Mouse Anti-TSHR Recombinant Antibody (clone 3BD10) (CAT#: PABL-353)

Figure 1 Immunoprecipitation of secreted, precursor-labeled antigen with mouse mAb 3BD10 to TSHR-289.

CHO cells expressing TSHR-289 were precursor labeled with methionine/cysteine and immunoprecipitated with mouse mAb 3BD10. The mAb 3BD10 interacted strongly with native TSHR-289 (N) in culture medium, but not at all which is the same material after reduction and alkylation (D, denatured).

Chazenbalk, G. D., Wang, Y., Guo, J., Hutchison, J. S., Segal, D., Jaume, J. C., ... & Rapoport, B. (1999). A mouse monoclonal antibody to a thyrotropin receptor ectodomain variant provides insight into the exquisite antigenic conformational requirement, epitopes and in vivo concentration of human autoantibodies. The Journal of Clinical Endocrinology & Metabolism, 84(2), 702-710.

Figure 2 Monoclonal antibody recognition of TSHR N terminal synthetic peptides.

Upper panel, TSHR 20-mer peptides encompassed cysteine loops 1 and 2 (peptide A; residues 22–41), neither loop (peptide B; residues 37–56), and an overlapping peptide containing only loop 2 (peptide AB; residues 30–49). Cysteine residues known to form disulfide bonds in the TSHR are shown as black disks. Residue R38 in loop 2, the most N-terminal contact residue for human TSAb M22 is depicted as a gray disk. Lower panel, ELISA with the peptides indicated in panel A. Binding of the indicated mAb with each peptide (mean ± SEM of triplicate assays, each measured in duplicate). Antibody concentrations were 5 μg/ml 3BD10, 5 μg/ml 2C11, and 1:4 dilution of 3E5 containing culture medium.

Hamidi, S., Chen, C. R., Murali, R., McLachlan, S. M., & Rapoport, B. (2013). Probing structural variability at the N terminus of the TSH receptor with a murine monoclonal antibody that distinguishes between two receptor conformational forms. Endocrinology, 154(1), 562-571.

Figure 3 Influence of TSHR N-terminal cysteine-bonded loop 1 on 3BD10 binding.

Flow cytometry was performed using CHO cells stably expressing the TSHR ECD tethered to the plasma membrane by a GPI anchor. A, Deletion of TSHR loop 1 (amino acid residues 22–30; D22–30-ECD-GPI). Fluorescence values obtained using mAb 3BD10 were expressed relative to the control mAb normalized to 100%. Two experiments used both 2C11 and CS-17 as controls, and a third experiment used CS-17. All mAbs were at 10 μg/ml. Each bar represents the mean ± SEM of values obtained in three experiments. B, Chimeric substitution of loop 1 (TSHR amino acids 25–30 replaced with the corresponding LHR residues) A1-ECD-GPI. These experiments tested mAb 3BD10 (10 μg/ml) and also 3E5 (undiluted culture medium) relative to the control mAb 2C11 (10 μg/ml), fluorescence with the latter normalized to 100%. Each bar represents values (mean ± SEM) obtained in three experiments. C, The mAb 3BD10 binding kinetics. Flow cytometry was performed with increasing concentrations of mAb 3BD10 and mAb 2C11 as a control. Both antibodies are of the same IgG1 isotype. Each point represents the mean ± SEM of fluorescence values obtained in five experiments. Data were analyzed by nonlinear regression.

Hamidi, S., Chen, C. R., Murali, R., McLachlan, S. M., & Rapoport, B. (2013). Probing structural variability at the N terminus of the TSH receptor with a murine monoclonal antibody that distinguishes between two receptor conformational forms. Endocrinology, 154(1), 562-571.