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 homo...

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Bibliographic Details
Published in:Cancer research (Chicago, Ill.) Ill.), 1986-02, Vol.46 (2), p.850-857
Main Authors: JOHNSON, V. G, SCHLOM, J, PATERSON, A. J, BENNETT, J, MAGNANI, J. L, COLCHER, D
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal
Antigens, Neoplasm - immunology
Antigens, Neoplasm - isolation & purification
Biological and medical sciences
Blood Group Antigens
Carbohydrate Sequence
Cytoplasm - immunology
Electrophoresis, Polyacrylamide Gel
Glycoproteins - immunology
Glycoproteins - isolation & purification
Host-tumor relations. Immunology. Biological markers
Humans
Medical sciences
Molecular Weight
Mucins - immunology
Mucins - isolation & purification
Neoplasm Proteins - immunology
Neoplasm Proteins - isolation & purification
Tumors
Ultracentrifugation
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Summary: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.
ISSN:0008-5472
1538-7445