Analysis of a human tumor-associated glycoprotein (TAG-72) identified by monoclonal antibody B72.3.
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.
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000276:immunology / Q000302:isolation & purification*; D001789:Blood Group Antigens; D002240:Carbohydrate Sequence; D003593:Cytoplasm / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D009077:Mucins / Q000276:immunology / Q000302:isolation & purification*; D009363:Neoplasm Proteins / Q000276:immunology / Q000302:isolation & purification*; D014461:Ultracentrifugation
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D001789:Blood Group Antigens; D006023:Glycoproteins; D009077:Mucins; D009363:Neoplasm Proteins