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.