Human Anti-TSHR Recombinant Antibody (clone K1-70)
CAT#: PABL-737 Autoantibody
Recombinant Human Antibody (K1-70) is capable of binding to TSH receptor (TSHR), expressed in HEK 293 cells. Expressed as the combination of a heavy chain (HC) containing VH from anti-TSH receptor (TSHR) mAb and CH1-3 region of human IgG1 and a light chain (LC) encoding VL from anti-TSH receptor (TSHR) proteins mAb and CL of human kappa light chain. Exists as a disulfide linked dimer of the HC and LC hetero-dimer under non-reducing condition. The blocking autoantibody K1-70 binds more N-terminally on the TSHR concave surface than either the stimulating autoantibody M22 or the hormone TSH, and this may reflect its different functional activity.
Published Data
Tested Data
Gene Expression
Figure 1 Crystal structure of TSHR260 in complex with thyroid stimulating monoclonal autoantibody (M22) Fab (TSHR-M22) and with the TSHR blocking monoclonal autoantibody (K1-70) Fab (TSHR-K1-70).
Cartoon diagrams of the complex structures are shown. TSHR is in cyan, light chains are in green and the heavy chains in blue. Disulphide-bonded cysteines are shown as balls and sticks, disulphide bonds are in yellow and cysteine residues are labelled in orange. The N- and C-termini are marked. M22 interactions with TSHR amino acid R255 are marked and shown as balls and sticks. In contrast there are no interactions between K1-70 and amino acid R255 or the C-terminus of the TSHR. Reproduced from Figure 5 of Furmaniak et al. [3] with kind permission from Springer Science and Business Media.
Furmaniak, J., Sanders, J., Miguel, R. N., & Smith, B. R. (2015). Mechanisms of action of TSHR autoantibodies. Hormone and Metabolic Research, 47(10), 735-752.
Figure 2 Bridge type TRAb ELISA using TSHR 260 alkaline phosphatase.
A. Binding of stimulating (K1-18 and M22) and blocking (K1-70) human monoclonal autoantibodies and B binding of K1 donor serum IgG (lower x-axis); NIBSC 90/672 (upper x-axis). K1-18 IgG (♦); K1-70 IgG (▪); M22 IgG (▴); control MAb (5B3 IgG) (×); K1 donor serum (□); NIBSC 90/672 (○).
Smith, B. R., Sanders, J., Evans, M., Tagami, T., & Furmaniak, J. (2009). TSH receptor–autoantibody interactions. Hormone and Metabolic Research, 41(06), 448-455.
Figure 3 Inhibition of porcine TSH (3 ng/mL)-mediated stimulation of cyclic AMP production by TSHR monoclonal antibodies with antagonist activity.
MAb-B2 IgG (m); K1–70 IgG (d); 5C9 IgG (■).
Sanders, J., Miguel, R. N., Furmaniak, J., & Smith, B. R. (2010). TSH receptor monoclonal antibodies with agonist, antagonist, and inverse agonist activities. In Methods in enzymology (Vol. 485, pp. 393-420). Academic Press.
Figure 3 Inhibition of porcine TSH (4 ng/mL)-mediated stimulation of cyclic AMP production by TSHR monoclonal antibodies with antagonist activity.
(A)Human MAb 5C9 (antagonist and inverse agonist; derived from the comparative model of 5C9 structure), (B) mouse MAb-B2 (antagonist; derived from the crystal structure solved at 3.3 A˚ resolution), (C) human MAb K1–70 (antagonist; derived from the crystal structure solved at 2.2 A˚ resolution), (D) human MAb M22 (agonist; derived from the crystal structure solved at 1.65 A˚ resolution). Position of the charged residues is indicated in the figures on the left-hand side of the panel and the position of aromatic
residues is indicated in the figures on the right-hand side of the panel. Acidic patches are shown in red and basic patches in blue (See Color Insert).
Sanders, J., Miguel, R. N., Furmaniak, J., & Smith, B. R. (2010). TSH receptor monoclonal antibodies with agonist, antagonist, and inverse agonist activities. In Methods in enzymology (Vol. 485, pp. 393-420). Academic Press.
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SDS-PAGE
Figure 1 Human Anti-TSHR Recombinant Antibody (PABL-737) in SDS-PAGE
SDS-PAGE analysis of PABL-737 in non-reduced (Lane 1) and β-mercaptoethanol-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively. And non-reduced protein migrates as about 180-245 kDa.
SEC-HPLC
Figure 2 Human Anti-TSHR Recombinant Antibody (PABL-737) in SEC-HPLC
The purity of PABL-737 was greater than 95% as determined by SEC-HPLC.
Column: 3 µm, 7.8 x 300 nm
Mobile phase: 150 mM Sodium Phosphate Buffer, pH 7.0
Detection: UV 280 nm
Injection: 10 µl
ELISA
Figure 3 Human Anti-TSHR Recombinant Antibody (PABL-737) in ELISA
ELISA analysis of PABL-737 was performed by coating with human TIM-1 / HAVCR1 protein, His tag. Then blocked with BSA and incubated with PABL-737. The HRP-conjugated goat anti-Mouse IgG as a secondary antibody (1: 2000). Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.
DB
Figure 4 Human Anti-TSHR Recombinant Antibody (PABL-737) in DB
Dot Blot analysis of PABL-737 was performed by coating with human TIM-1 / HAVCR1 protein, His tag.
PABL-737 incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-Mouse IgG as a secondary antibody (1: 2000)
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Normal Tissue
Figure 1 Thyroid gland
Glandular cells
Staining: High
Intensity: Strong
Quantity: 75%-25%
Location: Cytoplasmic/membranous
* Image credit: Image credit: Human Protein Atlas https://v21.proteinatlas.org/images/473/63994_B_3_5.jpg
Normal Tissue
Figure 2 Testis
Leydig cells
Staining: Medium
Intensity: Moderate
Quantity: 75%-25%
* Image credit: Image credit: Human Protein Atlas https://v21.proteinatlas.org/images/473/63994_A_6_6.jpg
RNA Expression
Figure 3 RNA cell line category: Cell line enhanced (JURKAT, MOLT-4, SCLC-21H, U-266/70)
Cell lines ordered by descending RNA expression order.
RNA Expression
Figure 3 RNA cell line category: Cell line enhanced (JURKAT, MOLT-4, SCLC-21H, U-266/70)
Cell lines ordered by descending RNA expression order.
* Image credit: Image credit: Human Protein Atlas https://v21.proteinatlas.org/ENSG00000165409-TSHR
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Specifications
- Immunogen
- Human thyroid stimulating hormone receptor
- Host Species
- Human
- Type
- Human IgG
- Specificity
- Human TSHR
- Species Reactivity
- Human
- Clone
- K1-70
- Applications
- Block
Product Property
- Purity
- >95% as determined by SDS-PAGE and HPLC analysis
- Concentration
- Please refer to the vial label for the specific concentration.
- Storage
- Centrifuge briefly prior to opening vial. Store at +4°C short term (1-2 weeks). Aliquot and store at -20°C long term. Avoid repeated freeze/thaw cycles.
Applications
- Application Notes
- The TSHR antibody has been reported in applications of Blocking, ELISA, Inhibition, Antagonist.
Blocking type human monoclonal autoantibody K1-70 was isolated from the same sample of patient peripheral blood lymphocytes as the thyroid stimulating monoclonal autoantibody K1-18.
Target
- Alternative Names
- TSHR; thyroid stimulating hormone receptor; LGR3; CHNG1; hTSHR-I; thyrotropin receptor; thyrotropin receptor-I, hTSHR-I; seven transmembrane helix receptor; thyroid stimulating hormone receptor, isoform 2
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Citations
-
Rotondo Dottore, Giovanna, et al. "Insights into the role of DNA methylation and gene expression in graves orbitopathy." The Journal of Clinical Endocrinology & Metabolism 108.5 (2023): e160-e168. https://doi.org/10.1210/clinem/dgac645This study investigated DNA methylation patterns and gene expression in orbital fibroblasts from patients with Graves orbitopathy (GO) compared to control subjects. The researchers examined global DNA methylation under basal conditions and following exposure to anti-thyrotropin receptor antibodies (M22) and various cytokines involved in GO pathogenesis. The findings revealed that M22 significantly increased global DNA methylation in both control and GO fibroblasts, which correlated with increased cell proliferation. Methylome analysis identified nearly 20,000 differently methylated DNA regions in GO fibroblasts, encompassing 3,957 genes and affecting CpG islands, shores, and shelves. Several genes, including IGF-1 receptor and various fibroblast growth factors, were differentially methylated, suggesting epigenetic mechanisms may contribute to the persistence of GO through altered gene expression.
Creative Biolabs provided the human monoclonal anti-TSHR antibody (M22) used in the study to simulate the autoimmune environment affecting orbital fibroblasts. This antibody played a crucial role in demonstrating how anti-TSHR antibodies can induce DNA methylation changes, alter gene expression, and increase cell proliferation - key features in GO pathophysiology. The ability of M22 to transform control fibroblasts to resemble GO fibroblasts offered an important in vitro model of GO pathogenesis, supporting the researchers' hypothesis that epigenetic mechanisms may be responsible for the maintenance of GO even after immune suppression.
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(Creative Biolabs Cat# PABL-737, RRID: AB_3111673)
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Related Diseases
Autoimmune Thyroid Disease
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Product Notes
This is a product of Creative Biolabs' Hi-Affi™ recombinant antibody portfolio, which has several benefits including:
• Increased sensitivity
• Confirmed specificity
• High repeatability
• Excellent batch-to-batch consistency
• Sustainable supply
• Animal-free production
See more details about Hi-Affi™ recombinant antibody benefits.
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