since the horseradish peroxidase system consumes dissolved oxygen during oxidation of the enol, alkaline phosphatase quantification may also be performed by accompanying the oxygen uptake rate.