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 Lewisa and Lewisb 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.


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).

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