PRESENCE OF HETEROGENEOUS THYROID-STIMULATING ANTIBODIES IN SERA FROMINDIVIDUAL GRAVES PATIENTS AS SHOWN BY SYNTHESIZED THYROTROPIN RECEPTOR PEPTIDE APPLICATION - EVIDENCE SHOWING 2 INDEPENDENT EPITOPES AND APOSSIBLE RECOGNITION OF 2 EPITOPIC REGIONS BY ONE ANTIBODY MOLECULE

To define the epitope(s) of stimulating thyrotropin receptor antibody (TSH-R-Sab), we synthesized 19 oligopeptides covering almost all amino acids of the extracellular domain of the human TSH-R and studied thes e effects on the inhibition of one TSH-R-Sab activity. Four of the 19 peptides encompassing residues 31-50 (P31-20), 91-119 (P91-29), 287-30 4 (P287-18) and 354-367 (P354-14) were found to show significant TSH-R -Sab inhibition and to have similar effects on the other three Graves' immunoglobulins. When these peptides were applied in combination with P354-14 only P287-18 revealed additional effects but the other two co mbinations did not. Furthermore, sequential addition of these peptide pairs confirmed the additional effects of P287-18 and P354-14. Sequent ial peptide-affinity gel studies were then performed. Most of the TSH- R-Sab activity in the unabsorbed fraction from P287-18 gel was absorbe d to a subsequent P354-14 gel and the eluted fraction from P287-18 mos tly remained unabsorbed by the P354-14 gel. On the other hand, most of the unabsorbed fraction from P91-29 gel remained unabsorbed even by t he subsequent P354-14 gel. When a P3 54-14 affinity gel-purified TSH-R -Sab immunoglobulin was labeled and evaluated for its binding to FRTL- 5 cells, additions of original immunoglobulin, P354-14 and P91-29 resu lted in significant inhibition of the binding but P287-18 did not affe ct either. From these results, it was concluded that most of the indiv idual Graves' immunoglobulins contain at least two heterogeneous moiet ies with TSH-R-Sab activity, one of which binds P354-14 and the other binds P287-18. Further, P354-14 and P91-29 were indicated to bind the same molecule of TSH-R-Sab immunoglobulin.