Kinetic immunochemical method for the simultaneous quantification of creatine kinase isoenzymes

We describe the development and evaluation of a kinetic immunochemical method for the simultaneous quantification of isoenzymes. Specifically, we use the inhibition of the M subunit of creatine kinase (CK; EC 2.7.3.2) by antibodies to quantify isoenzymes CK-MM and CK-MB in serum matrices. Nonlinear least-squares data-processing is used to compute the enzyme activities from the time-dependent response of absorbance vs time. Variables affecting the method, namely temperature, substrate concentration, and antibody concentration as well as their interactions, are evaluated by using response-surface methodology. For several concentrations of CK-MM and CK-MB in the range of diagnostic significance, least-squares fits of computed (y) vs expected (x) values yielded equations of y = 0.98x + (2.0 X 10(-5)) s-1 for CK-MM and y = 1.04x - (4.6 X 10(-5)) s-1 for CK-MB for rates between 0 and 3.5 X 10(-3) s-1 (CK-MM) and 0.5 X 10(-3) s-1 (CK-MB). The equations for comparison of kinetic results (y) with results by a kit method (x) were y = 0.97x + (6.5 X 10(-5)) s-1 for CK-MM and y = 1.02x + (4.3 X 10(-5)) s-1 for CK-MB. Pooled day-to-day relative standard deviations (CVs) for the kinetic method were 4.1% and 2.8% for CK-MM and CK-MB, respectively.