的ELISA基结合和竞争法快速测定配体 - 受体相互作用
Summary
The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.
Abstract
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.
Introduction
信号通路的全面了解需要有关配体 – 受体相互作用的详细知识。评估其特异性受体的特定配体的相互作用的大多数方法是昂贵的，耗时的，劳动密集型的，并且需要特定的设备和专门知识1。
本文介绍了两个快速和可靠的逐点的协议来调查的基础上的一种酶的配体 – 受体相互作用酶联免疫吸附测定（ELISA）：直接配体 – 受体相互作用测定（LRA）和竞争LRA。 ELISA是一个高度敏感的，具体的和随时可用的技术，几乎在每一个实验室常规使用。 ELISA可以执行并适于各种方式。所提出的协议对不同的λ干扰素（INFLs）和它们的受体之间的相互作用的调查进行了优化。
直接LRA允许一个quantificati关于配体 – 受体的相对于配体浓度结合和由此产生的结合曲线。使用适当的型号为配体-受体相互作用，可以将数据进一步分析以估计的解离常数（K D）。
在所提出的协议，通常使用希尔方程被施加到该配体 – 受体结合建模。尽管其它方法，例如表面等离子共振技术2,3-允许两个蛋白之间的结合亲和力的测定，这种技术常常是劳动密集型的，昂贵的，并且需要特殊的实验室设备。
竞争LRA能够抑制肽的筛选：在配体 – 受体结合相对于肽浓度定量。这产生描述的肽的抑制作用的剂量 – 反应曲线。数据可以被进一步分析以估计半数最大抑制浓度（IC 50 </子>）阻断肽。
两者的ELISA协议是易于使用，并且可以适合于广泛的研究问题。任何形式的重组蛋白可以用于可靠地并快速确定相互作用的部件。此外，竞争LRA可用于通过使用阻断肽，其目的是为了模仿或配体或受体，以确定配体和受体的关键相互作用位点。如果封闭肽显示有效和特异性抑制，肽占据配体的关键相互作用位点（如肽模拟物的受体）或配体（如肽模拟物的配位体）。
第一个协议描述不同INFLs的在K D值的决心和其受体α亚基， 即使用直接LRA白细胞介素28受体（IL28RA）。接着，第二协议显示如何确定一个20个氨基酸长的肽的能力抑制INFL-IL28RA相互作用。肽被设计成在其受体结合位点与IFNLs竞争，从而使相互作用的分子的理解。此外，该肽可以用于阻止在体外实验IL28RA以确定对下游信号效应4的影响。
Protocol
Representative Results
Discussion
ELISA是许多实验室标准和行之有效的方法。我们进一步修改和完善了先前公布的方法5,7。所表现出的一步一步的协议说明了如何能够以简单的方式被用于确定配体-受体相互作用的K个D取值。此外，阻挡肽，与配体 – 受体相互作用干扰的IC 50可被确定。
主要优点是快速安装，易于制备的试剂和熟悉的操作，因为大多数研究人员以前使用的ELISA协议。直接LRA协议是…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Prof. J. Stelling (Department of Biosystems Science and Engineering, ETH Zurich and Swiss Institute for Bioinformatics, Basel, Switzerland) for his critical review of the manuscript.
Materials
| Nunc-Immunoplate (F96 Maxi sorp) | Thermo Scientific | 442404 | ELISA plate |
| Sodium carbonate (Na2CO3) | Merck | 497-19-8 | For ELISA plate coating buffer |
| Sodium hydrogen carbomnate(NaHCO3) | Merck | 144-55-8 | For ELISA plate coating buffer |
| Bovine Serum Albumin (BSA) | Sigma | A7030-100G | 5% BSA in PBS for Blocking |
| rhIL-28Rα/IFNλR1 | R&D systems | 5260-MR | Recombinant human interlukin-28 Receptor alpha |
| rhIL-29/IFNλ1 | R&D systems | 1598-IL/CF | Recombinant human interlukin-29/Carrier free/C-terminal 10-His tag |
| rhIL-28A/IFNλ2 | R&D systems | 1587-IL/CF | Recombinant human interlukin-28A/Carrier free/C-terminal 6-His tag |
| rhIL-28B/IFNλ3 | R&D systems | 5259-IL/CF | Recombinant human interlukin-28B/Carrier free/C-terminal 6-His tag |
| 6X His Monoclonal antibody (Mouse) | Clontech | 631212 | Primary antiboy to capture His tagged Ligands |
| Goat anti-Mouse igG (H+L) | Jackson Immuno Research | 115-035-166 | Horseradish Peroxidase conjucated secondary antibody |
| BDoptEIA TMB reagent set | BD Biosciences | 555214 | ELISA – TMB substrate solution |
| Sulfuric acid (H2SO4) | Fulka | 84720 | 5N H2SO4 (Enzyme reaction stop solution) |
| Synergy/H1 – Microplate reader | BioTeK | ELISA plate reader |
References
- Schneider, P., Willen, L., Smulski, C. R. Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members. Methods in enzymology. 545, 103-125 (2014).
- Rossi, G., et al. Biosensor analysis of anti-citrullinated protein/peptide antibody affinity. Analytical biochemistry. 465, 96-101 (2014).
- van der Merwe, P. A., Barclay, A. N. Analysis of cell-adhesion molecule interactions using surface plasmon resonance. Curr Opin Immunol. 8, 257-261 (1996).
- Egli, A., et al. IL-28B is a key regulator of B- and T-cell vaccine responses against influenza. PLoS Pathog. 10, e1004556 (2014).
- Rosenbluh, J., et al. Positively charged peptides can interact with each other, as revealed by solid phase binding assays. Analytical biochemistry. 352, 157-168 (2006).
- Goutelle, S., et al. The Hill equation: a review of its capabilities in pharmacological modelling. Fundamental & clinical pharmacology. 22, 633-648 (2008).
- Levin, A., et al. Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration. PLoS One. 4, e4155 (2009).
- Egli, A., Santer, M. D., O’Shea, D., Tyrrell, D. L., Houghton, M. The impact of the interferon-lambda family on the innate and adaptive immune response to viral infections. Emerging infectious diseases. , e51 (2014).
- Gad, H. H., Hamming, O. J., Hartmann, R. The structure of human interferon lambda and what it has taught us. J Interferon Cytokine Res. 30, 565-571 (2010).
- Folch, B., Rooman, M., Dehouck, Y. Thermostability of salt bridges versus hydrophobic interactions in proteins probed by statistical potentials. Journal of chemical information and modeling. 48, 119-127 (2008).
- Yuzlenko, O., Lazaridis, T. Interactions between ionizable amino acid side chains at a lipid bilayer-water interface. The journal of physical chemistry. B. 115, 13674-13684 (2011).
- Tissot, A. C., Vuilleumier, S., Fersht, A. R. Importance of two buried salt bridges in the stability and folding pathway of barnase. Biochimie. 35, 6786-6794 (1996).
