Lanthanide-based time-resolved fluorescence of in cyto ligand-receptor interactions

Heather L. Handl, Josef Vagner, Henry I. Yamamura, Victor J. Hruby, Robert J. Gillies

Research output: Contribution to journalArticle

57 Scopus citations

Abstract

A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested peptide ligands modified with an Eu-DTPA chelate. These labeled ligands were used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. The sensitivity of time-resolved fluorescence is sufficient to detect attomoles of europium, allowing assays in 96-well plates, compared with 30-mm dishes for 125I binding assays to whole cells. We verified binding of Eu-DTPA-NDP-α-MSH to cells overexpressing the human melanocortin-4 receptor. The Eu-labeled ligand bound to these cells with an affinity similar to that of unlabeled NDP-α-MSH and was used to optimize a competitive binding assay. The lanthanide-based assays provided superior results with higher throughput and eliminated the need for radioactive waste disposal. This assay is appropriate for high-throughput screening of ligand libraries.

Original languageEnglish (US)
Pages (from-to)242-250
Number of pages9
JournalAnalytical Biochemistry
Volume330
Issue number2
DOIs
StatePublished - Jul 15 2004

Keywords

  • Binding assay
  • DELFIA
  • Lanthanide
  • Nonspecific binding
  • Time-resolved fluorescence

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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