Identification of Thyroid Blocking Antibodies and Receptor Epitopes in Autoimmune Hypothyroidism by Affinity Purification Using Synthetic TSH Receptor Peptides

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Abstract
To examine the interaction of immunoglobulins from patients with newly diagnosed hypothyroidism with the TSH receptor (TSHr), we tested protein-A purified IgG in an ELISA assay with a series of peptides representing the entire extracellular domain (ECD) of human TSHr. Antibodies bound, on average, 4.1 peptides (range 0-16) per patient, and antibodies from 26 of 30 patients (86.6%) demonstrated binding to at least one peptide. Six of the 20-mer peptides (61, 151, 181, 301, 361, 376) were most frequently recognized. These were used to construct affinity columns and separate IgGs from 10 patients into bound and unbound fractions. All fractions were tested for their ability to stimulate and inhibit cAMP generation in FRTL-5 cells. Inhibitory IgGs were purified from 9 patients (90%), suggesting that the incidence of blocking antibodies (TBAb) in autoimmune hypothyroidism is higher than previously reported. 7 of 10 patients had antibodies that recognized peptide 361 further supporting the importance of this epitope in TBAb binding. Anti-microsomal and anti-thyroglobulin antibodies did not co-purify with inhibitory antibodies, and were always in the unbound fractions. We found no correlation between the pattern of antibody binding or bioactivity with clinical manifestations of hypothyroidism. (1) The majority of patients with autoimmune hypothyroidism have antibodies against the TSHr-ECD that recognized linear epitopes. Most have antibodies directed at more than one site and the pattern is quite heterogeneous. (2) Six sites (noted above) are most frequently recognized. (3) Inhibitory antibodies are distinct from anti-microsomal and anti-thyroglobulin antibodies.

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