En ELISA baseret Indbinding og konkurrence metode til hurtigt at afgøre, ligand-receptor interaktioner
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
En omfattende forståelse af signalveje kræver detaljeret viden om ligand-receptor interaktion. De fleste metoder til vurdering af samspillet af en bestemt ligand med dens specifikke receptor er dyre, tidskrævende, arbejdskrævende og kræver særligt udstyr og ekspertise en.
I denne artikel beskrives to hurtige og pålidelige punkt-for-punkt-protokoller til at undersøge ligand-receptor interaktion baseret på en enzymbundet immunosorbentassay (ELISA): Den direkte ligandreceptorinteraktion assay (LRA) og konkurrencen LRA. ELISA er et yderst følsomt, specifik og let tilgængelige teknik, som rutinemæssigt anvendes i næsten alle laboratorier. ELISA kan udføres og tilpasses på forskellige måder. De præsenterede protokoller er optimeret til undersøgelse af samspillet mellem forskellige lambda interferoner (INFLs) og deres receptor.
Den direkte LRA giver mulighed for en quantificatipå af ligand-receptor binding med hensyn til ligandkoncentration og giver således en bindende kurve. Anvendelse af en passende model for ligand-receptor-interaktion, kan dataene analyseres yderligere at estimere dissociationskonstanten (KD).
I præsenterede protokol, er det almindeligt anvendte Hill-ligningen anvendes til at modellere ligand-receptor binding. Selv om andre metoder såsom overfladeplasmonresonans teknologi 2,3 muliggøre bestemmelsen af bindingsaffiniteterne mellem to proteiner, denne teknologi er ofte arbejdskrævende, kostbare og kræver særlig laboratorieudstyr.
Konkurrencen LRA muliggør screening af inhibitoriske peptider: ligand-receptor binding kvantificeres i forhold til peptid koncentration. Dette giver en dosis-respons-kurve, der beskriver den inhibitoriske virkning af peptidet. Dataene kan yderligere analyseres for at estimere halvmaksimal inhiberende koncentration (IC50 </sub>) for spærringen peptid.
Både ELISA-protokoller er nemme at bruge, og kan tilpasses til en bred vifte af forskningsspørgsmål. Rekombinante proteiner af enhver art kan anvendes til pålideligt og hurtigt bestemme interaktion dele. Derudover kan konkurrencen LRA anvendes til at bestemme kritiske interageringssteder på ligander og receptorer ved hjælp blokerende peptider, som er designet til at efterligne enten liganden eller receptoren. Hvis blokerende peptid viser effektiv og specifik inhibering, peptidet indtager en kritisk interaktion site af ligand (hvis peptidet efterligner receptor) eller af liganden (Hvis peptidet efterligner liganden).
Den første protokol beskriver KD værdi bestemmelse af forskellige INFLs og alfa-underenheden af deres receptor, dvs. interleukin-28 receptor (IL28RA) ved hjælp af direkte LRA. Dernæst den anden protokol viser, hvordan til at bestemme evnen af en 20 aminosyrer lang peptid tilhæmme INFL-IL28RA interaktioner. Peptidet er designet til at konkurrere med IFNLs på deres receptorbindingssted og dermed muliggør en molekylær forståelse af interaktionen. Endvidere kan dette peptid bruges til at blokere IL28RA i in vitro-forsøg for at fastslå virkningen på downstream signalering effekter 4.
Protocol
Representative Results
Discussion
ELISA er en standard og veletableret metode til mange laboratorier. Vi har yderligere modificeret og forbedret en tidligere publiceret metode 5,7. Den påvist trin-for-trin procedure viser, hvordan den kan anvendes på en enkel måde at bestemme Kd-værdier af ligand-receptor-interaktioner. Desuden kan bestemmes IC50 for et blokerende peptid, der interfererer med ligand-receptor interaktion.
Store fordele er den hurtige opsætning, nem fremstilling af reagenser og velke…
Disclosures
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 |
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