Immunoaffinity purification of creatine kinase-MB from human, dog, and rabbit heart with use of a monoclonal antibody specific for CK-MB.
We describe a simple, rapid immunoaffinity procedure for purifying the MB isoenzyme of creatine kinase. Immunoaffinity gel is prepared by linking a monoclonal antibody ("Conan-MB"), specific for this isoenzyme, to Sepharose 4B. Heart tissue is homogenized and fractionated with 40-70% saturated ammonium sulfate before it is applied to the immunoaffinity gel. CK-MB activity, retained on the gel, is then eluted with a high-pH diethylamine buffer (0.1 mol/L, pH 10.5). The purified CK-MB isoenzyme is stabilized by collection directly into tubes containing glycerol (to prevent dissociation of the enzyme subunits) and pH-neutralizing buffer. This procedure compares favorably in yield, specific activity, and technical ease with a multi-column purification method previously used in our laboratory. We have used the immunoaffinity procedure to purify to homogeneity CK-MB from human, dog, and rabbit heart, with yields of 50.0%, 53.1%, and 49.3% and specific activities of 540, 477, and 377 kU/g, respectively. The preparations are pure as judged by protein staining with silver nitrate after electrophoresis on sodium dodecyl sulfate/polyacrylamide gel.
Landt
Y
Y
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Vaidya
H C
HC
Porter
S E
SE
Dietzler
D N
DN
Ladenson
J H
JH
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D003402:Creatine Kinase / Q000276:immunology / Q000302:isolation & purification*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D009206:Myocardium / Q000201:enzymology*; D011817:Rabbits; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D003402:Creatine Kinase