Autoimmunity in ulcerative colitis: tropomyosin is not the major antigenic determinant of the Das monoclonal antibody, 7E12H12

Abstract

Ulcerative colitis (UC) has a proposed autoimmune pathogenesis. A 40-kD antigen (P40) has been isolated from UC colon, bound to immunoglobulin. Tropomyosin has been reported as the target antigen of a MoAb (7E12H12) raised against P40. We set out to investigate whether tropomyosin is the major antigenic determinant for 7E12H12. Formalin-fixed, paraffin-processed and cryostat sections of fresh frozen colon from patients with UC, Crohn's disease and normals, were immunostained with 7E12H12 and commercial anti-tropomyosin antibodies. In addition, the immunoreactivity of 7E12H12 with cytoskeletal components was examined on human endothelial cells (HUVEC) using anti-tropomyosin as a positive control. Con-focal microscopy was used to determine the subcellular localization of signal. An extract of total colonic protein from UC colon was prepared. Using a combination of Western and immunoblotting (dot-blots), the immunoreactivities of both tropomyosin (porcine and chicken) and colon protein extract with either 7E12H12 or commercial anti-tropomyosin were examined. Immunocytochemically, 7E12H12 localized to the apical and basolateral regions of plasma membrane, and to the supranuclear cytoplasm in colonic epithelium. Using anti-tropomyosin antibody it was not possible to identify the cytoskeleton in colonic epithelium. Cytoskeletal components were identifiable in HUVEC cultures with anti-tropomyosin antibody but not with 7E12H12. P40 antigen was identified in the colon protein extract by immunoblotting with 7E12H12. There was clear immunoreactivity between anti-tropomyosin antibody and both chicken and porcine tropomyosin, and the colon protein extract. 7E12H12 did not bind to either chicken or porcine tropomyosin in appropriately controlled systems. We conclude that the pattern of immunostaining with 7E12H12 is not cytoskeletal, and there is no reactivity in immunoblots, between tropomyosin and 7E12H12. Tropomyosin is not the major target antigen of this antibody in ulcerative colitis.