Production and certification of an enzyme reference material for creatine kinase isoenzyme 2 (CRM 608)

We describe the preparation of a lyophilized material containing purified human creatine kinase 2 (CK-MB), and the certification of its catalytic concentration. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the creatine kinase 2 catalytic concentration measurements. The enzyme was purified from human heart by ethanol precipitation and chromatography successively on DEAE-Sephacel and Blue-Sepharose. The purified enzyme had a specific activity of 998.4 U/mg and was > 99% pure on polyacrylamide gel electrophoresis. The material was examined for several possible contaminating enzymes, which were found to be absent. The purified creatine kinase 2 had two subunits (B and M) with molecular masses of 43,650 and 41,700 g/mol, respectively, and an isoelectric point at pH 5.8. The material was prepared by diluting the purified creatine kinase 2 in a matrix containing 25 mmol/L PIPES buffer, pH 7.2, 2 mmol/L ADP, 5 mmol/L 2-mercaptoethanol, 154 mmol/L sodium chloride and 50 g/L human serum albumin, dispensing it into vials and freeze-drying. The batch was shown to be homogeneous. The loss of enzyme activity on storage at -20 degrees C is predicted to be less than 0.18% per annum on the basis of accelerated degradation studies. The catalytic concentration of creatine kinase in samples of the reconstituted material is certified to be 67.2+/-1.8 U/L (1.12+/-0.03 microkat/L) when measured, at 30 degrees C, by the Recommended Method of the International Federation of Clinical Chemistry.