A monoclonal antibody to human brain-type creatine kinase. Increased avidity with mercaptans

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Abstract
A monoclonal antibody (subclass IgG1) was raised against human brain-type creatine kinase (CK-BB). This antibody did not cross-react with either muscle-type creatine kinase (CK-MM) or heart-type creatine kinase (CK-MB). The binding constant measured with native antibody was 6 .times. 108 M-1. In the presence of 2 mM-dithiothreitol this constant was some 40- to 50-fold greater. Partial reduction and alkylation showed that the increased binding was due to direct effect on the antibody and was associated with concomitant cleavage of the heavy-heavy interchain disulfide bonds. The binding constant measured with Fab'' fragments produced from reduced and alkylated antibody was similar to that shown by the native, unreduced antibody. The MW of the complex found in the absence of mercaptans was consistent with 1 antibody and 1 CK-BB molecule, whereas the MW estimated with reduced and alkylated antibody was consistent with a complex of 2 antibodies and 2 CK-BB molecules. Mercaptans may increase the flexibility of the hinge region of the antibody molecule, allowing the formation of a higher-order complex with increased avidity for the CK-BB dimer.

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