Monoclonal antibody against human colonic sulfomucin: immunochemical detection of its binding sites in colonic mucosa, colorectal primary carcinoma, and metastases

Previous studies using metabolic labeling of fresh colonic mucosa and colorectal carcinoma with [35S]sulfate followed by biochemical analysis demonstrated that the amount of a sulfated high-molecular-weight glycoprotein expressed in primary colorectal carcinoma was lower than that in normal mucosa, and that the amount further decreased in liver metastases. This suggested that this sulfated molecule represented a sulfomucin previously defined by histochemical reactivity with a cationic dye. We have extracted and partially purified this high-molecular-weight sulfated glycoprotein from normal human colonic mucosa. We immunized mice with the partially purified sulfomucin and generated hybridomas. One cloned hybridoma, designated as 91.9H, produced a monoclonal antibody strongly reactive with a component which migrated at an identical position as the metabolically [35S]sulfate-labeled high-molecular-weight glycoprotein after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reactive molecules appeared to have a polydisperse nature with a molecular weight ranging between 400,000 and 900,000. The [35S]sulfate-labeled high-molecular-weight glycoprotein was bound to Staphylococcus Protein A-agarose coated with this monoclonal antibody but did not bind to unconjugated Protein A-agarose. The immunoprecipitated substance also migrated at an apparent molecular weight range of 400,000 to 900,000. The reactivity of monoclonal antibody 91.9H with the extracts of normal mucosa, colorectal primary carcinoma, and metastasis was compared by dot blot assay on a nitrocellulose membrane. This antibody was more reactive with the extracts of mucosa adjacent to carcinoma tissues than with the carcinoma extracts. Primary tumors showed higher reactivity than metastases in most of the cases. These results strongly suggest that this antibody is specific to colonic sulfomucins or at least to mucins closely related to colonic mucins previously identified by metabolic labeling with [35S]sulfate.