Application of mouse monoclonal antibodies for identification of antigen regions of human thyroid peroxidase in adolescents with Graves' disease and non-toxic multinodular goiter by flow cytometry

Thyroid peroxidase (TPO) is the major thyroid autoantigen recognized by serum autoantibodies from patients with Graves' disease (GD) or Hashimoto's thyroiditis directed to two immunodominant conformational regions termed A and B. The epitopes of human TPO have been defined using a panel of mouse monoclonal antibodies (mAbs). The aim of this study was to estimate the expression of chosen surface antigen regions of TPO (1, 18, 30, 64 epitopes) on thyroid cells in 15 patients with non-toxic multinodular goiter (NTMG) and 15 patients with GD. The thyrocytes were identified by indirect method: in the first stage we added mouse monoclonal autoantibodies specific for TPO regions and in the second stage we conjugated this complex with rabbit anti-mouse antibodies IgG (Fab')2 with FITC. All investigations were performed by flow cytometry using Coulter EPICS XL apparatus. The percentages of thyrocytes with expression of epitopes 1, 18, 30, 64 TPO were measured in relation to the respective anti-TPO concentrations: 50-1600 μg/ml. The analysis of epitopes located in immunodominant regions (IDR) of TPO revealed higher percentages of thyrocytes in cases with GD in comparison to NTNG. The most predominant difference was observed for mAb 64 epitope (48 vs 7%, p < 0.019; 39 vs 5%, p < 0.017) at the concentration of 100-200 μg/ml mAbs. The expression of 18 epitope on thyrocytes was also statistically higher in Graves' patients than in the NTMG (14 vs 6%, p < 0.025) at concentration of 400 μg/ml mAbs. However, this expression was much less pronounced. In all the cases, the percentages of thyrocytes with epitopes 1 and 30 were in low detection (8-15% of positive cells). In conclusion, our findings suggest that the elevated expression of TPO epitopes 18 and 64 in young patients with thyroid autoimmune diseases increase stimulation and activation of thyroid cells during inflammatory reaction within the thyroid gland. In addition, predominant expression of 64 TPO epitope that recognizes B domain in GD patients could be a useful marker of the immune process in the thyroid gland.