CHARACTERIZATION OF THE ANTIBODY-RESPONSE TO THE EXTRACELLULAR REGIONOF RECOMBINANT THYROTROPIN RECEPTOR

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogeni c in a number of autoimmune thyroid diseases including Graves' disease . We have characterised polyclonal antisera to TSH-R for antibodies wh ich may mimic those present in autoimmune thyroid disease. For immunis ations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react w ith the full length membrane receptor in transfected mammalian cells b y flow cytometry showing the presence of antibody capable of recognisi ng the native functional receptor. The properties of the generated ant ibodies have been compared after two injections or following a multipl e immunisation protocol with the receptor in adjuvant. High titre anti sera were readily generated after the short injection protocol and fur ther immunisations did not lead to any change in antibody titers. Anal ysis of the epitopes recognised using synthetic peptides confirmed pre vious observations that tile immunodominant determinants localise to t he amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibod y as assessed by inhibition of TSH mediated cAMP stimulation. There wa s an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determina nts on TSH-R. Our results support previous observations on the difficu lties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in a utoimmune disorders such as Graves' disease or primary myxoedema.