A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions

Kim, Beomkyu; Tarchevskaya, Svetlana S; Eggel, Alexander; Vogel, Monique; Jardetzky, Theodore S (2012). A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions. Analytical biochemistry, 431(2), pp. 84-9. San Diego, Calif.: Elsevier 10.1016/j.ab.2012.09.010

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The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE-receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the IgE-receptor interaction. The TR-FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR-FRET assay can also be used to follow the kinetics of IgE-Fc-FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR-FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.

Item Type:	Journal Article (Original Article)
Division/Institute:	04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Institute for Immunology [discontinued]
UniBE Contributor:	Eggel, Alexander, Vogel, Monique
ISSN:	0003-2697
Publisher:	Elsevier
Language:	English
Submitter:	Factscience Import
Date Deposited:	04 Oct 2013 14:37
Last Modified:	05 Dec 2022 14:11
Publisher DOI:	10.1016/j.ab.2012.09.010
PubMed ID:	22995065
Web of Science ID:	000311065500002
URI:	https://boris.unibe.ch/id/eprint/15093 (FactScience: 222286)