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 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 (2.0M) prior to the luminescent enhancement step. [Nle(4),d-Phe(7)]-alpha-melanocyte-stimulating hormone (NDP-alpha-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.
Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA.
Channa R
CR
De Silva
Josef
J
Vagner
Ronald
R
Lynch
Robert J
RJ
Gillies
Victor J
VJ
Hruby
D000595:Amino Acid Sequence; D002460:Cell Line; D005063:Europium / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015200:Fluoroimmunoassay / Q000379:methods*; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008024:Ligands; D009074:Melanocyte-Stimulating Hormones / Q000737:chemistry / Q000378:metabolism; D043887:Receptor, Cholecystokinin B / Q000737:chemistry / Q000378:metabolism; D044105:Receptor, Melanocortin, Type 4 / Q000737:chemistry / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis*; D013194:Staining and Labeling; D013997:Time Factors
D005456:Fluorescent Dyes; D006573:Heterocyclic Compounds, 1-Ring; D028581:Lanthanoid Series Elements; D008024:Ligands; C470072:MC4R protein, human; D043887:Receptor, Cholecystokinin B; D044105:Receptor, Melanocortin, Type 4; D011956:Receptors, Cell Surface; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D005063:Europium; D009074:Melanocyte-Stimulating Hormones