Hill Katie L, Hamers Timo, Kamstra Jorke H, Willmore William G, Letcher Robert J
Ecotoxicology and Wildlife Health Division, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Ottawa, Canada.
Department of Biology, Carleton University, Ottawa, Canada.
MethodsX. 2017 Oct 20;4:404-412. doi: 10.1016/j.mex.2017.10.004. eCollection 2017.
Thyroid hormones (THs) are involved in the regulation of many physiological processes in vertebrates. Competition for TH binding sites on serum transport proteins can interfere with delivery of THs to target tissues, and this is a potential mechanism of action of exogenous thyroidogenic substances. To date, detailed accounts of methods for competitive binding with THs on TH transport proteins (human or wildlife) are sparse. In the limited number of published studies on radio-labelled TH-TH transport protein interactions, method descriptions were brief and with insufficient details for successful replication. Furthermore, upon review of these methodologies, we identified several opportunities for optimization. The present study addresses the methodological deficiencies and describes, in detail, a fully optimized and validated competitive T4 radio-ligand binding assay with human transthyretin (TTR) and albumin (ALB). •Significant improvements were made over previous methods, including better maintenance of protein stability and enhanced measurement of competition between different ligands.•Sample size was reduced to allow use of small pre-packed size exclusion chromatography columns, which eliminates the rinsing step during the separation procedure.•The assay was parameterized for use with T4 and human TTR and ALB.
甲状腺激素（THs）参与脊椎动物多种生理过程的调节。血清转运蛋白上甲状腺激素结合位点的竞争会干扰甲状腺激素向靶组织的传递，这是外源性甲状腺生成物质的一种潜在作用机制。迄今为止，关于在甲状腺激素转运蛋白（人类或野生动物）上与甲状腺激素竞争性结合方法的详细报道很少。在已发表的关于放射性标记甲状腺激素 - 甲状腺激素转运蛋白相互作用的有限研究中，方法描述简短且细节不足，难以成功重复。此外，在审查这些方法时，我们发现了几个优化的机会。本研究解决了方法上的缺陷，并详细描述了一种针对人甲状腺素转运蛋白（TTR）和白蛋白（ALB）进行的完全优化和验证的竞争性T4放射性配体结合测定法。•相对于以前的方法有显著改进，包括更好地维持蛋白质稳定性以及增强对不同配体之间竞争的测量。•样本量减少，以便使用小型预装尺寸排阻色谱柱，从而消除了分离过程中的冲洗步骤。•该测定法针对T4以及人TTR和ALB进行了参数化设置。
