Sensitive quantification of isoforms of canine MM creatine kinase with an immunoblot procedure suitable for large...

To quantify individual isoforms of canine myocardial MM creatine kinase (CK) we developed an immunoblot procedure suitable for analysis of large numbers of samples. Isoforms in plasma were separated by agarose electrophoresis, immobilized on nitrocellulose, detected with anti-MM followed by a labeled second antibody, and quantified by well counting. The amount of non-CK protein was reduced with sequential ethanol precipitation. Reproducibility of the assay developed was high with a standard deviation of 5.9% of mean values for each isoform. Variation of results after serial dilutions of samples constituted with 33.3% of each of the three isoforms was modest with a standard deviation of 3.5%. The method developed provided sensitive, discrete resolution of individual MM CK isoforms under nondenaturing conditions and should prove useful for improved, objective definition of the time of onset of irreversible injury to myocardial tissue rendered ischemic and hence improved stratification of patients studied in clinical trials concerned with protection and salvage of myocardium.