Optimization of time-resolved fluorescence assay for detection of europium–tetraazacyclododecyltetraacetic acid-labeled ligand–receptor interactions

Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as di...

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Bibliographic Details
Published in:Analytical biochemistry 2010-03, Vol.398 (1), p.15-23
Main Authors: De Silva, Channa R., Vagner, Josef, Lynch, Ronald, Gillies, Robert J., Hruby, Victor J.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding assay
Cell Line
DELFIA
DOTA derivatives
Europium - chemistry
Fluorescent Dyes - chemistry
Fluoroimmunoassay - methods
Heterocyclic Compounds, 1-Ring - chemistry
High-Throughput Screening Assays
Humans
Lanthanide
Lanthanoid Series Elements - chemistry
Ligands
Melanocortin receptor
Melanocyte-Stimulating Hormones - chemistry
Melanocyte-Stimulating Hormones - metabolism
Receptor, Cholecystokinin B - chemistry
Receptor, Cholecystokinin B - metabolism
Receptor, Melanocortin, Type 4 - chemistry
Receptor, Melanocortin, Type 4 - metabolism
Receptors, Cell Surface - analysis
Solid-phase peptide synthesis
Staining and Labeling
Time Factors
Time-resolved luminescence
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Summary:Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)–DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0 M) prior to the luminescent enhancement step. [Nle 4, d-Phe 7]-α-melanocyte-stimulating hormone (NDP-α-MSH) labeled with Eu(III)–DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)–DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)–DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2009.10.031