Creatine kinase B-subunit activity in human serum. I. Development of an immunoinhibition method for routine determination of S-creatine kinase B-sununit activity

Abstract

The properties of an inhibiting antibody directed against the M-subunits of human creatine kinase (EC 2.7.3.2, CK) were investigated in the reaction system recommended by the Scandinavian Committee on Enzymes (S.C.E.). At 37° C the rate of immunoinhibition of human CK M-subunit dependent activity corresponded to a t 1 2 of 38 s giving a k value of −2 per cent · s −1 when samples were incubated in the S.K.E. CK reagent A in the presence of antibody. Under the selected conditions immunoinhibition of S-CK M-subunit activity up to 1800 U/l was 99 per cent completed within 5 minutes using undiluted samples. No inhibition of CK B-subunit activity occurred at the chosen concentration of antibody. The inhibition data were verified using human sera and electrophoretically homogeneous preparations of human CK isoenzymes BB and MM. Sample adenylate kinase (EC 2.7.4.3, AK) was found to constitute a potential source of falsely increased S-CK B activity. As reported in the accompanying paper a S-CK B value of 15 U/l was used as a discrimination value in the diagnosis of acute myocardial infarction. The S.C.E. CK method incorporates a combination of two AK inhibitors adenosine 5'-monophosphate and P 1, P 5-diadenosine 5'-pentaphosphate. However, a frequency analysis of sample AK activities demonstrated that AK activities of more than 8 U/l will occur in about 10 per cent of the cases. Consequently, it was deemed necessary to measure the individual sample blank AK rates. The routine procedure developed thus included three separate measurements: determination of total S-CK and of S-CK B activities in the absence and presence of antibody, respectively. As the immunoinhibition was only 99 per cent complete a value corresponding to one per cent of the total S-CK activity was subtracted from the measured S-CK B activity. Likewise, the individual sample AK activity had to be measured and subtracted from the apparent S-CK B activity. The within-series precision of this S-CK B method at the levels of 10 U/l and 20 U/l corresponded to C.V. values of 10 and 5 per cent, respectively. Day-to-day precision at the level of 417 U/l corresponded to a C.V. of 4 per cent. The day-to-day precision of a control, total CK activity 625 U/l, gave a mean value of 53% ± 3% of CK B activity.