Immobilized enzyme assay of creatine kinase with amperometric detection

Serum creatine kinase-MB isoenzyme was determined by an immunoinhibition-amperometric technique in a coupled reaction scheme in which ADP and ATP are recycled and hydrogen peroxide is a product of the reaction. Two enzymes in the scheme, glycerokinase and glycerol-3-phosphate oxidase, were co-immobilized on collagen membrane at the surface of a platinum electrode polarized at 700 mV. The rate of production of hydrogen peroxide in the reaction was measured amperometrically at the electrode and related to the activity of creatine kinase. Calibration curves prepared with human creatine kinase-MB isoenzyme standards were linear from 10 to 500 U liter-1 with a detection limit of 8 U liter-1. Total creatine kinase in serum was also determined by the present method. The precision of the method was 3.1% relative standard deviation at the 10 U liter-1 level of creatine kinase. Interference from adenylate kinase was eliminated by the addition of diadenosine-5'-pentaphosphate to samples.