Binding characteristics of antibodies to the TSH receptor

Oda, Yasuo; Sanders, Jane; Roberts, Seren; Maruyama, Mina; Kato, Ryoji; Pérez, Mario Noa; Petersen, V B; Wedlock, D. Neil; Furmaniak, Jadwiga; Smith, Bernard Rees

doi:10.1677/jme.0.0200233

Cited by 42 publications

(28 citation statements)

References 45 publications

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“…Intact IgG was undetectable by SDS-PAGE in the Fab preparations. mAb IgG and Fab preparations were labelled with 125 I as described previously (22). labeled TSH binding to TSHR-coated tubes (kit from RSR Ltd, Cardiff, United Kingdom) (23) (inhibition range 11%-98%).…”

Section: Production and Labeling Of Mabsmentioning

confidence: 99%

“…In addition, assays using Chinese hamster ovary (CHO) cells expressing hTSHR (approximately 50,000 receptors per cell) (22) were carried out according to the method of Latif et al (27). Briefly, CHO cells were seeded into 96-well plates (30,000 cells per well) and incubated for 24 hours in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Ltd., Paisley PA4 9RF, UK) containing 10% fetal calf serum.…”

Section: Analysis Of Mab Thyroid Stimulating Activitiesmentioning

confidence: 99%

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Thyroid-Stimulating Monoclonal Antibodies

Sanders¹,

Jeffreys²,

Depraetere³

et al. 2002

Thyroid

107

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Thyrotropin (TSH) receptor monoclonal antibodies (TSHR mAbs) were obtained from cDNA-immunized NMRI mice. Three mAb immunoglobulin Gs (IgGs) (TSmAbs 1-3) that had distinct V(H )and V(L) region sequences stimulated cyclic adenosine monophosphate (cAMP) production in isolated porcine thyroid cells greater than 10x basal and as little as 20 ng/mL (0.13 nmol/L) of TSmAb 1 IgG caused a 2x basal stimulation. TSmAb 1 and 2 Fab fragments were also effective stimulators and thyroid-stimulating activities of the IgGs and Fabs were confirmed using TSHR transfected Chinese hamster ovary (CHO) cells. The TSmAbs also inhibited (125)I-labeled TSH binding to TSHR-coated tubes by 50% or more at concentrations of 1 microg/mL or less and gave 15%-20% inhibition at 20-50 ng/mL. (125)I-labeled TSmAbs bound to TSHR-coated tubes with high affinity (approximately 10(10) L/mol) and this binding was inhibited by TSHR autoantibodies with both TSH agonist and antagonist activities. Inhibition of labeled TSmAb binding by Graves' sera correlated well with inhibition of TSH binding (r = 0.96; n = 18; p < 0.001 for TSmAb 2). The TSmAbs have considerable potential as (1) new probes for TSHR structure-function studies, (2) reagents for new assays for TSHR autoantibodies, and (3) alternatives to recombinant TSH in various in vivo applications.

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Section: Production and Labeling Of Mabsmentioning

confidence: 99%

Section: Analysis Of Mab Thyroid Stimulating Activitiesmentioning

confidence: 99%

Thyroid-Stimulating Monoclonal Antibodies

Sanders¹,

Jeffreys²,

Depraetere³

et al. 2002

Thyroid

107

View full text Add to dashboard Cite

show abstract

“…Concentrations of hamster mAb used were saturating unless otherwise indicated. Monoclonal TSHR-Abs M1 and M4, used in our cleavage assay, were mouse IgG 1 (Oda et al 1998(Oda et al , 2000 kindly supplied by Dr B Rees Smith (RSR Limited, Cardiff, UK). 2C11 was a mouse IgG 1 TSHR-mAb (Shepherd et al 1999) purchased from Serotec (Raleigh, NC, USA).…”

Section: Cells and Tshr-mabsmentioning

confidence: 99%

Antibody-induced modulation of TSH receptor post-translational processing

Ando¹,

Latif²,

Davies³

2007

Journal of Endocrinology

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The post-translational processing of the TSH receptor (TSHR) includes intra-molecular cleavage with the loss of a 50 amino acid ectodomain region and the formation of two subunits (a and b), followed by likely a subunit shedding. TSHR antibodies (TSHR-Abs), which are directed at the ectodomain, may influence thyroid function by stimulating or inhibiting TSHR signaling or may bind without any such influence (the neutral group of antibodies). When we examined the characteristics of a series of monoclonal TSHR-Abs, we found that many were able to inhibit receptor cleavage and enhance TSHR expression. This was especially apparent with the neutral type of TSHR-Abs directed to the cleaved region of the ectodomain (aa 316-366). Indeed, such inhibition appeared to be epitope dependent with TSHR-Abs directed to regions after residues 335-354 showing no such activity. We propose that this aberrant process, whereby TSHR-Abs influence antigen processing, is a novel mechanism for the maintenance and exacerbation of autoimmune thyroid disease.

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“…Recombinant human TSHR expressed in CHO-K1 cells (4 3 10 5 TSHR per cell as assessed by Scatchard analysis of TSH binding) were isolated, solubilized with 50 mM NaCl, 10 mM Tris-HCl pH 7.5, 1% Triton-X-100, and centrifuged (90000g for 2 hours at 4°C) as previously described (9). TSHR mouse monoclonal antibody (3F3 IgG; [9]) was coupled to CNBr-activated sepharose 4B (Amersham Pharmacia Biotech, St. Albans, UK) and the gel packed into a column.…”

Section: Tshr Preparationsmentioning

confidence: 99%

“…Recombinant TSHR produced in Chinese hamster ovary (CHO) cells and extracted with detergents is usually present in two main forms: a 100-kd precursor form consisting of the full-length 84-kd peptide chain and high mannose sugar residues and a 120-kd mature form consisting of the fulllength peptide chain and complex-type sugars (7)(8)(9)(10)(11). Recent studies have shown that only the 120-kd form of the TSHR is principally responsible for binding TSH and we now describe a study of the interaction of TRAb with the two forms of the receptor.…”

Section: Introductionmentioning

confidence: 99%

Reactivity of Thyrotropin Receptor Autoantibodies with the Thyrotropin Receptor on Western Blots

Maruyama

Sanders²,

Oda

et al. 2001

Thyroid

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Affinity purified recombinant human thyrotropin receptor (TSHR) was run on sodium dodecyl sulfate (SDS) gels and subjected to a renaturing and blotting procedure. Twenty sera from thyrotropin receptor autoantibodies (TRAb)-positive patients with a history of hyperthyroidism and 20 sera with high levels of TSH blocking activity were analyzed. Four of 20 sera with blocking-type of TRAb (i.e., TSH antagonist activity) were able to recognize the mature, fully glycosylated 120-kd form of the receptor on blots of gels run under reducing conditions. No sera recognized the 100-kd high mannose precursor form of the TSHR. Three of the four recognized a 74-kd band and 2 of the 4 recognized a 50-kd band. These bands are probably proteolytic cleavage fragments of the mature 120-kd TSHR. In the absence of reducing agent the same 4 of 20 sera described above together with a further serum sample (i.e., 5/20 in total) reacted with the 120-kd form of the receptor. No specific reaction with the TSHR was observed on Western blots with the remaining 15 sera with TSH blocking activity, nor with 20 sera from patients with a history of hyperthyroidism, nor with sera from 10 healthy blood donors, 10 Hashimoto sera (negative for TRAb) and 10 systemic lupus erythematosus sera. No clear differences were observed in the TRAb positive sera that were reactive and nonreactive on Western blots in terms of their ability to inhibit TSH binding or to immunoprecipitate 125I-labeled TSHR. Overall, our results indicate that the mature 120-kd form of the TSHR that is principally responsible for binding TSH is also responsible for binding TRAb (when this binding can be detected). These observations together with immunoprecipitation and TSH binding inhibition studies, emphasize the close relationship between the receptor's binding sites for TSH and TRAb.

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Binding characteristics of antibodies to the TSH receptor

Cited by 42 publications

References 45 publications

Thyroid-Stimulating Monoclonal Antibodies

Thyroid-Stimulating Monoclonal Antibodies

Antibody-induced modulation of TSH receptor post-translational processing

Reactivity of Thyrotropin Receptor Autoantibodies with the Thyrotropin Receptor on Western Blots

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