Antibodies to porcine eye muscle in patients with graves’ ophthalmopathy: Identification of serum immunoglobulins directed against unique determinants by immunoblotting and enzyme-linked immunosorbent assay

The immunological mechanisms involved in Graves’ ophthalmopathy are not known. To explore the pathophysiology of this disorder, we used an enzyme-linked immunosorbent assay to detect antibodies that bound to the 100, 000 × g sediment fraction of porcine eye muscle. Serum from patients with Graves’ ophthalmopathy had enhanced immunoglobulin binding to porcine eye muscle compared to serum from control subjects (P < 0.0001) or Graves’ disease patients without ophthalmopathy (P < 0.001); however, several individual normal serum samples also had elevated binding activity. Incubation of serum from some Graves’ patients with skeletal muscle or liver tissue resulted in reduction in immunoglobulin binding to porcine eye muscle. Thyroglobulin and TSH reduced binding only at high concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed some obvious differences in protein bands between porcine eye muscle and skeletal muscle. Serum from many normal subjects and patients with autoimmune thyroid disease formed bands with skeletal muscle protein fractions on immunoblotting, and no specific bands were found using serum from Graves’ ophthalmopathy patients. Immunoblots of Graves’ patients’ serum after reaction with either porcine skeletal or eye muscle showed no reactivity with thyroglobulin (200K and 320K) or thyroid microsomal antigen (105K). Instead, immunoblots of eye muscle and serum from Graves’ patients, with or without eye disease, showed two bands at 64K and 73K, which were shared by serum from normal subjects. In addition, several unique eye muscle determinants were detected in serum from 6 of 13 Graves’ disease patients analyzed. These unique bands usually had mol wt of less than 50K, and 5 of the 6 patients whose serum reacted with these determinants had significant ophthalmopathy. Our findings support the presence of potential antigenic differences between eye muscle and skeletal muscle accounting forc the immunological specificity of eye muscle as a target in Graves’ ophthalmopathy.