IDENTIFICATION OF EPITOPES AND AFFINITY PURIFICATION OF THYROID-STIMULATING AUTOANTIBODIES USING SYNTHETIC HUMAN TSH RECEPTOR PEPTIDES

We prepared a series of overlapping peptides (29 in total, 20 amino ac ids each) containing the sequence of the entire extracellular domain o f the human TSH receptor. Three peptides (181-200, 376-394, and EC3 (6 29-639)) bound IgG from patients with Graves' disease in an enzyme lin ked immunoassay. Peptide 181-200 bound IgG from 9 of 10, EC3 from 8 of 10, and 376-394 from 6 of 10 patients respectively, compared to 0 of 9 controls. We affinity purified TSHr auto-antibodies from four Graves ' patients using the three above noted peptides bound to epoxy-activat ed sepharose. Thyroid stimulating activity was enriched in the bound f raction from at least two of the three peptide affinity columns in eac h of the four patients, although the pattern of affinity enrichment di ffered between patients. One patient was found to possess a combinatio n of stimulatory and inhibitory TSHr antibodies and, after affinity pu rification, the anti-374-394 and anti-EC3 fractions were enriched in s timulatory activity, suggesting that those regions of the receptor wer e epitopes for stimulatory antibodies. However, affinity purification against peptide 181-200 produced an IgG preparation that was not stimu latory, but was a potent thyroid inhibitor. Thus, we have not only par tially purified TSHr auto-antibodies, but also successfully separated stimulatory and inhibitory antibodies from a single patient using comb ination TSHr peptide affinity.