Lanthanide-based time-resolved fluorescence of in cyto ligand-receptor interactions
Lanthanide-based time-resolved fluorescence of in cyto ligand-receptor interactions
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DOI：
10.1016/j.ab.2004.04.012
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发表时间：
2004-07-15
影响因子：
2.9
通讯作者：
Gillies, RJ
中科院分区：
文献类型：
--
作者：
Handl, HL;Vagner, J;Gillies, RJ
镧系元素为基础的配体-受体相互作用的测定提供了一个有吸引力的替代传统的放射性标记的测定灵敏度，吞吐量和生物危害。我们设计并测试了用Eu-DTPA螯合物修饰的肽配体。将这些标记的配体用于竞争性结合测定，其结果与使用传统放射性标记的结合测定获得的结果相当。时间分辨荧光的灵敏度足以检测到阿托摩尔的铕，允许在96孔板中进行测定，与30 mm培养皿相比，I-125结合测定全细胞。我们验证了Eu-DTPA-NDP-α-MSH与过表达人黑皮质素-4受体的细胞的结合。Eu标记的配体以与未标记的NDP-α-MSH相似的亲和力结合到这些细胞上，并用于优化竞争性结合测定。基于镧系元素的测定提供了具有更高通量的上级结果，并且消除了对放射性废物处置的需要。该测定法适用于配体文库的高通量筛选。(C)2004年爱思唯尔公司All rights reserved.
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 I-125 binding assays to whole cells. We verified binding of Eu-DTPA-NDP-alpha-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-alpha-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. (C) 2004 Elsevier Inc. All rights reserved.