ANALYSIS OF THE IGG AUTOANTIBODY REPERTOIRE IN ENDOCRINE OPHTHALMOPATHY USING THE MEGABLOT TECHNIQUE

Purpose. Normal sera contain a large number of naturally-occurring aut oantibodies which can complicate the differentiation of disease-associ ated autoantibodies from the complex background of ''autoimmune noise' ' (i.e. naturally-occurring autoantibodies). The aim of this study was to analyze the human IgG autoantibody repertoire of sera from patient s suffering from endocrine ophthalmopathy and to compare those to sera from healthy subjects using the recently developed MegaBlot technique . This new method allows the simultaneous global and quantitative scre ening for reactivities of antibodies with a large number of antigens. Methods. Sera (n = 17: endocrine ophthalmopathy; n = 10: healthy subje cts) were tested against Western blots of SDS-PAGE preparations of pro teins from human extraorbital eye muscle. Digital image analysis was p erformed (ScanPacK(TM), Biometra, Germany). The blots were subsequentl y analyzed by our new MegaBlot technique. Results. Both the sera of pa tients suffering from endocrine ophthalmopathy and the sera of healthy subjects revealed reactivities of antibodies against eye muscle prote ins (up to 15 bands/blot). The MegaBlot procedure could differentiate between the banding patterns of patients suffering from endocrine opht halmopathy and healthy subjects. The staining patterns of both groups were significantly different (p < 0.02). A 64 kDa protein was recogniz ed by autoantibodies in the sera of both healthy subjects and patients suffering from endocrine ophthalmopathy. Conclusions. This study show s that naturally-occurring antibodies in sera of healthy subjects reco gnize many antigenic proteins, but despite that, the MegaBlot techniqu e can differentiate between the complex staining patterns of both grou ps. Our new method can be a valuable tool in the evaluation of autoant ibody repertoires in autoimmune diseases such as endocrine ophthalmopa thy and can help to find and to identify the disease-associated autoan tibodies.