Differences in TSH Receptor Binding and Thyroid-stimulating Properties between TSH and Graves' IgG. Slowly-acting TSH Receptor Antibody Moieties in Graves' Sera Affect Assay Data

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We analyzed TSH receptor (TSHR) effects, both binding and thyroid-stimulation, of TSH and Graves' IgG. A new TRAb assay system utilizes rhTSHR coated tubes and is comprised of two step incubation, the first incubation with patient serum followed by a second incubation with ¹²⁵I-bTSH. We called TRAb measured by this method as hTRAb. ¹²⁵I-bTSH binding capacity of the tube was found close to saturation at 1 hr with 200 μl of ¹²⁵I-bTSH. Up to 5 hr of first incubation for hTRAb assay revealed significant increases in all hTRAb activities. hTRAb was not affected by second incubation time or dose of ¹²⁵I-bTSH. When 1 step incubation with ¹²⁵I-bTSH and Graves' serum was performed, hTRAb again increased significantly with time. A simple competitive equilibrium model could not be applied to these ligands. Second, Graves' IgG and bTSH were compared for in vitro thyroid-stimulation sequentially up to 24 hr, measuring cAMP generation from cultured porcine thyrocytes. While bTSH yielded peak cAMP generation by 8 hr, TSAb revealed more cAMP generation by 24 hr than at 8 hr. We concluded that individual Graves' sera contain heterogeneous TRAb of variable avidities, and that slow-acting TRAb, which may lack biological activity, can be detected by prolonged incubation.