NEW ASSAYS FOR THE MEASUREMENT OF SERUM ANTIBODIES REACTIVE WITH EYE MUSCLE MEMBRANE-ANTIGENS CONFIRM THEIR SIGNIFICANCE IN THYROID-ASSOCIATED OPHTHALMOPATHY

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophores is (SDS-PAGE) and Western blotting are widely used to detect serum ant ibodies in patients with autoimmune disorders, this procedure unfolds and denatures proteins and may alter antibody binding sites, We have u sed nondenaturing methods for the purification of a 64-kDa eye muscle (EM) membrane antigen associated with thyroid-associated ophthalmopath y (TAO). Pig EM membrane proteins were prepared from crude homogenates by high-speed centrifugation and solubilized by hand homogenization. The 64-kDa protein was further purified by isoelectric focusing perfor med in the absence of SDS, detergents, reducing agents, and urea, Sera from patients with active TAO of recent onset and thyroid autoimmunit y without ophthalmopathy were tested for reactivity against purified n ative 64-kDa protein in inmunoblotting, Tests were positive in 64% of patients with TAO, in 37.5% of those with Graves' hyperthyroidism with out eye disease, in 11% of patients with Hashimoto's thyroiditis witho ut eye disease, and in 13% of normal subjects, Many of the same sera w ere also tested for cytotoxic activity against human EM cells in an an tibody-dependent cell-mediated cytotoxicity (ADCC) assay, ADCC tests w ere positive in 69% of patients with TAO but in no normal subject, The specificity and sensitivity of these two tests in TAO surpass those f or all other published results for orbital tissue reactive autoantibod ies. Although there was a tendency for a relationship between reactivi ty to the 64-kDa protein and cytotoxic activity against EM cells in AD CC there were many exceptions and overall the relationship between the two tests was not significant, Studies are necessary to determine the identity of the antigen(s) that is targeted by cytotoxic antibodies i n ADCC.