Antibodies to normal human colon membranes: preparation, characterization and tissue distribution

Rabbit antisera were raised to a membrane fraction of normal human colonic epithelium. After absorption, two antisera appeared to show restricted epithelial specificity when tested on routine paraffin wax-embedded histological sections by the indirect immunoperoxidase technique. The reaction was intense on epithelial cells of large and small intestine, and positive on stomach and duodenum, bile ducts in liver, gall bladder, pancreas and salivary gland. A very weak reaction was also seen in the bronchus and lung. There was no reaction with stromal, vascular or muscle components. All other tissues tested were negative, including hepatocytes, ectodermally-derived glandular epithelia, urogenital tissues and lymphoid organs. The antigen was also detected in 21 primary and metastatic large bowel carcinomata. By immunofluorescence, the antisera reacted with the colon adenocarcinoma-derived HT29 cell line and with primary colon epithelium explant cultures, but not with cultured fibroblasts. By immunoblotting of HT29 whole cell lysates, a triplet of polypeptides of approximate molecular weight range 55 000 to 60 000 were detected. This specificity appeared to be unrelated to previously described normal or tumour-associated antigens by criteria of tissue distribution, immunolocalization, molecular weight, and either absorption or radiobinding assays, or both.