Isolation and properties of a human pancreatic adenocarcinoma-associated antigen, DU-PAN-2

Abstract

This work describes the molecular properties of a human pancreatic adenocarcinoma-associated mucin-like antigen defined by a murine monoclonal antibody (DU-PAN-2). DU-PAN-2 antigen is a large molecule, readily detected in the body fluids of patients with pancreatic adenocarcinoma and sensitive to neuraminidase treatment and alkaline reduction. The antigen binding activity of the DU-PAN-2 immunoglobulin M antibody is markedly reduced when coupled to an insoluble matrix. Therefore, the antigen was partially purified from the serum and ascites of patients with pancreatic adenocarcinoma by ammonium sulfate fractionation and Affi-Gel-Blue chromatography to remove most of the serum proteins. Noncovalently associated proteins were further separated on CsCl/CsBr density gradients and noncovalently associated lipids removed by organic solvent extraction. DU-PAN-2 antigen was monitored by a solid-phase competition radioimmunoassay. We have been able to detect antigen reactivity and analyze its electrophoretic pattern following transfer from 1% agarose gel onto nitrocellulose paper and immunoblotting with DU-PAN-2 antibody. Antigen was also labeled metabolically with various radioactive monosaccharides and sulfates. Radioimmunoprecipitation of labeled antigen with DU-PAN-2 antibody showed two distinct broad antigen bands consistent with the immunoblotting signals. The heavily glycosylated and polydisperse nature of this antigen and the results of various enzyme treatments suggest that the DU-PAN-2 epitope is expressed on a mucin-like molecule.