Epitope heterogeneity of thyrotropin receptor-blocking antibodies in graves' patients as detected with wild-type versus chimeric thyrotropin receptors

The stable transfectants of wild-type (W25) and mutant thyrotropin-receptor (TSH-R) allow detection of the bioactivities of TSH-R antibodies in Graves ' patients. A mutant Chinese hamster ovary (CHO) cell line (Mcl+2) transfec ted with a chimeric construct, where residues 8 to 165 of the TSH-R are rep laced with residues 10 to 166 of the lutropin/choriogonadotropin (LH/CGR) r eceptor, lacks the cyclic adenosine monophosphate (cAMP) response to most t hyrotropin stimulating antibodies (TSAb), yet retains the response to TSH a nd acquires the response to LH/CG. We compared Mc1+2 cells with wild-type W 25 cells for their ability to detect TSAb as well as thyrotropin-blocking a ntibodies (TBAb) in Graves' sera. Eighteen normal and 39 Graves' sera were tested for TSAb and TBAb levels by in vitro bioassays using W25 and Mc1+2 c ells. In addition, these sera were also tested for thyrotropin-binding inhi bitory activity (TBII) by a radioreceptor assay. Eighteen (47%) Graves' ser a had TBAb activity measured with W25 cells but not with Mc1+2 cells. These TBAbs were, therefore, a population of antibodies with functional epitopes on the N-terminus of the extracellular domain. This TBAb activity by W25 c ells exhibited a high degree of correlation with TBII levels by a radiorece ptor assay (r = 0.70, p = 0.001). Ten (25.6%) Graves' sera had positive TBA b activity in both W25 and Mc1+2 cells; moreover, their activity in both as says was similar (r = 0.83, P < 0.001). The TBAb activity in these sera, ho wever, did not correlate with TBII activity. Eleven (28%) Graves' sera had no TBAb activity. Overall, thyroid-stimulating antibodies were detected in 87% and 28% of the 39 Graves' sera by W25 and Mc1+2 cells, respectively. Th us, using the 2 cell lines, at least 2 distinct populations of TBAbs were d etected. One is detected in a similar fashion by both W25 and Mc1+2 cell li nes and likely interacts with the epitopes residing in the unaltered C-term inus of the TSH-R. The other is reactive in W25 cells only, indicating the loss of TBAb epitope in the chimeric receptor located in the N-terminus of the TSH-R. Furthermore, our results indicate that the TBAb binding epitope in 8-165 residues of the native TSH-R is highly associated with TBII activi ty in Graves' disease. These results indicate that patients with Graves' di sease harbor TBAbs with epitope heterogeneity and favor the notion that the re are different sites and mechanisms by which TBAbs act in Graves' patient s. It remains to be determined whether or not TBAb subtyping will have a us eful predictive role in the management of patients with Graves' disease.