Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions.
Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions.
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DOI：
10.1016/j.ab.2009.10.031
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发表时间：
2010-03-01
影响因子：
2.9
通讯作者：
Hruby VJ
中科院分区：
文献类型：
--
作者：
De Silva CR;Vagner J;Lynch R;Gillies RJ;Hruby VJ
基于镧系元素的发光配体结合测定上级传统的放射性标记测定，这是由于在高通量筛选中提高了灵敏度和可负担性，同时消除了放射性的使用。尽管使用镧系元素（III）配位的螯合剂如DTPA衍生物取得了显著进展，但由于镧系元素（III）离子从络合物中的不完全释放，解离增强的镧系元素荧光免疫测定（DELFIA）尚未成功地与更稳定的螯合剂如DOTA衍生物一起使用。在这里，一个修改和优化的DELFIA程序纳入酸处理协议被引入用于与Eu（III）-DOTA标记的肽。在发光增强步骤之前，使用盐酸（2.0M）观察到Eu（III）离子从DOTA标记的配体的完全释放。合成了Eu（III）-DOTA标记的NDP-α-MSH，并使用改进的方案评估了与过表达人黑素皮质素-4受体（hMC 4 R）的细胞的结合亲和力。结合数据表明Eu（III）-DOTA连接的肽以与其DTPA类似物类似的亲和力结合至这些细胞。改良的DELFIA程序进一步用于监测Eu（III）-DOTA标记的异二价肽与表达hMC 4 R和CCK-2（胆囊收缩素）受体两者的细胞的结合。改进的测定法提供了上级结果，并且适合于配体文库的高通量筛选。
Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improved sensitivity and affordability in high throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as DTPA derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIA) have not yet been successfully used with more stable chelators, e.g. DOTA derivatives, due to the incomplete release of lanthanide(III) ions from the complex. Here, a modified and an 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. NDP-α-MSH labeled with Eu(III)-DOTA was synthesized and the binding affinity to cells overexpressing the human melanocortin-4 receptors (hMC4R) 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 hMC4R and CCK-2 (Cholecystokinin) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries.
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DOI：
10.1073/pnas.77.10.5754
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
SAWYER, TK;SANFILIPPO, PJ;HADLEY, ME
通讯作者：
HADLEY, ME
影响因子：
4.3
作者：
Comby, Steve;Imbert, Daniel;Bunzli, Jean-Claude G.
通讯作者：
Bunzli, Jean-Claude G.
影响因子：
--
作者：
Kashem, Mohammed A.;Nelson, Richard M.;Jakes, Scott
通讯作者：
Jakes, Scott
影响因子：
2.3
作者：
Kovacs, Z.;De Leon-Rodriguez, L. M.
通讯作者：
De Leon-Rodriguez, L. M.
影响因子：
2.2
作者：
Wilkinson, D
通讯作者：
Wilkinson, D