Patients with autoimmune thyroid disease frequently have high affinity antibodies to thyroid peroxidase (TPO), although the role they play in disease pathogenesis is not known. We have previously prepared 37 monoclonal anti-TPO IgG kappa Fab fragments from two patients with Hashimoto's thyroiditis and demonstrated the similarity of these Fab sequences to those published previously, mainly derived from patients with Graves' disease. In this paper, we described epitope mapping of these Fabs using a previously characterized panel of murine monoclonal antibody (mAb) and show that the Fabs bind to two neighboring epitopes on native TPO. Although the epitope-mapping method differs from that used to characterize previously published TPO-reactive Fab sequences, it indicates a similarly restricted response to neighboring epitopes in both Graves' disease and Hashimoto's thyroiditis. The epitope mapping included mAb 47, which binds to a linear TPO peptide of known sequence in addition to native TPO. Although TPO-reactive Fab did not inhibit the binding of mAb 47, mAb 47 did inhibit the binding of Fab, indicating the likely site of the immunodominant region on native TPO. These results confirm the restricted nature of TPO antibody and further delineate the immunodominant region of native TPO as defined by the mAb.
Summary There is disagreement concerning the expression of thyroid peroxidase (TPO) in thyroid cancer, some studies finding qualitative as well as quantitative differences compared to normal tissue. To investigate TPO protein expression and its antigenic properties, TPO was captured from a solubilizate of thyroid microsomes by a panel of murine anti-TPO monoclonal antibodies and detected with a panel of antihuman TPO IgGκ Fab. TPO protein expression in 30 samples of malignant thyroid tissue was compared with TPO from adjacent normal tissues. Virtual absence of TPO expression was observed in 8 cases. In the remaining 22 malignant thyroid tumours the TPO protein level varied considerably from normal to nearly absent when compared to normal thyroid tissue or tissues from patients with Graves' disease (range less than 0.5 to more than 12.5 µg mg -1 of protein). When expressed TPO displayed similar epitopes, to that of TPO from Graves' disease tissue. The results obtained by the TPO capturing method were confirmed by SDS-PAGE and Western blot analysis with both microsomes and their solubilizates. The present results show that in about two-thirds of differentiated thyroid carcinomas, TPO protein is expressed, albeit to a more variable extent than normal; when present, TPO in malignant tissues is immunologically normal.
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