Autoantibodies interacting with purified native thyrotropin receptor

Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatog raphy from membrane extracts of stably transfected L cells. An ELISA test w as devised to study anti-TSHR autoantibodies directly. Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conform ational epitopes only present on the native receptor. The use of deglycosyl ated TSHR and of purified receptor ectodomain (alpha-subunit) showed that t he autoantibodies recognized only the protein backbone moiety of the recept or and that their epitopes were localized entirely in its ectodomain. Autoantibodies were detected in 45 of 48 subjects with untreated Graves' di sease and in 26 of 47 healthy volunteers. The affinity for the receptor was similar in the two groups (K-d = 0.25-1 x 10(-10) M) and the autoantibodie s belonged to the IgG class in all cases. Although the concentration of aut oantibodies was higher in Graves' disease patients (3.50 +/- 0.36 mg.L-1) t han in control subjects (1.76 +/- 0.21) (mean +/- SEM), then was an overlap between the groups. Receptor-stimulating autoantibodies (TSAb) were studied by measuring cAMP s ynthesis in stably transfected HEK 293 cells. Their characteristics (recogn ition of alpha-subunit, of deglycosylated TSHR, nonrecognition of bacterial ly expressed denatured receptor) were similar to those of the antibodies de tected by the ELISA test. TSAb were only found in individuals with Graves' disease. The ELISA test measures total anti-TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific e pitopes involved in receptor activation Our observations thus disprove the hypothesis according to which Graves' disease is related to the appearance of anti-TSHR antibodies not present in normal subjects. Actually, anti-TSHR antibodies exist in many euthyroid subjects, in some cases even at concent rations higher than those found in patients with Graves' disease. What dist inguishes the latter from normal subjects is the existence of subpopulation (s) of antibodies directed against specific epitope(s) of the receptor invo lved in its activation.