Detection and Quantification of CWD Prions in Fixed Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion
Detection and Quantification of CWD Prions in Fixed Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion
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DOI：
10.1038/srep25098
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发表时间：
2016-05-09
影响因子：
4.6
通讯作者：
Hoover, Edward A.
中科院分区：
文献类型：
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作者：
Hoover, Clare E.;Davenport, Kristen A.;Hoover, Edward A.
慢性消耗性疾病（CWD）的传统诊断检测依赖于通过蛋白质印迹、ELISA或免疫组织化学（IHC）对错误折叠的CWD朊蛋白（PrPCWD）的免疫检测。这些技术需要单独的样本采集（冷冻和固定），这可能会导致差异，因为朊病毒组织分布和检测灵敏度的变化限制了检测，特别是在早期和亚临床感染中。在这里，我们利用实时震动诱导转换（RT-QuIC）的力量来放大，检测和量化固定石蜡包埋（FPE）组织切片中朊病毒淀粉样蛋白的接种活性。我们表明，FPE RT-QuIC在PrPCWD负荷较低的组织（包括IHC阴性组织）中具有比IHC更高的检测灵敏度。我们还采用淀粉样蛋白形成动力学产生一个半定量估计朊病毒浓度在一个给定的FPE组织。我们报告说，FPE RT-QuIC有能力加强对朊病毒和其他潜在蛋白质错误折叠疾病状态中疾病相关PrPRES的诊断和调查检测。
Traditional diagnostic detection of chronic wasting disease (CWD) relies on immunodetection of misfolded CWD prion protein (PrPCWD) by western blotting, ELISA, or immunohistochemistry (IHC). These techniques require separate sample collections (frozen and fixed) which may result in discrepancies due to variation in prion tissue distribution and assay sensitivities that limit detection especially in early and subclinical infections. Here, we harness the power of real-time quaking induced conversion (RT-QuIC) to amplify, detect, and quantify prion amyloid seeding activity in fixed paraffin-embedded (FPE) tissue sections. We show that FPE RT-QuIC has greater detection sensitivity than IHC in tissues with low PrPCWD burdens, including those that are IHC-negative. We also employ amyloid formation kinetics to yield a semi-quantitative estimate of prion concentration in a given FPE tissue. We report that FPE RT-QuIC has the ability to enhance diagnostic and investigative detection of disease-associated PrPRES in prion, and potentially other, protein misfolding disease states.