Production and certification of an enzyme reference material for pancreatic α-amylase (CRM 476)

Gemma Gubern, Francesca Canalias, F. Javier Gella, Elizabeth Colinet, Christos Profilis, Derek H. Calam, Ferruccio Ceriotti, J. Dufaux, Anthony G. Hadjivassiliou, Jean Marc Lessinger, Klaus Lorentz, Anne Vassault

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10 Citations (Scopus)

Abstract

We describe the preparation of a lyophilized material containing purified human pancreatic α-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was 299% pure on polyacrylamidegel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic α-amylase had a molar mass of 57 500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified α-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/l and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20°C. The certified value for α-amylase catalytic concentration in the reconstituted reference material is 555 U/l ± 11 U/l when measured by the specified method at 37°C. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control or for calibration of α-amylase catalytic concentration measurements.
Original languageEnglish
Pages (from-to)145-162
JournalClinica Chimica Acta
Volume251
DOIs
Publication statusPublished - 30 Jul 1996

Keywords

  • Enzyme activity
  • Refeence material
  • Standardization

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