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An ELISA Based Binding and Competition Method to Rapidly Determine 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.

SUBMITTER: Syedbasha M

PROVIDER: S-EPMC4828978 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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

Syedbasha Mohameedyaseen M Linnik Janina J Santer Deanna D O'Shea Daire D Barakat Khaled K Joyce Michael M Khanna Nina N Tyrrell D Lorne DL Houghton Michael M Egli Adrian A

Journal of visualized experiments : JoVE 20160314 109

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 th ...[more]

PMID: 27023275

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Project description:BackgroundMas-related G-protein coupled receptor member X2 (MrgX2) directly mediates drug-induced pseudo allergic reactions. Skin mast cell MrgX2 is upregulated in severe chronic urticaria (CU). Mast cells and leukocytes are key effector cells in allergic reactions and undergo degranulation upon stimulation. It is unknown whether circulating MrgX2 expression can be detected occurs in the whole blood of CU patients and reflects pseudo-allergic reaction. There is no effective method for its detection. Therefore, an enzyme-linked immuno-sorbent assay (ELISA) for MrgX2 was developed.MethodsMonoclonal and polyclonal MrgX2 specific antibodies were obtained from rabbits and mice immunized by MrgX2 peptides prepared. Indirect ELISA and Dot blot were used to determine antibody titers before a sandwich ELISA for MrgX2 was established. The whole blood from healthy subjects and CU patients was used to detect MrgX2 concentrations. The use of feasibility of this MrgX2-ELISA as a clinical detection tool was explored and diagnostic purposes was assessed.ResultsThe sandwich antibody ELISA method for MrgX2 was established with good linearity regression (R2 = 0.9910). The lowest detection limit was 3.125 ng/mL. The quantification limit was 6.25 ng/mL. The sandwich ELISA for MrgX2 have good stability and high specificity. The initial truncation value of MrgX2 was 60.91 ng/mL (95% confidence interval). The whole blood MrgX2 concentrations in CU patients (median 98.01 ± 4.317 ng/mL, n = 75) was significantly increased compared to healthy subjects (58.09 ± 1.418 ng/mL, n = 75), with significant difference (p < 0.0001) and higher accuracy of (AUC = 0.8795). Comprehensive the frequency analysis of MrgX2 expression in 75 CU patients reference frequency distribution and ROC curve analysis, determined the threshold for CU patients as 71.23 ng/mL, with 81.33% sensitivity and 90.67% specificity.ConclusionMrgX2-ELISA provides a useful and convenient method for detecting MrgX2 in whole blood samples. The MrgX2-ELISA will help improve the understanding of the role of MrgX2 in regulating chronic urticaria.

Dataset Information

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

Publications

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

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