A reference method for measurement of alkaline phosphatase activity in human serum

We present an official AACC reference method for the measurement of alkaline phosphatase, the culmination of optimization experiments conducted by a group of independent laboratories. The details of this method and evaluation of factors affecting the measurement are described. A metal ion buffer has been incorporated that maintains optimal and constant concentrations of zinc(II) and magnesium(II) ions. Final reaction conditions are: pH (30 degrees C), 10.40 +/- 0.05; 2-amino-2-methyl-1-propanol buffer, 0.35 mol/L; 4-nitrophenyl phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L; zinc sulfate, 1.0 mmol/L; and N-(2-hydroxyethyl)ethylenediaminetriacetic acid, 2.0 mmol/L.