Biochemical and immunological properties of the human carcinoma-associated CAR-3 epitope defined by the monoclonal antibody AR-3.
The monoclonal antibody AR-3 reacts with an epitope (CAR-3) carried on a high-molecular-weight glycoprotein associated with carcinomas of the pancreas, stomach, colon, uterus, and ovary. This study reports the partial purification and characterization of CAR-3-bearing molecule. The antigen was quantified by a double determinant immunoradiometric assay. CAR-3 antigen was purified by a three-step procedure, consisting of perchloric acid extraction, molecular sieving on Sepharose CL-4B, and affinity chromatography on AR-3 antibodies coupled to Sepharose 4B. Following this procedure CAR-3 antigen was purified about 400-fold with a 36% yield. Treatment of the CAR-3 antigen with 16 mM metaperiodate or with 1 N NaOH resulted in complete loss of activity. Antigenicity survived enzymatic treatments known to destroy proteins. The epitope was found to be carried on a molecule with a molecular weight of greater than 400,000 with a density of 1.45 g/ml, metabolically labeled with [35S]sulfate, [3H]glucosamine, and [35S]methionine. It is concluded that CAR-3 epitope is expressed on a carbohydrate moiety linked to a sulfo-mucin-like molecule via an O-glycosidic bond. Cross-competition experiments showed that CAR-3 epitope is strictly related or in close topographic proximity to Lewis(a) and Lewis(b) antigens. Cross-double determinant immunoradiometric assay experiments indicated that the same mucin carrying CAR-3 bears also CA 19-9, CA 125, and BW 494 epitopes.
Prat
M
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Department of Biomedical Sciences and Oncology, School of Medicine, University of Torino, Italy.
Medico
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E
Rossino
P
P
Garrino
C
C
Comoglio
P M
PM
D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000302:isolation & purification; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology; D000939:Epitopes / Q000032:analysis*; D006017:Glycolipids / Q000276:immunology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009077:Mucins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D000939:Epitopes; D006017:Glycolipids; D009077:Mucins