Biotin-avidin interaction-based screening assay for Alzheimer's β-peptide oligomer inhibitors
Biotin-avidin interaction-based screening assay for Alzheimer's β-peptide oligomer inhibitors
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DOI：
10.1016/j.ab.2006.04.036
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发表时间：
2006-09-15
影响因子：
2.9
通讯作者：
LeVine, Harry, III
中科院分区：
文献类型：
--
作者：
LeVine, Harry, III
病理性错误折叠蛋白质（如阿尔茨海默氏β-肽（A β））的寡聚体形成抑制剂的体外测试受到缺乏适当灵敏的高通量方法测量寡聚体的限制。即使开发了寡聚体特异性抗体和单位点抗体测定，也需要多种对照来排除由于化合物与抗体识别的肽上的表位或与抗体本身的相互作用而导致的假阳性，并且免疫试剂是昂贵的。用于测量亚纳摩尔浓度的阿尔茨海默氏β-肽残基1-42 [A β（1-42）]寡聚体的非放射性非免疫学方法，该方法结合了生物素-抗生物素蛋白相互作用，该方法一直是筛选测定的主力，在此应用于单位点中性抗生物素蛋白捕获/标记的链霉抗生物素蛋白检测配置，以特异性地识别多聚体（> 20 kDa）N-α-生物素基-A β（1-42）（bio-A β 42）的寡聚体，但不是单体bio-A β 42。生物素与中性亲和素和链霉亲和素相互作用的高亲和力和特异性排除了不含生物素化合物的干扰。所述试剂是廉价的，并且可以应用于任何错误折叠/寡聚肽或蛋白质，其可以在单个位点被生物素化。(c)2006年爱思唯尔公司All rights reserved.
In vitro testing for inhibitors of oligomer formation of pathologically misfolded proteins such as Alzheimer's beta-peptide (A beta) has been limited by the lack of a suitably sensitive high-throughput method for measuring oligomers. Even with the development of oligomer-specific antibodies and a single-site antibody assay, there are multiple controls required to rule out false positives due to compound interactions with the epitopes on the peptide that are recognized by the antibodies or with the antibodies themselves, and the immunoreagents are expensive. A non-radioactive non-immunological method for the measurement of subnanomolar concentrations of Alzheimer's beta-peptide residues 1-42 [A beta(1-42)] oligomers incorporating the biotin-avidin interaction that has been a workhorse for screening assays is applied here in a single-site NeutrAvidin capture/labeled streptavidin detection configuration to specifically recognize multimeric (> 20 kDa) oligomers of N-alpha-biotinyl-A beta(1-42) (bio-A beta 42) but not monomeric bio-A beta 42. The high affinity and specificity of the biotin interaction with NeutrAvidin and streptavidin obviate interference by non-biotin-containing compounds. The reagents are inexpensive and can be applied to any misfolding/oligomerizing peptide or protein that can be biotinylated at a single site. (c) 2006 Elsevier Inc. All rights reserved.