Alkaline phosphatase catalyzes the hydrolysis of phosphate esters under alkaline environments, resulting in the formation of inorganic phosphate and an alcohol. In mammals, this enzyme is found mainly in the liver and bones.  Elevated serum alkaline phosphatase levels are known as hyperalkaline phosphatasemia, whichbeen associated with a variety of symptoms including malignant biliary obstruction, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic lymphoma and sarcoidosis. Simple, direct and automation-ready procedures for measuring alkaline phosphatase activity in serum are becoming popular in Research and Drug Discovery.

Chemediate’s Assay Kit is designed to measure alkaline phosphatase activity directly in biological samples without pretreatment. The improved method utilizes phosphorylated fluorescein as the substrate that is hydrolyzed by alkaline phosphatase into a yellow colored and fluorescent product (maximal absorbance around 490 nm, Ex/Em=490/510 nm). The rate of the reaction is directly proportional to the enzyme activity.  The new assay was 50-100 fold more sensitive in an ELISA assay as compared to the conventional assay using the substrate of p-nitrophenol phosphate (PNPP).
Assay Principle