Investigation of antigenic determinants in thyroid peroxidase by synthesis of possible sequential peptides

One way to locate antigenic determinants in thyroid peroxidase (TPO) is to evaluate the binding of chemically synthesized peptides to autoantibodies. A major epitope of TPO involved in thyroid autoimmunity has been reported to be at residues 590 to 675. Therefore, we synthesized three peptides (P1, residues 603-610; P2, 615-630; and P3, 654-665) in the most antigenic part of this range, as judged by antigenicity analysis, and measured their antigenicity by enzyme-linked immunosorbent assay (ELISA). Amino groups protected with fluorenyl-methoxycarbonyl were used in the synthesis. Sera from 42 untreated patients with Hashimoto's disease and 30 untreated patients with Graves' disease were tested for binding with each peptide. In addition, 6 sera with antibodies against denatured and reduced microsomal antigen were tested. Sera obtained from 22 normal subjects were used as a control. Binding of sera from the patients with autoimmune thyroid disease to the synthetic peptides was weak, about 1% of binding to thyroid microsomes prepared from Graves' thyroid. However, the ELISA index of a mixture of P1, P2, and P3 was significantly higher in the patients with Hashimoto's disease than in the healthy subjects. For both P2 and P3, the ELISA index of the sera with antibodies against denatured and reduced TPO was higher than that of the sera from normal subjects. These results suggested 1) that the antigenic epitopes of TPO recognized by autoantibodies may be conformational and discontinuous, and 2) that antibodies against these linear portions of TPO may exist in patients with Hashimoto's disease.