Exploitable results Certification and Production of a Purified Human CK-2 (CK-MB). Preparation as an enzyme reference material. General Information The objective of the proposed work is to prepare a CK-MB reference material in order to harmonize respective measurements within the European Community. The measurement of creatine kinase isoenzyme 2, is widely used for diagnosing myocardial infarction, and important clinical decisions depend on the results of this test; harmonization is necessary since the various assay categories put on the market produce discrepant results. Achievements : A lyophilized material containing purified human creatine kinase-2, has been prepared and its catalytic concentration certified (CRM 608). About 1.4 mg of creatine kinase-2 was purified from human heart by ethanol precipitation and chromatography successively on DEAE-Sephacel(r) and Blue-Sepharose(r). The purified enzyme had a specific activity of 1001 U/mg protein 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 (M and B) with relative molar masses of 43 650 g/mol (B) and 41 700 (M) g/mol and an isoelectric point at pH 5.8. The material was prepared by diluting the purified creatine kinase-2 in a matrix containing PIPES buffer 25 mmol/l, ADP 2 mmol/1, 2-mercaptoethanol 5 mmol/1, NaC1 154 mmol/1, human albumin 50 g/1, pH 7.2, by dispensing in vials and by freeze-drying. There was no significant variation between vials in their content of catalytic activity. The loss of enzyme activity and of enzyme mass on storage at -20 C is predicted to be less than 0.18% and 0.04% respectively per annum on the basis of accelerated degradation studies. The Catalytic concentrations of creative kinase - 2 in samples of the reconstituted material is certified to be (1.12 ± 0.03) µkat/l or (67.2 ± 1.8) u/l when measured by the IFCC recommended method at 30 C. The material can be used to verify the comparability of results from different laboratories, for quality control purposes, as a reference for manufacturers of reagents, or for calibration of the creatine kinase-2 catalytic concentration measurements. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
