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Purification and characterization of Fab fragments with rapid reaction kinetics against myoglobin

Myoglobin is an early biomarker for acute myocardial infarction. Recently, we isolated the antibody IgG-Myo2-7ds, which exhibits unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid dissociation kinetics are thought to be premature IgG forms that are produced during the early stage of in vivo immunization. In the present study, we identified the epitope region of the IgG-Myo2-7ds antibody to be the C-terminal region of myoglobin, which corresponds to 144–154 aa. The Fab fragment was directly purified by papain cleavage and protein G affinity chromatography and demonstrated kinetics of an association constant of 4.02 × 104 M−1 s−1 and a dissociation constant of 2.28 × 10−2 s−1, which retained the unique reaction kinetics of intact IgG-Myo2-7ds antibodies. Because a rapid dissociation antibody can be utilized for antibody recycling, the results from this study would provide a platform for the development of antibody engineering in potential diagnostic areas such as a continuous monitoring system for heart disease. Fab fragments of rapid reaction antibody were produced and the epitope site was identified. Purified Fab exhibits the unique reversible binding pattern.