Standardization of creatine kinase-MB (CK-MB) mass assays: the use of recombinant CK-MB as a reference material.
BACKGROUND
The AACC assembled a committee to identify and validate a standard creatine kinase MB isoenzyme (CK-MB) material to improve the comparability of CK-MB mass assays.


METHODS
Three protocols were used. In protocol I, various CK-MB materials prepared in different matrices were screened as candidate standards. In protocol II, participating manufacturers calibrated their systems with concentrates of human heart CK-MB and then tested 20 patient samples to evaluate calibration bias. In protocol III, participating manufacturers calibrated their immunoassay systems using recombinant CK-MB2 (rCK-MB2) diluted into their respective sample diluents and measured 50 samples.


RESULTS
Candidate materials showed high recovery in stripped human serum, but bias improved only from 59% to 38%. These data led to the use of human heart CK-MB diluted in each manufacturer's sample diluent. This strategy reduced bias from 31% to 15%. Because human heart CK-MB is difficult to provide, a lyophilized source of CK-MB2 was identified. rCK-MB2 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase HPLC, intrinsic protein fluorescence, circular dichroism, agarose gel electrophoresis, immunoreactivity studies, high and low temperature stability, and reconstituted stability to be equivalent to human heart CK-MB. Calibration of immunoassay systems with rCK-MB2 added into each respective manufacturer's sample diluent showed a 13% between-manufacturer bias.


CONCLUSIONS
Lyophilized rCK-MB2 was determined suitable for use as a reference material for CK-MB mass assays.
Christenson
R H
RH
Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. rchriste@umaryland.edu
Vaidya
H
H
Landt
Y
Y
Bauer
R S
RS
Green
S F
SF
Apple
F A
FA
Jacob
A
A
Magneson
G R
GR
Nag
S
S
Wu
A H
AH
Azzazy
H M
HM
D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000032:analysis / Q000302:isolation & purification / Q000592:standards*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D007527:Isoenzymes; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results
D007527:Isoenzymes; D011994:Recombinant Proteins; D003402:Creatine Kinase