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

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.