Semi-automated direct colorimetric measurement of creatine kinase isoenzyme MB activity after extraction from serum by use of a CK-MB-specific monoclonal antibody.
This semi-automated colorimetric assay for the MB isoenzyme of creatine kinase (EC 2.7.3.2) is based on a monoclonal antibody ("Conan-MB") specific for this isoenzyme and is a modification of a previously published method (Vaidya et al., Clin Chem 1986;32:657-63). A 0.64-cm bead coated with 2 to 3 micrograms of antibody is incubated with 100 microL of serum and 10 microL of 0.2 mol/L beta-mercaptoethanol for 1 h at room temperature, to extract CK-MB. The beads are washed with de-ionized water and incubated with CK substrate for 45 min at 37 degrees C. A solution containing trans-1,2-diaminocyclohexane-N,N,N', N'-tetraacetic acid, p-iodonitrotetrazolium violet, and diaphorase is added and the resulting colored product is measured at 492 nm. The standard curve is linear to 200 U of CK-MB per liter, and analytical recovery is 97-113%. Total assay CV for low (9.7 U/L) and high (50.7 U/L) quality-control materials was 14.1% (n = 1878) and 11.6% (n = 1842), respectively. CK-MB activity correlated well (r = 0.978, n = 226) with CK-MB measured by a two-site mass immunoassay, and 99.4% of samples with CK-MB greater than or equal to 12 U/L (n = 347) were verified by electrophoresis on agarose.
Landt
Y
Y
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Vaidya
H C
HC
Porter
S E
SE
Whalen
K
K
McClellan
A
A
Amyx
C
C
Parvin
C A
CA
Kessler
G
G
Nahm
M H
MH
Dietzler
D N
DN
D000327:Adsorption; D000911:Antibodies, Monoclonal; D001322:Autoanalysis; D003124:Colorimetry; D004396:Coloring Agents; D003402:Creatine Kinase / Q000097:blood*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007118:Immunoassay; D007527:Isoenzymes; D008863:Microspheres; D009203:Myocardial Infarction / Q000201:enzymology; D011137:Polystyrenes; D011786:Quality Control; D013778:Tetrazolium Salts
D000911:Antibodies, Monoclonal; D004396:Coloring Agents; D007527:Isoenzymes; D011137:Polystyrenes; D013778:Tetrazolium Salts; C013574:iodonitrotetrazolium; D003402:Creatine Kinase