Thyroid peroxidase (TPO) is responsible for the thyroid hormone biosynthesis (1). TPO is also one of the autoantigens in disorders such as Hashimoto’s thyroiditis and Graves’ disease, which are the most common human autoimmune diseases (2, 3). Autoantibodies to TPO are polyclonal and recognise discontinuous immunodominant regions (IDRs) on the molecule (4, 5). The major part of the autoantibody response to TPO is directed towards two regions, which were defined with a panel of murine monoclonal antibodies (moabs), termed IDR-A and -B (4). These IDR regions of TPO have not been unequivocally identified so far. Several techniques have been used in elucidating the location of IDR on the TPO molecule. Chimeric molecules of TPO-myeloperoxidase (6, 7) allowed exclusion of two major areas of TPO from involvement in IDRs of TPO. Studies on large recombinant fragments of TPO suggested the involvement of amino acids 590-622 and 709-721 (8). Deletion of large segment of TPO suggested the importance of sequence 386-652 (9). Studies on proteolytic fragments of TPO suggested an involvement of C-terminal amino acids (742-848) in building up the IDRs (10, 11). This finding have been questioned by others (12) as the recombinant TPO with truncated C-terminal (1-741) was precipitated by four monoclonal antibodies as well as TPO 1-771 and the full TPO ectodomain. The participation of the EGF-like domain (796-841) has been excluded as being a part of the IDR (13), while some evidences has been presented for the involvement of the CCP-like domain (739-795). Footprinting experiments suggested the participation of Lys 713, but it location at the fringe of IDR is possible (14). Other studies with recombinant TPO fragments suggested the involvement of a junction region between the MPO- and CCP-like domain of TPO (13, 15). An earlier work using recombinant TPO fragments (16) suggested the involvement of amino acid sequence 513-633 in building up the IDR, the importance of the sequence 589-633 was underlined. We have described that anti-TPO peptide antibodies to part of the sequence (599-617) described by Arscott et al. (16) strongly inhibit autoantibodies and IDR-B specific moabs binding to TPO (17). Using the model of TPO and the known localization of one of the IDR-A specific moab 47, we obtained antibodies to peptides covering the whole surface between and around the moab 47 and the sequence 599-617 (18), and found that a mixture of rabbit antibodies to this region inhibit binding of IDR-A and -B specific recombinant fab fragments and autoantibodies to TPO up to 90%. Bresson and co-workers (19) in an elegant paper described that four regions are taking part in building up the IDR for one human monoclonal antibody to TPO (moab T13), by replacement of 8 to 10 amino acids sequence. These results are in apparent conflict with our results as they do not involve the sequences described by us (17, 18). Strong inhibition of autoantibodies, moabs, and recombinant fabs binding to TPO by rabbit peptide antibodies are clear cut, however, interpretation of these results might be more complicated and we have addressed these questions in the present work.

The molecular model of TPO, based upon the homologous structure of MPO, has been described previously (17). All the synthetic peptide sequences used in this study (tab. 1) correspond to sequences in the MPO- and CCP-like domain of TPO. The location and solvent accessibility of some of these peptides such as P6, P14, P15, P16, and P17 has been described (17). The other peptides were selected by inspection of the model to cover the TPO surface around and between the epitope for moab 47 (713-721) and our peptide P14 (599-617). All peptides were synthesized by F-moc chemistry with C-terminal amides and a cysteine residue at the N- or C-terminus for coupling to carrier protein as described earlier (17). All peptides were checked for purity by mass spectrometry.

Mouse moabs to TPO were obtained from Dr. J. Ruf (4). Serum from patients with thyroid autoimmune disease was obtained from the Warsaw Outpatient Endocrine Clinic. Pooled serum from normal healthy individuals (n = 20) was used as a control. Pooled sera from 20 patients with thyroid autoimmune disease, positive for TPO antibodies, were used as positive control for experiments with human sera. Autoantibodies to TPO were measured by ELISA, standardized to the WHO/MRC international standard 66/387 (17). Peptides were conjugated to maleimide activated keyhole limpet hemocyanin (KLH) (1 mg peptide/1 mg KLH) and further purified by chromatography on Sephadex G-100 in PBS (17). At least two New Zealand White rabbits per peptide were injected according to the described schedule (17). All antisera were tested for reactivity to human proteins (albumin, IgG, thyroglobulin), bovine albumin, and egg albumin. All antisera were also tested for reactivity with human myeloperoxidase and lactoperoxidase.

All experiments with animals were approved by the Warsaw Ethical Committee for Experiments on Animals no. 55/01.

Human TPO was prepared from pooled Graves’ thyroid tissue as described (20). TPO preparations used for ELISA were further purified by affinity chromatography on protein L-Sepharose. A column containing 2 ml of protein L-Sepharose (Actigen) was washed with 20ml of Tris-buffered saline (TBS) pH 8.0 containing 0.05% deoxycholate (DOC) followed by TPO solubilisation in the same buffer. The column was incubated for 1 h at room temperature, washed with TBS containing 0.05% DOC and the non-retained fraction collected and concentrated for use in the ELISA experiments. This step removes almost all IgG contamination from the TPO preparation.

All ELISA tests were performed as described previously (17). In short, microtitre plates (Nunc) were coated with purified human TPO (or other protein at 1 μg/ml), 100 μl of diluted rabbit anti-peptide serum added and incubated for 1 h at room temperature. After washing three times with PBST, HRP-conjugated goat anti-rabbit autoantibodies were added to the wells and incubated for 1 h at room temperature. The plates were developed with TMB solution and the optical density (OD) was measured at 450 nm.