Method Article

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

DOI:

10.3791/53575

⸱

March 14th, 2016

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Applied Microbiology Research, Department of Biomedicine, University of Basel, 2Department of Biosystems Science and Engineering, ETH Zurich, and Swiss Institute of Bioinformatics, 3Swiss Institute of Bioinformatics, 4Li Ka Shing Institute for Virology, University of Alberta, 5Regional Infectious Diseases Unit, University of Edinburgh, 6Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 7Infection Biology, Department of Biomedicine, University of Basel, 8Clinical Microbiology, University Hospital Basel

In This Article

Summary

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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

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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

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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 u....

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Protocol

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1. Reagent Preparation

  1. 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 µm polyethersulfone (PES) membrane filter and store at RT until usage.
  2. Prepare washing solution by adding 0.05% v/v Tween 20 in Phosphate buffered saline (PBS).
  3. Prepare a 5% Bovine Serum Albumin (BSA) (blocking solution) in PBS solution by dissolving 5 g BSA (≥98%) in 100 ml PBS and store at 4 °C.
  4. Recombinant Receptor, Ligands and Blocking Peptides
    1. Reconstitutethe recombinant human interleukin receptor ....

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Results

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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

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Discussion

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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.......

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Disclosures

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A.E. was supported by a research grants from the "SNSF Ambizione Score" program (PZ00P3_154709), "Forschungsfond, Förderung strategischer Projekte" University of Basel, Stiftungsinfektionskrankheiten Basel, and Bangeter Rhyner Stiftung. J.L. acknowledges support by an iPhD fellowship of the SystemsX.ch initiative in systems biology program (9th call).

Acknowledgements

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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.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Nunc-Immunoplate (F96 Maxi sorp)Thermo Scientific442404ELISA plate
Sodium carbonate (Na2CO3)Merck497-19-8For ELISA plate coating buffer
Sodium hydrogen carbomnate(NaHCO3)Merck144-55-8For ELISA plate coating buffer
Bovine Serum Albumin (BSA)SigmaA7030-100G5% BSA in PBS for Blocking
rhIL-28Rα/IFNλR1R&D systems5260-MRRecombinant human interlukin-28 Receptor alpha
rhIL-29/IFNλ1R&D systems1598-IL/CFRecombinant human interlukin-29/Carrier free/C-terminal 10-His tag
rhIL-28A/IFNλ2R&D systems1587-IL/CFRecombinant human interlukin-28A/Carrier free/C-terminal 6-His tag
rhIL-28B/IFNλ3R&D systems5259-IL/CFRecombinant human interlukin-28B/Carrier free/C-terminal 6-His tag
6x His Monoclonal antibody (Mouse)Clontech631212Primary antiboy to capture His tagged Ligands
Goat anti-Mouse igG (H+L)Jackson Immuno Research115-035-166Horseradish Peroxidase conjucated secondary antibody
BDoptEIA TMB reagent setBD Biosciences555214ELISA - TMB substrate solution
Sulfuric acid (H2SO4)Fulka847205 N H2SO4 (Enzyme reaction stop solution)
Synergy/H1 - Microplate readerBioTeKELISA plate reader

References

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  1. 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).
  2. Rossi, G., et al.

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Tags

ELISALigand Receptor InteractionBinding AffinityCompetition AssayCytokine ReceptorDissociation ConstantIC50 ValueInterferon LambdaReceptor Alpha SubunitBlocking Peptide

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