Development of a lanthanide-based assay for detection of receptor-ligand interactions at the delta-opioid receptor.
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 five peptide ligands for the delta-opioid receptor that were modified with a europium (Eu)-containing chelate. These labeled ligands were tested for their binding affinities and compared with the unlabeled parental ligands. The Eu-diethylenetriaminepentaacetic acid (DTPA)-[D-Pen(2),l-Cys(5)] enkephalin (DPLCE) ligand bound to Chinese hamster ovary (CHO) cells overexpressing the human delta-opioid receptor with affinity similar to the unlabeled ligand. This ligand was used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. These lanthanide-based assays provide superior results with higher throughput and eliminate the need for radioactive waste disposal; hence, they are appropriate for high-throughput screening of ligand libraries.
Handl
Heather L
HL
Department of Biochemistry and Biophysics, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA.
Vagner
Josef
J
Yamamura
Henry I
HI
Hruby
Victor J
VJ
Gillies
Robert J
RJ
D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D004745:Enkephalins / Q000737:chemistry; D005063:Europium / Q000737:chemistry; D005453:Fluorescence; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008024:Ligands; D009842:Oligopeptides / Q000138:chemical synthesis; D010455:Peptides / Q000737:chemistry; D017465:Receptors, Opioid, delta / Q000737:chemistry*; D013997:Time Factors
D004745:Enkephalins; D028581:Lanthanoid Series Elements; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D017465:Receptors, Opioid, delta; C060209:deltorphin II, Ala(2)-; D005063:Europium