Production and certification of an enzyme reference material for adenosine deaminase 1 (BCR 647)

Abstract

Background: We describe the preparation of a lyophilised reference material containing purified human adenosine deaminase 1 and the certification of its catalytic concentration. Methods: The enzyme was purified from human erythrocytes. Results: The enzyme was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts (<0.4%) of alanine aminotransferase, aspartate aminotransferase and l-lactate dehydrogenase were detected in the purified fraction. The purified adenosine deaminase had a molar mass of 41 600 g/mol and an isoelectric pH at 4.7, 4.85 and 5.0. The material was prepared by diluting the purified adenosine deaminase in a matrix containing 50 mmol/l Tris–HCl buffer pH 7.4 and 30 g/l human serum albumin; dispensing in vials and freeze-drying. The batch was homogeneous and the predicted loss of adenosine deaminase activity per year on the basis of accelerated degradation studies was 0.006% at −20°C and 0.04% at 4°C. The certified value for adenosine deaminase catalytic concentration in the reconstituted reference material is (2.55±0.09) μkat/l when measured by the method that uses adenosine as substrate and glutamate dehydrogenase as auxiliary enzyme at 37°C. Conclusions: The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the adenosine deaminase catalytic concentration measurements.