Estimation of Serum TSH Receptor Autoantibody Concentration and Affinity

Abstract

We have used the human monoclonal TSH receptor (TSHR) autoantibody (M22) as a labeled ligand in competition with individual patient TSHR autoantibodies (TRAb) to estimate their serum concentrations and affinities. TSHR coated tubes, (125)I-labeled M22 IgG and Fab, and patient sera IgG and Fab were used in these studies. In 15 patients with Graves' disease, TRAb concentrations ranged from 50 to 500 ng/mL of serum (5- 60 parts per million of total serum IgG) and TRAb IgG affinities from 3.0 +/- 1.0-6.7 +/- 1.54-10(10) L/mol (mean +/- SD; n=3). Fab fragment affinities were similar to those of intact IgG. Serum TRAb with blocking (TSH antagonist; 4 patients) activity had similar affinities (3.0 +/- 0.25-7.2 +/- 2.2-10(10) L/mol) to TRAb IgG from patients with Graves' disease, but blocking TRAb concentrations were higher (1.7 - 27 mg/mL of serum). The concentrations of TRAb that we observed in the sera of the 15 Graves' patient (0.33 - 3.3 nmol/L) can be compared with that of circulating TSH. In particular, a serum TSH concentration of 100mU/L (0.7 nmol/L) is in the same range as the concentrations of TRAb we observed. Such a TSH concentration (similar to that observed after injection of 0.9 mg of recombinant human TSH) would be expected to cause a similar degree of thyrotoxicosis as seen in Graves' disease. Consequently, the thyroid-stimulating potencies (i.e., activity per mol) of patient serum TRAb and human TSH appear to be of a similar magnitude in vivo as well as in vitro. Overall, our results indicate that serum TRAb affinities are high and show only limited variations between different sera whereas concentrations of the autoantibodies vary widely.

Crystal Structure of the TSH Receptor in Complex with a Thyroid-Stimulating Autoantibody

“…It also appears that M22 binds the TSHR in a ''lock and key'' fashion with essentially no movement in the atoms of M22 residues when TSHR binding occurs. Consequently, loss of free energy through induction of a conformational change in M22 does not occur during binding and this is consistent with the observed high affinity of the antibody-receptor interaction (6,7,12). In contrast to M22 binding considerable conformational changes occur in FSH on receptor binding (3).…”

“…In our study we used an insect cell expression system to produce part of the extracellular region of the TSHR (amino acids 1-260) which has the ability to bind M22 with high affinity and in particular with a very slow dissociation rate (12). This feature was helpful in producing TSHR260-M22 Fab complexes that remained intact during several rounds of purification.…”

“…M22 Fab was analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10) under reducing conditions to assess purity and biological activity tested in adenasine 3 0 ,5 0 -cyclic monophosphate (cyclic AMP) stimulation assays using Chinese hamster ovary (CHO) cells that express the TSHR (7). In addition, the ability of M22 Fab to inhibit 125 I-TSH or 125 I-M22 binding to TSHR-coated tubes was assessed (11,12).…”

Thyrotropin-Blocking Autoantibodies and Thyroid-Stimulating Autoantibodies: Potential Mechanisms Involved in the Pendulum Swinging from Hypothyroidism to Hyperthyroidism or Vice Versa

“…Moreover, in previous studies on whole serum, TBAb concentrations associated with hypothyroidism are far higher than TSAb concentrations that can cause hyperthyroidism (39,40). Therefore, if the concentration and affinity of TBAb K1-70 is higher than TSAb K1-18, the latter should dilute out more rapidly, the reverse of what is observed.…”

TSH Receptor Antibodies

“…The reason for this is not clear although studies with 125 I-labeled ligands indicate that M22 Fab binds to the TSHR more rapidly than intact IgG (53). Further, dissociation of receptor-bound M22 Fab or IgG occurs slowly, particularly when compared with TSH (53).…”