Affinity purification and diagnostic use of TSH receptor autoantibodies from human serum

Morgenthaler, Nils G.; Minich, Waldemar B.; Willnich, Marita; Bogusch, Thomas; Hollidt, Jörg M.; Weglöhner, Wolfgang; Lenzner, Cornelia; Bergmann, Andreas

doi:10.1016/j.mce.2003.09.018

Cited by 34 publications

(23 citation statements)

References 19 publications

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“…Their epitopes on the TSHR are likely to be close and intimately associated with each other, because the smaller Fabs of the KSAb1 and KSAb2 also compete for binding to the receptor. Additionally, our finding that these epitopes also overlap with the determinants associated with strong antagonism for TSH binding to the TSHR is in agreement with previous studies on the close relationship of the receptor epitopes associated with receptor stimulation and TSH antagonism (23,38).…”

Section: Discussionsupporting

confidence: 93%

Monoclonal Pathogenic Antibodies to the Thyroid-Stimulating Hormone Receptor in Graves’ Disease with Potent Thyroid-Stimulating Activity but Differential Blocking Activity Activate Multiple Signaling Pathways

Gilbert

Gianoukakis

Salehi

et al. 2006

The Journal of Immunology

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The thyroid target Ag for disease-inducing autoantibodies in Graves’ disease is the receptor for thyroid-stimulating hormone (TSH), but little is known about the molecular basis of this pathogenic Ab response. We describe the characteristics of two high- affinity mAbs developed from an experimental murine model of hyperthyroid Graves’ disease that exhibit potent thyroid-stimulating activity. Nanogram concentrations of the IgG mAbs KSAb1 and KSAb2 and their Fab induce full stimulation of the TSH receptor that is matched by the ligand TSH and, thus, act as full agonists for the receptor. However, KSAb1 and KSAb2 display differential activities in their ability to block TSH-mediated stimulation of the receptor, indicating subtle differences in their biological properties. In displacement studies, IgG and Fabs of KSAb1 and KSAb2 compete with Graves’ disease autoantibodies as well as thyroid-blocking Abs present in some hypothyroid patients, indicating a close relationship between these autoimmune determinants on the receptor. In passive transfer studies, single injections of microgram quantities of KSAb1 or KSAb2 IgG led to rapid elevation of serum thyroxine and a hyperthyroid state that was maintained for a number of days. The thyroid glands showed evidence of cell necrosis, but there was no accompanying mononuclear cell infiltrate. In studying their receptor activation pathways, both KSAb1 and KSAb2 provoked phosphorylation of the intracellular ERK1/2 pathway in primary thyrocytes, indicating that multiple signaling pathways may participate in the pathogenesis of Graves’ disease. In summary, our findings emphasize the similarities of the experimental mouse model in reproducing the human disorder and provide improved means for characterizing the molecular basis of this pathogenic response.

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Section: Discussionsupporting

confidence: 93%

Monoclonal Pathogenic Antibodies to the Thyroid-Stimulating Hormone Receptor in Graves’ Disease with Potent Thyroid-Stimulating Activity but Differential Blocking Activity Activate Multiple Signaling Pathways

Gilbert

Gianoukakis

Salehi

et al. 2006

The Journal of Immunology

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“…Thus, the previous concept that the epitopes for blocking antibodies are largely confined to the C-terminus of the ectodomain is oversimplified. Our findings are more in keeping with recent TRAB epitope studies, which indicate that binding sites for stimulating and blocking autoantibodies lie close together, not on distinct or distant parts of the TSHR molecule (Morgenthaler et al 2003, Ando et al 2004, Costagliola et al 2004, Sanders et al 2004, 2005, Schott et al 2005.…”

Section: Discussionsupporting

confidence: 91%

Effects of genetic immunization of Swiss outbred mice with human thyroid stimulating hormone receptor cDNA plasmids harboring gain-of-function mutations

Goh²,

Kee³

et al. 2007

Journal of Molecular Endocrinology

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Animal models of Graves' disease have been generated in recent years with various vaccination protocols using wild-type TSH receptor. In this study, we report the findings of genetic immunization of Swiss outbred mice with three different mutated human TSH receptor plasmids, each containing one constitutive activating mutation located at the ectodomain (S281N), exoloop (I486F), and transmembrane segment (D633H) respectively. Although the overall rate of thyrotoxicosis in the mice was !10%, anti-TSH receptor antibodies could be detected in many animals by flow cytometry, radioreceptor assay, and functional bioassays using recombinant human TSH receptor. Mice injected with plasmids harboring activated mutants (S281N and D633H) showed production of predominantly stimulating antibodies, whilst those treated with wild-type receptor plasmids generated mainly blocking sera. Most of these antibodies displaced radiolabeled bovine TSH, and their epitopes, independent of functional characteristics, were mapped to the first 271 amino acids of the TSH receptor. This supports recent findings that binding of stimulatory or blocking antibodies lie in close proximity within the leucine-rich repeat region.

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“…None of the mAb or even a mixture of them could improve the assay results obtained by 125 I-bTSH application, which suggests the heterogeneity and complexity of epitope recognition among TRAb. On the other hand, Morgenthaler et al have succeeded in the affinity purification of human TRAb to 3420-fold [30]. The purified TRAb was useful for the TRAb assay, and virtually identical results were obtained whether the labeled TRAb were TSAb or thyroidstimulation blocking antibody (TSBAb).…”

Section: Discussionmentioning

confidence: 99%

“…However, TRAb in the unabsorbed fractions showed an excellent linear correlation with the original TRAb activities. In general, heterogeneity among TRAb appeared to be rather limited as suggested by Morgenthaler et al [30]. The quality of TSHR-absorbed TRAb was then studied measuring the unabsorbed fractions by the 1 step assay subsequently.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2006

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Abstract. The aim of this study was to develop an assay to selectively detect high-affinity components among TRAb. Using an rhTSHR-coated tube system, a 1 step TRAb assay method was developed that included 1) co-incubation with 125 I-bTSH, 2) a 50 ml serum sample, 3) an increased incubation volume (450 ml), and 4) a 1 hour incubation time. Sixtyone TRAb positive Graves' sera were studied. When the regular TRAb assay (Reg) results were quantitatively compared to the 1 step assay (1 step) results, certain dispersions and overestimations using the latter were seen. Further, some 1 step positive results were observed in the low Reg range. Overestimations were considered mostly due to the differences between TRAb standard and patients' serum TRAb in the binding competition against co-incubated 125 I-bTSH, which was shown from a modified assay mimicking the 1 step conditions. Therefore, the 1 step results were decided to be expressed by % inhibition against 125 I-bTSH. As for data dispersions, TRAb absorptions during the regular 1 st incubation were studied. Individually, the absorption rates varied from 11 to 69%, and higher absorptions were observed in lower Reg range, especially in those negative by the 1 step. Observed 1 step positive results in the low Reg range were of interest, and 1 step/Reg ratios were calculated. The ratios with 1 step negative samples were significantly lower than those of 1 step positive samples. In conclusion, the 1 step assay was proved to detect a particular and biologically active TRAb, especially in those with low TRAb. The clinical significance of the 1 step results should be of future interest.

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Affinity purification and diagnostic use of TSH receptor autoantibodies from human serum

Cited by 34 publications

References 19 publications

Monoclonal Pathogenic Antibodies to the Thyroid-Stimulating Hormone Receptor in Graves’ Disease with Potent Thyroid-Stimulating Activity but Differential Blocking Activity Activate Multiple Signaling Pathways

Monoclonal Pathogenic Antibodies to the Thyroid-Stimulating Hormone Receptor in Graves’ Disease with Potent Thyroid-Stimulating Activity but Differential Blocking Activity Activate Multiple Signaling Pathways

Effects of genetic immunization of Swiss outbred mice with human thyroid stimulating hormone receptor cDNA plasmids harboring gain-of-function mutations

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

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