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Biochemical and Immunological Properties of the Human Carcinoma-associated CAR-3 Epitope Defined by the Monoclonal Antibody AR-3¹

Department of Biomedical Sciences and Oncology, School of Medicine, University of Torino, Torino, Italy

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 >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 topografic 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.

¹ This research was supported by grants from the National Research Council (CNR PFTBMS 87.00768.57), the Ministry of Public Education and the Italian Association for Cancer Research (AIRC).

² To whom requests for reprints should be addressed, at Department of Biomedical Sciences and Oncology, Section of Histology, University of Torino, C.so M. D'Azeglio 52, 10126 Torino, Italy.

Received 1/ 4/88. Revised 8/ 1/88. Revised 12/ 1/88. Accepted 12/12/88.

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