Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets
Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets
复制标题
DOI：
10.1073/pnas.97.10.5468
复制
发表时间：
2000-05-09
影响因子：
11.1
通讯作者：
Kretzschmar, H
中科院分区：
文献类型：
--
作者：
Bieschke, J;Giese, A;Kretzschmar, H
对克雅氏病(CJD)等普恩病毒疾病的明确诊断依赖于病理性普恩蛋白(PrPSc)的检测。然而，到目前为止，还没有检测脑脊液中PrPSc的方法。基于一种适用于单分子检测的共聚焦双色荧光相关光谱的建立，我们发展了一种高灵敏的PrPSc检测方法。用荧光染料标记的特异性抗体探针标记病理性的Prion蛋白聚集体，产生强烈的荧光靶标，在共聚焦扫描装置中通过双色荧光强度分布分析来测量这些靶标。在诊断模型系统中，PrPSc聚集体被检测到2 PM的PrPSc浓度，对应于大约2fM的聚集体浓度，这比蛋白质印迹分析的灵敏度高一个数量级以上。在CJD患者的一些脑脊液样本中也可以检测到PrPSc特异性信号，但在对照样本中检测不到，这为CJD和其他普恩病毒疾病的快速和特异性检测提供了基础。此外，该方法可用于其他疾病相关淀粉样聚集体的敏感检测，如阿尔茨海默病。
A definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) relies on the detection of pathological prion protein (PrPSc). However, no test for PrPSc in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule detection, we developed a highly sensitive detection method for PrPSc. Pathological prion protein aggregates were labeled by specific antibody probes tagged with fluorescent dyes, resulting in intensely fluorescent targets, which were measured by dual-color fluorescence intensity distribution analysis in a confocal scanning setup. In a diagnostic model system, PrPSc aggregates were detected down to a concentration of 2 pM PrPSc, corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sensitive than Western blot analysis. A PrPSc-specific signal could also be detected in a number of CSF samples from patients with CJD but not in control samples, providing the basis for a rapid and specific test for CJD and other prion diseases. Furthermore, this method could be adapted to the sensitive detection of other disease-associated amyloid aggregates such as in Alzheimer's disease.