Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease
Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease
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DOI：
10.1186/1471-2202-11-124
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发表时间：
2010-10-05
期刊：
影响因子：
2.4
通讯作者：
Landegren, Ulf
中科院分区：
文献类型：
--
作者：
Kamali-Moghaddam, Masood;Pettersson, Frida Ekholm;Landegren, Ulf
背景：蛋白质聚集在多种神经退行性疾病中起着重要作用。例如，磷酸化tau和Aβ多肽的不可溶聚集体是阿尔茨海默病病理的基石。因此，可溶性蛋白聚集体是潜在的诊断和预后相关疾病的生物标记物。检测聚集的物种需要灵敏的工具来有效地将它们与相同蛋白质的单体区分开来。在这里，我们建立了一种通过同时识别聚集体中存在的三个相同的决定因素来特异和灵敏地检测Aβ原纤维的邻近连接试验(PLA)。聚乳酸是一种多功能技术，它结合了多靶点识别的要求和将信号从被检测的靶分子转化为可扩增的DNA链的能力，提供了非常高的特异性和灵敏度。结果：对于Aβ原纤维的特异性检测，我们使用了对改良的聚乳酸中的Aβ原纤维具有选择性的单抗mAb158，其中相同的单抗用于检测所需的三类亲和试剂。这些试剂用于固相反应中可溶抗体聚集体的检测，仅可检测0.1pg/mlAβ原纤维，动态范围大于六个数量级。与相同抗体的夹心ELISA法相比，聚乳酸将抗体原纤维检测的敏感度提高了25倍。结论：邻近连接法是一种通用的蛋白质分析技术，可以提供对Aβ聚集物的高灵敏度和特异性的检测，并暗示其他与阿尔茨海默病和其他神经退行性疾病相关的蛋白质聚集物。
Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A beta peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A beta protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.Results: For specific detection of A beta protofibrils we have used a monoclonal antibody, mAb158, selective for A beta protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Ab aggregates in solid- phase reactions, allowing detection of just 0.1 pg/ml A beta protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Ab protofibril detection by up to 25- fold. The assay was used to measure soluble Ab aggregates in brain homogenates from mice transgenic for a human allele predisposing to A beta aggregation.Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of A beta aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.