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

Abstract

Abstract. 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 125 I-bTSH. We called TRAb measured by this method as hTRAb. 125 I-bTSH binding capacity of the tube was found close to saturation at 1 hr with 200 ml of 125 I-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 125 I-bTSH. When 1 step incubation with 125 I-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.

Evaluation of a 1 step TRAb Assay for the Detection of High-affinity Components to hTSHR: Evidences Indicating Superiority of the Assay in the Lower TRAb Range

“…Two step incubations were performed as described previously [6,18]. The 1 st incubation was performed by adding 100 ml of serum sample or standard substance to 200 ml of assay buffer containing anti-hTSH antibody to an rhTSHR-coated tube.…”

“…As described previously [18], TRAb activities were also expressed as % inhibition of 125 I-bTSH binding, which was calculated as; {1-(bound count by sample/ maximal bound count)} × 100 (%). Normal ranges were defined as less than 1.5 IU/L for quantitative expression and 15% for % inhibition, respectively.…”

“…As described previously [18], 50 ml of serum sample, 200 ml of assay buffer and 200 ml of 125 I-bTSH solution were mixed together and then poured into an rhTSHR-coated tube. After 1 hour incubation, the tube was washed and directly counted for bound radioactivity.…”

“…Through extensive analyses of the 2 nd generation TBII assay system, we reported recently that 1) TSH receptor (TSHR) actions of Graves' TRAb are very different from those of bTSH, 2) the 2 step incubation TRAb assay is not of competitive binding assay and 125 I-bTSH in the 2 nd incubation can not displace any of the previously bound TRAb to the hTSHR, 3) Graves' IgG effects on the TSHR increase gradually depending on the TSHR reaction time, and 4) individual Graves' IgGs should contain heterogeneous TRAb components with variable avidities to the TSHR [18]. We propose that highly sensitive TRAb assays may detect lowaffinity TRAb, which may lack biological thyroid stimulatory activity.…”

“…We propose that highly sensitive TRAb assays may detect lowaffinity TRAb, which may lack biological thyroid stimulatory activity. This may be one reason responsible for the clinically unmatched TRAb-positive results in some euthyroid or inactive patients with Graves' disease, such as those under remission or with euthyroid Graves' ophthalmopathy [18].…”