Name: an elisa based binding and competition method to rapidly determine ligand-receptor interactions
Uploaded: 2016-03-14T13:00:00
Description: Watch this Scientific Journal Video about An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions at JoVE.com

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.

1. Reagent Preparation
<ol>
	<li>To prepare carbonate coating buffer, dissolve 0.36 g Na2CO3 and 0.84 g NaHCO3in 100 ml distilled water; sterile filter the buffer by using a vacuum driven 0.22 &#181;m polyethersulfone (PES) membrane filter and store at RT until usage.</li>
	<li>Prepare washing solution by adding 0.05% v/v Tween 20 in Phosphate buffered saline (PBS).</li>
	<li>Prepare a 5% Bovine Serum Albumin (BSA) (blocking solution) in PBS solution by dissolving 5 g BSA (&#8805;98%) i...

<ol>
	<li>Schneider, P., Willen, L., Smulski, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+and+techniques+to+study+ligand-receptor+interactions+and+receptor+activation+by+TNF+superfamily+members.">Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members.</a> Methods in enzymology. 545, 103-125 (2014).</li><li>Rossi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensor+analysis+of+anti-citrullinated+protein/peptide+antibody+affinity.">Biosensor analysis of anti-citrullinated protein/peptide antibody affinity.</a> Analytical biochemistry. 465, 96-101 (2014).</li><li>van der Merwe, P. A., Barclay, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell-adhesion+molecule+interactions+using+surface+plasmon+resonance.">Analysis of cell-adhesion molecule interactions using surface plasmon resonance.</a> Curr Opin Immunol. 8, 257-261 (1996).</li><li>Egli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-28B+is+a+key+regulator+of+B-+and+T-cell+vaccine+responses+against+influenza.">IL-28B is a key regulator of B- and T-cell vaccine responses against influenza.</a> PLoS Pathog. 10, e1004556 (2014).</li><li>Rosenbluh, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positively+charged+peptides+can+interact+with+each+other,+as+revealed+by+solid+phase+binding+assays.">Positively charged peptides can interact with each other, as revealed by solid phase binding assays.</a> Analytical biochemistry. 352, 157-168 (2006).</li><li>Goutelle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hill+equation:+a+review+of+its+capabilities+in+pharmacological+modelling.">The Hill equation: a review of its capabilities in pharmacological modelling.</a> Fundamental &amp; clinical pharmacology. 22, 633-648 (2008).</li><li>Levin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+derived+from+HIV-1+integrase+that+bind+Rev+stimulate+viral+genome+integration.">Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration.</a> PLoS One. 4, e4155 (2009).</li><li>Egli, A., Santer, M. D., O'Shea, D., Tyrrell, D. L., Houghton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+the+interferon-lambda+family+on+the+innate+and+adaptive+immune+response+to+viral+infections.">The impact of the interferon-lambda family on the innate and adaptive immune response to viral infections.</a> Emerging infectious diseases. , e51 (2014).</li><li>Gad, H. H., Hamming, O. J., Hartmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+human+interferon+lambda+and+what+it+has+taught+us.">The structure of human interferon lambda and what it has taught us.</a> J Interferon Cytokine Res. 30, 565-571 (2010).</li><li>Folch, B., Rooman, M., Dehouck, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermostability+of+salt+bridges+versus+hydrophobic+interactions+in+proteins+probed+by+statistical+potentials.">Thermostability of salt bridges versus hydrophobic interactions in proteins probed by statistical potentials.</a> Journal of chemical information and modeling. 48, 119-127 (2008).</li><li>Yuzlenko, O., Lazaridis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+ionizable+amino+acid+side+chains+at+a+lipid+bilayer-water+interface.">Interactions between ionizable amino acid side chains at a lipid bilayer-water interface.</a> The journal of physical chemistry. B. 115, 13674-13684 (2011).</li><li>Tissot, A. C., Vuilleumier, S., Fersht, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+two+buried+salt+bridges+in+the+stability+and+folding+pathway+of+barnase.">Importance of two buried salt bridges in the stability and folding pathway of barnase.</a> Biochemistry. 35, 6786-6794 (1996).</li></ol>

ELISA is a standard and well-established method for many laboratories. We have further modified and improved a previously published method 5,7. The demonstrated step-by-step protocol shows how it can be used in a simple way to determine the KD values of ligand-receptor interactions. In addition, the IC50 of a blocking peptide that interferes with the ligand-receptor interaction can be determined.
Major advantages are the rapid setup, easy preparation of reagents and famil...

A comprehensive understanding of signaling pathways requires detailed knowledge about the ligand-receptor interaction. Most methods for assessing the interaction of a particular ligand with its specific receptor are expensive, time consuming, labor intensive and require specific equipment and expertise 1.
This article describes two fast and reliable point-by-point protocols to investigate the ligand-receptor interaction based on an enzyme linked immunosorbent assay (ELISA): the direct ligand-receptor interaction assay (LRA) and the competition LRA. ELISA is a highly sensitive, specific and readily available technique, routinely used in almost every laboratory. ELISA can be performed and adapted in various fashions. The presented protocols are optimized for the investigation of the interaction between different lambda interferons (INFLs) and their receptor.
The direct LRA allows for a quantification of ligand-receptor binding with respect to ligand concentration and thus yields a binding curve. Using an appropriate model for the ligand-receptor interaction, the data can be further analyzed to estimate the dissociation constant (KD).
In the presented protocol, the commonly used Hill equation is applied to model the ligand-receptor binding. Although other methods such as the surface plasmon resonance technology 2,3 allow the determination of the binding affinities between two proteins, this technology is often labor intensive, expensive, and requires special laboratory equipment.
The competition LRA enables the screening of inhibitory peptides: The ligand-receptor binding is quantified with respect to peptide concentration. This yields a dose-response curve describing the inhibitory effect of the peptide. The data can be further analyzed to estimate the half maximal inhibitory concentration (IC50) of the blocking peptide.
Both ELISA protocols are easy to use and can be adapted to a broad range of research questions. Recombinant proteins of any kind can be used to reliably and fast determine the interaction parts. In addition, the competition LRA can be used to determine critical interaction sites of ligands and receptors by using blocking peptides, which are designed to mimic either the ligand or the receptor. If the blocking peptide shows efficient and specific inhibition, the peptide occupies a critical interaction site of the ligand (if the peptide mimics the receptor) or of the ligand (if the peptide mimics the ligand).
The first protocol describes the KD value determination of different INFLs and the alpha subunit of their receptor, i.e., the interleukin-28 receptor (IL28RA) using the direct LRA. Next, the second protocol shows how to determine the capability of a 20 amino acid long peptide to inhibit the INFL-IL28RA interactions. The peptide is designed to compete with IFNLs at their receptor binding site and thus enables a molecular understanding of the interaction. Furthermore, this peptide can be used to block IL28RA in in vitro experiments to determine the impact on downstream signaling effects4.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Nunc-Immunoplate (F96 Maxi sorp)</td>
			<td style="width:164px;">Thermo Scientific</td>
			<td style="width:119px;">442404</td>
			<td style="width:359px;">ELISA plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sodium carbonate (Na2CO3)</td>
			<td style="width:164px;">Merck</td>
			<td style="width:119px;">497-19-8</td>
			<td>For ELISA plate coating buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Sodium hydrogen carbomnate(NaHCO3)</td>
			<td style="width:164px;">Merck</td>
			<td style="width:119px;">144-55-8</td>
			<td>For ELISA plate coating buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">A7030-100G</td>
			<td>5% BSA in PBS for Blocking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">rhIL-28R&#945;/IFN&#955;R1</td>
			<td style="width:164px;">R&#38;D systems</td>
			<td style="width:119px;">5260-MR</td>
			<td>Recombinant human interlukin-28 Receptor alpha</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">rhIL-29/IFN&#955;1</td>
			<td style="width:164px;">R&#38;D systems</td>
			<td style="width:119px;">1598-IL/CF</td>
			<td>Recombinant human interlukin-29/Carrier free/C-terminal 10-His tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">rhIL-28A/IFN&#955;2</td>
			<td style="width:164px;">R&#38;D systems</td>
			<td style="width:119px;">1587-IL/CF</td>
			<td>Recombinant human interlukin-28A/Carrier free/C-terminal 6-His tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">rhIL-28B/IFN&#955;3</td>
			<td style="width:164px;">R&#38;D systems</td>
			<td style="width:119px;">5259-IL/CF</td>
			<td>Recombinant human interlukin-28B/Carrier free/C-terminal 6-His tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">6X His Monoclonal antibody (Mouse)</td>
			<td style="width:164px;">Clontech</td>
			<td style="width:119px;">631212</td>
			<td>Primary antiboy to capture His tagged Ligands</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Goat anti-Mouse igG (H+L)</td>
			<td style="width:164px;">Jackson Immuno Research</td>
			<td style="width:119px;">115-035-166</td>
			<td>Horseradish Peroxidase conjucated secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">BDoptEIA TMB reagent set</td>
			<td style="width:164px;">BD Biosciences</td>
			<td style="width:119px;">555214</td>
			<td>ELISA - TMB substrate solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sulfuric acid (H2SO4)</td>
			<td style="width:164px;">Fulka</td>
			<td style="width:119px;">84720</td>
			<td>5N H2SO4 (Enzyme reaction stop solution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Synergy/H1 - Microplate reader</td>
			<td style="width:164px;">BioTeK</td>
			<td style="width:119px;"></td>
			<td>ELISA plate reader</td>
		</tr>
	</tbody>
</table>

an elisa based binding and competition method to rapidly determine ligand-receptor interactions

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.

The dissociation constants between INFL1-3 and their receptor alpha subunit IL28RA were determined using the direct LRA. The results are shown in Figure 3: The fraction of occupied binding sites is plotted against the logarithm of the respective IFN concentration. The Scatchard plot of the data is shown in the bottom right corner. The results illustrate that the direct LRA yields a binding curve, which can be further analyzed to estimate the KD value. The K<sub...

Watch this Scientific Journal Video about An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions at JoVE.com

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

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.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

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