TY - JOUR
T1 - Production and certification of an enzyme reference material for pancreatic α-amylase (CRM 476)
AU - Gubern, Gemma
AU - Canalias, Francesca
AU - Gella, F. Javier
AU - Colinet, Elizabeth
AU - Profilis, Christos
AU - Calam, Derek H.
AU - Ceriotti, Ferruccio
AU - Dufaux, J.
AU - Hadjivassiliou, Anthony G.
AU - Lessinger, Jean Marc
AU - Lorentz, Klaus
AU - Vassault, Anne
PY - 1996/7/30
Y1 - 1996/7/30
N2 - 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.
AB - 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.
KW - Enzyme activity
KW - Refeence material
KW - Standardization
U2 - https://doi.org/10.1016/0009-8981(96)06302-4
DO - https://doi.org/10.1016/0009-8981(96)06302-4
M3 - Article
SN - 0009-8981
VL - 251
SP - 145
EP - 162
JO - Clinica Chimica Acta
JF - Clinica Chimica Acta
ER -