Presence of heterogeneous thyroid-stimulating antibodies in sera from individual Graves' patients as shown by synthesized thyrotropin receptor peptide application: evidence showing two independent epitopes and a possible recognition of two 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 these 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), 287-304 (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 combinations did not. Furthermore, sequential addition of these peptide pairs confirmed the additional effects of P287-18 and P354-14. Sequential peptide-affinity gel studies were then performed. Most of the TSH-R-Sab activity in the unabsorbed fraction from P287-18 gel was absorbed to a subsequent P354-14 gel and the eluted fraction from P287-18 mostly remained unabsorbed by the P354-14 gel. On the other hand, most of the unabsorbed fraction from P91-29 gel remained unabsorbed even by the subsequent P354-14 gel. When a P354-14 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 resulted in significant inhibition of the binding but P287-18 did not affect either. From these results, it was concluded that most of the individual Graves' immunoglobulins contain at least two heterogeneous moieties 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.
Sugawa
H
H
Department of Laboratory Medicine, Kyoto University, School of Medicine, Japan.
Akamizu
T
T
Kosugi
S
S
Ueda
Y
Y
Ohta
C
C
Okuda
J
J
Mori
T
T
D000042:Absorption; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D002846:Chromatography, Affinity; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006111:Graves Disease / Q000276:immunology*; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating / Q000097:blood* / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D051381:Rats; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000276:immunology*; D013961:Thyroid Gland / Q000276:immunology
D000939:Epitopes; D018828:Immunoglobulins, Thyroid-Stimulating; D010446:Peptide Fragments; D011989:Receptors, Thyrotropin