ANTIBODIES AGAINST D-1, A RECOMBINANT 64-KDA MEMBRANE-PROTEIN, ARE ASSOCIATED WITH OPHTHALMOPATHY IN PATIENTS WITH THYROID AUTOIMMUNITY

We have tested for serum antibodies reactive with D-1, a recombinant 6 5-kDa human thyroid protein which is also expressed in eye muscle, in patients with thyroid autoimmunity and ophthalmopathy by immunofluores cence and SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Wester n blotting. We also measured antibodies to a 64-kDa pig eye muscle mem brane protein which is identified by SDS PAGE and Western blotting, co rrelating the two reactivities. While antibodies to D-1, expressed in Chinese hamster ovary (CHO) cell membrane, were detected in approximat ely 40% of patients with ophthalmopathy, in both tests the greatest pr evalence, by immunofluorescence, 73%, was demonstrated in patients wit h Graves' hyperthyroidism without clinically evident eye disease, alth ough only 50% of these patients were positive in immunoblotting. When the two tests for anti-D-1 antibodies were compared, immunofluorescenc e appeared to be the more specific and immunoblotting appeared to be t he more sensitive. The greatest prevalence of antibodies reactive with a 64-kDa pig eye muscle protein, 71%, was in patients with TAO of les s than 1 year duration; tests were positive in 49% of patients with mo re chronic ophthalmopathy and in 50% of patients with Graves' hyperthy roidism without evident eye disease. Antibodies reactive with D-1 were detected in 17% of normals by immunofluorescence and 24% by immunoblo ts, while antibodies reactive with the 64-kDa pig eye muscle protein w ere detected in only 10% of the normal subjects tested. Lesser prevale nces of antibodies to the two 64-kDa proteins in patients with establi shed eye disease suggest that such antibodies may be an early abnormal ity in patients with Graves' hyperthyroidism who are predisposed to de velop ophthalmopathy. Although the association was not close, reactivi ty against D-1 by immunoblotting, but not immunofluorescence, was sign ificantly correlated with reactivity to a 64-kDa eye muscle membrane p rotein by immunoblotting. On the other hand, when sera containing anti bodies reactive with both D-1 and the 64-kDa eye muscle protein were i ncubated with CHO (D-1) cell membrane, reactivity against D-1 was abso rbed while that against the eye muscle protein was not. The precise re lationship between the two 64-kDa proteins can only be clarified by cl oning the 64-kDa protein from an eye muscle expression library and com paring the sequences with those of D-1. (C) 1994 Academic Press, Inc.