Comparison of a Novel Homogeneous Cyclic Amp Assay and a Luciferase Assay for Measuring Stimulating Thyrotropin-Receptor Autoantibodies
| Autor: | Christian Wüster, George J. Kahaly, Paul D. Olivo, Michael Kanitz, Yie-Hwa Chang, Tanja Diana |
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| Rok vydání: | 2019 |
| Předmět: |
Forskolin
business.industry Activator (genetics) Endocrinology Diabetes and Metabolism Ligand binding assay Chinese hamster ovary cell 030209 endocrinology & metabolism Molecular biology Thyrotropin receptor 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine chemistry Cell culture 030220 oncology & carcinogenesis Medicine Bioassay Luciferase business Translational Thyroidology / Research Article |
| Zdroj: | Eur Thyroid J |
| ISSN: | 2235-0640 |
| Popis: | Objective: Stimulating thyrotropin-receptor antibodies (TSAb) cause Graves’ disease (GD). We tested a novel homogeneous fluorescent 3′,5′ cyclic adenine monophosphate (cAMP) assay for the detection of TSAb in a bioassay. Methods: Chinese hamster ovary (CHO) cell lines expressing either a chimeric (MC4) or wild-type (WT) TSH-R were incubated with the adenyl cyclase activator forskolin, a human TSAb monoclonal antibody (M22), and with sera from GD patients. Intracellular cAMP levels were measured using a Bridge-It® cAMP assay, and the results were compared with a luciferase-based bioassay. Results: Both cell lines were stimulated with forskolin concentrations (0.006–200 µM) in a dose-dependent manner. The linear range in the MC4 and WT cells was 0.8–25 and 3.1–50 µM, respectively. Levels of cAMP and luciferase in forskolin-treated MC4 and WT cells were positively correlated (r = 0.91 and 0.84, both p < 0.001). The 50% maximum stimulatory concentration of forskolin was more than 16-fold higher for the CHO-WT cells than the CHO-MC4 cells in the cAMP assay and 4-fold higher in the luciferase assay. Incubation of both cell lines with M22 (0.006–50 ng/mL) resulted in a dose-dependent increase in cAMP levels with linear ranges for the MC4 and WT cells of 0.8–12.5 and 0.2–3.125 ng/mL, respectively. Comparison of cAMP and luciferase levels in M22-treated MC4 and WT cells also showed a positive correlation (r = 0.88, p < 0.001 and 0.75, p = 0.002). A positive correlation was also noted when using patient samples (r = 0.96, p < 0.001) that were all TSH-R-Ab binding assay positive. Conclusion: The novel, rapid, simple-to-perform cAMP assay provides TSAb-mediated stimulatory results comparable to a luciferase-based bioassay. |
| Databáze: | OpenAIRE |
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