IDENTIFICATION OF THYROID BLOCKING ANTIBODIES AND RECEPTOR EPITOPES IN AUTOIMMUNE HYPOTHYROIDISM BY AFFINITY PURIFICATION USING SYNTHETIC TSH RECEPTOR PEPTIDES

To examine the interaction of immunoglobulins from patients with newly diagnosed hypothyroidism with the TSH receptor (TSHr), we tested prot ein-A purified IgG in an ELISA assay with a series of peptides represe nting the entire extracellular domain (ECD) of human TSHr. Antibodies bound, on average, 4.1 peptides (range 0-16) per patient, and antibodi es from 26 of 30 patients (86.6%) demonstrated binding to al least one peptide. Six of the 20-mer peptides (61, 151, 181, 301, 361, 376) wer e most frequently recognized. These were used to construct affinity co lumns and separate IgGs from 10 patients into bound and unbound fracti ons. All fractions were tested for their ability to stimulate and inhi bit cAMP generation in FRTL-5 cells. Inhibitory IgGs were purified fro m 9 patients (90%), suggesting that the incidence of blocking antibodi es (TBAb) in autoimmune hypothyroidism is higher than previously repor ted. 7 of 10 patients had antibodies that recognized peptide 361 furth er supporting the importance of this epitope in TBAb binding. Anti-mic rosomal and anti-thyroglobulin antibodies did not co-purify with inhib itory antibodies, and were always in the unbound fractions. We found n o correlation between the pattern of antibody binding or bioactivity w ith clinical manifestations of hypothyroidism. Conclusions: (1) The ma jority of patients with autoimmune hypothyroidism have antibodies agai nst the TSHr-ECD that recognized linear epitopes. Most have antibodies directed at more that one site and the pattern is quite heterogeneous . (2) Six sites (noted above) are most frequently recognized. (3) Inhi bitory antibodies are distinct from anti-microsomal and anti-thyroglob ulin antibodies.