IDENTIFICATION OF ANTIGENIC EPITOPES OF 1D ANTIGEN RECOGNIZED BY ANTIBODIES IN THE SERUM OF PATIENTS WITH THYROID-ASSOCIATED OPHTHALMOPATHY

Random fragments of full-length 1D cDNA encoding the 64-kDa antigen we re cloned and expressed in Escherichia coli as fusion proteins. Antige nic peptides were detected by colony blotting with sera from patients with thyroid-associated ophthalmopathy (TAO) and corresponding cDNA in serts were sequenced, Four antigenic peptides were characterized and p ositioned with respect to the 1D amino acid sequence. One antigenic pe ptide (G-8) was found to be close to the C-terminal end of 1D, and thr ee others (G-2, G-4, and G-5) were clustered at sites between amino ac ids 76 and 205. The immunoreactivities of a panel of sera were tested against full-length 1D and the four peptides. A panel consisting of se ra from (1) 15 patients with TAO, (2) 9 patients with Graves' hyperthy roidism without ophthalmopathy, (3) 6 patients with Hashimoto's thyroi ditis without ophthalmopathy, and (4) 12 normal subjects were tested a gainst the four peptides and full-length 1D protein by Western blottin g. Sera from patients with TAO also reacted more often with G-4 and/or G-5 peptides than did those from normal subjects [7 of 15 (47%) sera from TAO patients versus 1 of 12 (8%) sera from normal subjects; P < 0 .05], Based on positioning of peptides onto the full-length 1D sequenc e and the reaction pattern of the serum panel, six immunogenic epitope s were identified on the 1D molecule, (C) 1995 Academic Press, Inc.