PMID- 22995065
OWN - NLM
STAT- MEDLINE
DCOM- 20130403
LR  - 20211021
IS  - 1096-0309 (Electronic)
IS  - 0003-2697 (Print)
IS  - 0003-2697 (Linking)
VI  - 431
IP  - 2
DP  - 2012 Dec 15
TI  - A time-resolved fluorescence resonance energy transfer assay suitable for 
      high-throughput screening for inhibitors of immunoglobulin E-receptor 
      interactions.
PG  - 84-9
LID - S0003-2697(12)00457-5 [pii]
LID - 10.1016/j.ab.2012.09.010 [doi]
AB  - The interaction of immunoglobulin E (IgE) antibodies with the high-affinity 
      receptor, FcepsilonRI, 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 FcepsilonRIalpha proteins. The assay can readily monitor classic 
      competitive inhibitors that bind either IgE-Fc or FcepsilonRIalpha in equilibrium 
      competition binding experiments. Furthermore, the TR-FRET assay can also be used 
      to follow the kinetics of IgE-Fc-FcepsilonRIalpha 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.
CI  - Copyright (c) 2012 Elsevier Inc. All rights reserved.
FAU - Kim, Beomkyu
AU  - Kim B
AD  - Department of Structural Biology, Stanford University School of Medicine, 
      Stanford, CA 94305, USA.
FAU - Tarchevskaya, Svetlana S
AU  - Tarchevskaya SS
FAU - Eggel, Alexander
AU  - Eggel A
FAU - Vogel, Monique
AU  - Vogel M
FAU - Jardetzky, Theodore S
AU  - Jardetzky TS
LA  - eng
GR  - R01 AI038972/AI/NIAID NIH HHS/United States
GR  - R21 NS074067/NS/NINDS NIH HHS/United States
GR  - R37 AI038972/AI/NIAID NIH HHS/United States
GR  - AI-18939/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20120918
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (Antibodies, Anti-Idiotypic)
RN  - 0 (Receptors, IgE)
RN  - 0 (anti-IgE antibodies)
RN  - 37341-29-0 (Immunoglobulin E)
SB  - IM
MH  - *Antibodies, Anti-Idiotypic/immunology
MH  - Fluorescence
MH  - Fluorescence Resonance Energy Transfer/methods
MH  - High-Throughput Screening Assays/methods
MH  - Humans
MH  - *Immunoglobulin E/immunology/metabolism
MH  - Kinetics
MH  - *Receptors, IgE/analysis/chemistry
PMC - PMC3583350
MID - NIHMS408894
EDAT- 2012/09/22 06:00
MHDA- 2013/04/04 06:00
PMCR- 2013/12/15
CRDT- 2012/09/22 06:00
PHST- 2012/08/09 00:00 [received]
PHST- 2012/09/09 00:00 [accepted]
PHST- 2012/09/22 06:00 [entrez]
PHST- 2012/09/22 06:00 [pubmed]
PHST- 2013/04/04 06:00 [medline]
PHST- 2013/12/15 00:00 [pmc-release]
AID - S0003-2697(12)00457-5 [pii]
AID - 10.1016/j.ab.2012.09.010 [doi]
PST - ppublish
SO  - Anal Biochem. 2012 Dec 15;431(2):84-9. doi: 10.1016/j.ab.2012.09.010. Epub 2012 
      Sep 18.