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Demonstration of Two Molecular Variants of Carcinoembryonic Antigen by Concanavalin A Sepharose Affinity Chromatography¹

Department of Clinical Chemistry [S. R. H., T. M. C], Roswell Park Memorial Institute, and Departments of Biochemistry and Experimental Pathology, [T. M. C.] State University of New York at Buffalo, Buffalo, New York 14263

The carcinoembryonic antigen (CEA) active glycoproteins from perchloric acid extract of liver-metastasized primary colon tumor have been separated by concanavalin A Sepharose (Con A Sepharose) chromatography. The CEA activities separated by Con A Sepharose chromatography were designated as loosely bound and tightly bound which, respectively, eluted on the Con A Sepharose column between 0.12 and 0.15 M and 0.3 M -methylmannose in a linear gradient of -methylmannose. Further purification of these activities by Sephadex G-200, Bio-Gels A-1.5m and P-300 yielded two variants of glycoproteins (B₁ and C₂) with CEA activity. Both purified preparations of CEA had similar immunochemical properties. Their A₂₈₀/A₂₆₀ ratios were 1.30 and 1.56, respectively. The purified loosely bound CEA (B₁) had immunological, chromatographic, and electrophoretic properties similar to those of ¹²⁵I-CEA, whereas the tightly bound CEA (C₂) had a lower molecular weight (120,000 to 140,000). Further, specificity of these two CEA's was established by their reactions in immunoelectrophoresis with preparations of specific goat anti-CEA antiserum obtained from other investigators. The results indicate the practical use of Con A Sepharose affinity chromatography for the separation and characterization of glycoprotein tumor antigens.

¹ This investigation was supported in part by USPHS Research Grant CA-15437 from the National Cancer Institute; Contract CB-33858 with the Division of Cancer Biology and Diagnosis, National Cancer Institute; and a grant from Hoffmann-La Roche, Inc.

Received 12/ 2/74. Accepted 7/22/75.