High throughput screening of β-amyloid secretion inhibitors using homogenous time-resolved fluorescence
High throughput screening of β-amyloid secretion inhibitors using homogenous time-resolved fluorescence
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DOI：
10.2174/1386207043328256
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发表时间：
2004-12-01
影响因子：
1.8
通讯作者：
Imbimbo, BP
中科院分区：
文献类型：
--
作者：
Albrecht, H;Zbinden, P;Imbimbo, BP
已经开发了一种使用均匀时间分辨荧光的基于细胞的测定方法，用于高通量筛选推定的β-淀粉样蛋白（Abeta）产生抑制剂。在该测定中，通过简单地添加两种市售抗体复合物来检测总Abeta。第一种是生物素化的单克隆抗体（4G 8），其特异性识别包含A β肽的残基17-24的表位，与铕穴状化合物-链霉亲和素缀合物复合。第二种是多克隆抗体（BioS-N），针对的是Abeta肽的N-末端，与别藻蓝蛋白-抗兔抗体缀合物复合。两种复合物与Abeta肽的结合使铕穴状化合物（荧光供体）和别藻蓝蛋白（荧光受体）紧密接近，因此在337 nm激发时产生荧光共振能量转移信号。然后使用Discovery（TM）或ViewLux（TM）读数器检测所得荧光信号（665 nm）。通过所提出的方法检测Abeta在约1 nM的浓度下是可能的。该方法用于检测从稳定转染的人神经胶质瘤细胞系（H4）分泌的Abeta，该细胞系过度表达突变形式的人淀粉样蛋白前体蛋白（APP 695 NL），并开发用于机器人自动化。在优化的条件下，在384孔格式中实现了超过5的信号背景比和约0.7的Z'因子。对56，913种潜在的Abeta产生抑制剂的高通量筛选导致鉴定出新的非细胞毒性和细胞渗透性化合物，其效力在亚微摩尔范围内。
A cell-based assay using homogeneous time-resolved fluorescence has been developed for high throughput screening of putative beta-amyloid (Abeta)production inhibitors. In this assay, total Abeta is detected by simply adding two commercially available antibody complexes. The first was a biotinylated monoclonal antibody (4G8), specifically recognizing an epitope comprising the residues 17-24 of the Abeta peptide, complexed with europium cryptate-streptavidin conjugate. The second was a polyclonal antibody (BioS-N), raised against the N-terminus of the Abeta peptide, complexed with an allophycocyanin-anti rabbit antibody conjugate. Binding of the two complexes to the Abeta peptide brought europium cryptate (fluorescence donor) and allophycocyanin (fluorescence acceptor) into close proximity, consequently a fluorescent resonance energy transfer signal was produced upon excitation at 337 nm. The resulting fluorescence signal (665 nm) was then detected using a Discovery(TM) or a ViewLux(TM) reader. Detection of Abeta by the proposed method is possible at concentrations of approximately 1 nM. The method was employed for the detection of Abeta secreted from a stable transfected human neuroglioma cell line (H4) overexpressing a mutated form of the human amyloid precursor protein (APP695NL) and developed for robotic automation. At optimized conditions, signal-to-background ratios exceeding 5 and Z' factors around 0.7 were achieved in a 384-well format. High throughput screening of 56,913 potential Abeta production inhibitors led to identification of new non-cytotoxic and cell permeable compounds with potencies in the submicromolar range.