Optimization of time-resolved fluorescence assay for detection of europium–tetraazacyclododecyltetraacetic acid-labeled ligand–receptor interactions
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- 作者：Channa R. De Silva ; Josef Vagner ; Ronald Lynch ; Robert J. Gillies ; Victor J. Hruby
- 关键词：DELFIA ; Lanthanide ; DOTA derivatives ; Time-resolved luminescence ; Binding assay ; Solid-phase peptide synthesis ; Melanocortin receptor
- 刊名：Analytical Biochemistry
- 出版年：2010
- 出版时间：1 March 2010
- 年：2010
- 卷：398
- 期：1
- 页码：15-23
- 全文大小：576 K
文摘
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.