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Analysis of a Human Tumor-associated Glycoprotein (TAG-72) Identified by Monoclonal Antibody B72.3

Laboratory of Tumor Immunology and Biology, National Cancer Institute [V. G. J., J. S., A. J. P., J. B., D. C.], and National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases [J. L. M.], NIH, Bethesda, Maryland 20892

Monoclonal antibody B72.3 binds a high-molecular-weight tumor-associated glycoprotein identified as TAG-72. This study reports the partial purification and characterization of TAG-72 from a xenograft of a human carcinoma cell line, LS-174T, which expresses high levels of this antigen. The tumor homogenate was initially fractionated by Sepharose CL-4B chromatography. The high-molecular-weight TAG-72, found in the exclusion volume, was then subjected to two sequential passages through B72.3 antibody affinity columns. At each step of the procedure, TAG-72 content was quantitated using a competition radioimmunoassay, and the degree of purification was expressed as the ratio of antigen in units to total protein. The three-step procedure produced a purification of TAG-72 with minimal contamination by other proteins as shown by polyacrylamide gel electrophoresis, followed by staining with Coomassie Blue or periodic acid/Schiff reagent. The density of affinity-purified TAG-72, as determined by cesium chloride gradient ultracentrifugation, was found to be 1.45 g/ml. This density determination, together with the high molecular weight of TAG-72, its resistance to Chondroitinase digestion, the presence of blood group-related oligosaccharides, and sensitivity to shearing into lower-molecular-weight forms suggest that TAG-72 is a mucin-like molecule.

¹ To whom requests for reprints should be addressed.

Received 7/31/85. Revised 11/ 1/85. Accepted 11/ 4/85.

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