Abstract
Although antibodies to plasma isoenzymes have been induced, a radioimmunoassay (RIA) has not previously been developed in part because of lack of specificity and loss of enzymatic activity incurred during radioactive labelling. A RIA for human MB CK ('myocardial'), a creatine kinase isoenzyme, would provide a specific and quantitative test for myocardial infarction, and perhaps more important, it would aid in elucidating the nature of MB CK protein turnover in the circulation independent of enzymatic activity. Assuming that MB, but not MM (the only other isoenzyme present appreciably in blood from patients with myocardial infarction) would bind to B subunit antibody, we harvested BB antiserum from rabbits immunized with human brain BB purified by chromatography. BB, MB, and MM for use as antigens in the RIA were labelled with radioactive N-succinimidyl 3-(4-hydroxyphenyl-propionate), a procedure that avoided oxidation of the antigen and yielded 200 000 cpm/μg and 97% of initial CK enzymatic activity. After incubation in 1.6 M Tris, pH 7.4, 2% albumin, and 20 mM CH3CH2SH, labelled BB and MB bound to anti-BB (93 and 56%) and precipitated in 44% (NH4)2SO4, but 125I-MM in 5000-fold excess did not. Unlabelled MB (20 pg/ml) but not MM (up to 5000 pg/ml) competitively displaced precipitable counts. Development of a sensitive and specific RIA based on this phenomenon accurately detected MB (20 pg/ml) despite excess MM (5000-fold excess) in plasma samples constituted with standard. In patients with myocardial infarction, MB CK determined by the RIA was elevated in all cases. © 1978.