Quantitation of creatine kinase MB using an agarose electrophoresis method; importance of enzyme reagent concentration.

Quantification of CK-MB activity in serum using agarose electrophoresis requires that CK enzyme reagent be layered over the gel in order to develop enzyme activity. We have shown that when commercial enzyme reagents were used, and reconstituted in a normal fashion, that the CK-MM isoenzyme activity is underestimated which results in an overestimation of CK-MB activity. Underestimation of CK-MM occurs because the enzyme reagent are diluted approximately five-fold by the agarose, which results in the non-linear development of enzyme activity under sub-optimal conditions. Reconstitution of these commercial enzyme reagents in a concentrated manner (5 times more concentrated) compensates for the dilution of the reagent by the agarose and allows for the linear development with time of CK-MM and CK-MB enzyme activity. However, use of five-fold concentrated enzyme reagent does not totally prevent underestimation of CK-MM enzyme activity, and care must be taken in limiting the amount of enzyme activity placed on the agarose gel.