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

Channa R. De Silva, Josef Vagner, Ronald Lynch, Robert J. Gillies, Victor J. Hruby

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

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. [Nle4,d-Phe7]-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.

Original languageEnglish (US)
Pages (from-to)15-23
Number of pages9
JournalAnalytical Biochemistry
Volume398
Issue number1
DOIs
StatePublished - Mar 1 2010

Keywords

  • Binding assay
  • DELFIA
  • DOTA derivatives
  • Lanthanide
  • Melanocortin receptor
  • Solid-phase peptide synthesis
  • Time-resolved luminescence

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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