An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions.
An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions.
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DOI：
10.3791/53575
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发表时间：
2016-03
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通讯作者：
Mohameedyaseen Syedbasha;J. Linnik;D. Santer;D. O’Shea;K. Barakat;M. Joyce;N. Khanna;D. Tyrrell-D.-Tyrr
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作者：
Mohameedyaseen Syedbasha;J. Linnik;D. Santer;D. O’Shea;K. Barakat;M. Joyce;N. Khanna;D. Tyrrell-D.-Tyrr
信号通路的全面理解需要关于配体-受体相互作用的详细知识。本文介绍了两种快速可靠的逐点酶联免疫吸附试验（ELISA）的配体-受体相互作用的调查协议：直接配体-受体相互作用试验（LRA）和竞争LRA。作为一个案例研究，提出了基于ELISA的不同λ干扰素（IFNLs）与其受体（IL 28 RA）的α亚基之间相互作用的分析：直接LRA用于测定解离常数受体和IFN配体之间的KD值，以及用于测定寡肽的抑制能力的竞争LRA，其被设计为在IFNLs的受体结合位点与IFNLs竞争。描述了估计KD和半数最大抑制浓度（IC 50）值的分析步骤。最后，讨论突出了所提出的方法的优点和缺点，以及如何使配体-受体相互作用的分子更好的理解的结果。
A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.