AUTOIMMUNITY IN ULCERATIVE-COLITIS - TROPOMYOSIN IS NOT THE MAJOR ANTIGENIC DETERMINANT OF THE DAS MONOCLONAL-ANTIBODY, 7E12H12

Ulcerative colitis (UC) has a proposed autoimmune pathogenesis. A 40-k D antigen (P40) has been isolated from UC colon, bound to immunoglobul in. Tropomyosin has been reported as the target antigen of a MoAb (7E1 2H12) raised against P40. We set out to investigate whether tropomyosi n is the major antigenic determinant for 7E12H12. Formalin-fixed, para ffin-processed and cryostat sections of fresh frozen colon from patien ts with UC, Crohn's disease and normals, were immunostained with 7E12H 12 and commercial anti-tropomyosin antibodies. In addition, the immuno reactivity of 7E12H12 with cytoskeletal components was examined on hum an endothelial cells (HUVEC) using anti-tropomyosin as a positive cont rol. Con-focal microscopy was used to determine the subcellular locali zation of signal. An extract of total colonic protein From UC colon wa s prepared. Using a combination of Western and immunoblotting (dot-blo ts), the immuno-reactivities of both tropomyosin (porcine and chicken) and colon protein extract with either 7E12H12 or commercial anti-trop omyosin were examined. Immunocytochemically, 7E12H12 localized to the apical and basolateral regions of plasma membrane, and to the supranuc lear 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 ant i-tropomyosin antibody but not with 7E12H12. P40 antigen was identifie d in the colon protein extract by immunoblotting with 7E12H12. There w as clear immunoreactivity between anti-tropomyosin antibody and both c hicken and porcine tropomyosin, and the colon protein extract, 7E12H12 did not bind to either chicken or porcine tropomyosin in appropriatel y controlled systems. We conclude that the pattern of immunostaining w ith 7E12H12 is not cytoskeletal, and there is no reactivity in immunob lots, between tropomyosin and 7E12H12. Tropomyosin is not the major ta rget antigen of this antibody in ulcerative colitis.