Isolation of Antibodies that are Specific for Human Carboxypeptidase B but not for the Zymogen Procarboxypeptidase B by Affinity Chromatography

Abstract

A major theory for the pathogenesis of acute pancreatitis is that the normally inactive precursors of pancreatic proteases become activated, with subsequent destruction of enzymes, polypeptide hormones and other proteins in tissue and blood. To investigate the role of proteases in the disease, we developed radioimmunoassays for a number of these enzymes. However, experiments with serum samples fractionated by gel filtration showed that the amount of immunoreactive protein is not directly related to the amount of active enzyme against which the antiserum was directed. For example, procarboxypeptidase B effectively crossreacts about 30% with carboxypeptidase B using antiserum to active enzyme. To obtain antibodies that are specific for active enzyme, we treated the antiserum with resin containing immobilized procarboxypeptidase B. Stepwise addition of the resin produced antiserum with progressively less reactivity towards the zymogen relative to the enzyme, with the final material having less than 1% crossreactivity. The titer of enzyme-specific antiserum was 1% that of the original; however, it could be estimated that 10% of the original antibodies were specific for the active enzyme. To increase the yield of enzyme-specific antibodies, we are currently trying to obtain a mouse hybridoma that produces the desired antibody.