Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.
Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.
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DOI：
10.1371/journal.ppat.1001217
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发表时间：
2010-12-02
期刊：
影响因子：
6.7
通讯作者：
Caughey B
中科院分区：
文献类型：
--
作者：
Wilham JM;Orrú CD;Bessen RA;Atarashi R;Sano K;Race B;Meade-White KD;Taubner LM;Timmes A;Caughey B
有效诊断和管理朊病毒疾病的一个主要问题是缺乏快速高通量测定法来测量低水平的朊病毒。此类测量通常需要在动物身上进行长时间的生物测定。基于朊病毒将通常可溶性蛋白酶敏感形式的朊病毒蛋白转化为蛋白酶抗性和/或淀粉样纤维形式的能力，已经开发了高度灵敏但通常是非定量的朊病毒检测方法。在这里，我们描述了一种使用称为实时震动诱导转化测定（RT-QuIC）的新朊病毒播种测定来估计朊病毒相对数量的方法。基础反应混合了先前描述的振动诱导转化（QuIC）和淀粉样蛋白播种测定（ASA）方法的各个方面，并涉及细菌表达的重组 PrPC 的富含 α 螺旋形式的朊病毒种子转化为富含 β 折叠的淀粉样蛋白原纤维形式。 RT-QuIC 与动物生物测定一样灵敏，但可以在 2 天或更短的时间内完成。与终点稀释动物生物测定类似，该方法涉及测试样品的系列稀释液并统计估计接种剂量 (SD)，在 50% 的重复反应 (SD50) 中给出阳性反应。 263K 受瘙痒病影响的仓鼠的脑组织的 SD50 值为 1011-1012/g，使得 RT-QuIC 的敏感性与终点稀释生物测定相似。对患有传染性水貂脑病的仓鼠鼻腔灌洗液的生物测定阳性分析得出 SD50 值为 103.5-105.7/ml，表明鼻腔释放大量可快速检测到的朊病毒感染性。 263K 受瘙痒病影响的仓鼠的脑脊液中含有朊病毒 SD50 值为 102.0–102.9/ml。 RT-QuIC 测定还区分了鹿慢性消耗性疾病和羊痒病脑样本与正常对照样本。原则上，终点稀释定量可应用于多种类型的朊病毒和淀粉样蛋白接种测定。终点稀释 RT-QuIC 提供了一种灵敏、快速、定量和高通量的朊病毒接种活性测定方法。朊病毒疾病是哺乳动物致命的传染性神经退行性疾病，涉及宿主朊病毒蛋白的错误折叠。为了更好地管理这些疾病，我们需要能够检测和量化生物样本中的传染性颗粒或朊病毒。然而，当前的测试缺乏医学、农业、野生动物生物学和研究领域许多重要应用所需的灵敏度、速度和/或定量能力。为了解决这个问题，我们开发了一种高度灵敏、快速且定量的新型朊病毒检测方法。该测定利用微量传染性朊病毒在试管反应中播种大量过量正常朊病毒蛋白的错误折叠的能力。通过测试一系列样品稀释度并确定接种活性的损失（即终点稀释度）来实现定量。类似的分析长期以来一直用于通过接种动物来量化朊病毒。然而，此类生物测定需要数月或数年的时间才能进行，并且需要大量动物且昂贵。我们的新方法提供了一种更实用的检测和量化朊病毒的方法。到目前为止，我们已将这种检测方法应用于绵羊、鹿和仓鼠的朊病毒，并发现受感染仓鼠的鼻腔和脑脊液中的朊病毒含量高得惊人。
A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrPC to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD50). Brain tissue from 263K scrapie-affected hamsters gave SD50 values of 1011-1012/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD50 values of 103.5–105.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD50 values of 102.0–102.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity. Prion diseases are deadly infectious neurodegenerative disorders of mammals which involve the misfolding of host prion protein. To better manage these diseases, we need to be able to detect and quantify the infectious particles, or prions, in biological samples. However, current tests lack the sensitivity, speed and/or quantitative capabilities required for many important applications in medicine, agriculture, wildlife biology and research. To address this problem, we have developed a new prion assay that is highly sensitive, rapid, and quantitative. This assay takes advantage of the ability of miniscule amounts of infectious prions to seed the misfolding of large excesses of normal prion protein in test tube reactions. Quantitation is achieved by testing a range of sample dilutions and determining loss of seeding activity, i.e. the end-point dilution. Similar analyses have long been used to quantify prions by inoculation into animals; however, such bioassays take months or years to perform and are both animal-intensive and expensive. Our new method provides a more practical means of detecting and quantifying prions. So far, we have applied this assay to prions from sheep, deer, and hamsters, and have found surprisingly high levels of prions in the nasal and cerebral spinal fluids of infected hamsters.
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在培养细胞中的合成和拓扑的动力学方面，crap和细胞prion蛋白不同。
DOI：
10.1083/jcb.110.3.743
发表时间：
1990-03
期刊：
The Journal of cell biology
影响因子：
--
作者：
Borchelt DR;Scott M;Taraboulos A;Stahl N;Prusiner SB
通讯作者：
Prusiner SB
影响因子：
2.6
作者：
Konold, Timm;Moore, S. Jo;Bellworthy, Susan J.;Simmons, Hugh A.
通讯作者：
Simmons, Hugh A.
影响因子：
6.7
作者：
Colby DW;Wain R;Baskakov IV;Legname G;Palmer CG;Nguyen HO;Lemus A;Cohen FE;DeArmond SJ;Prusiner SB
通讯作者：
Prusiner SB
影响因子：
4.8
作者：
Callahan, NA;Xiong, LW;Caughey, B
通讯作者：
Caughey, B
影响因子：
48
作者：
Atarashi, Ryuichiro;Moore, Roger A.;Caughey, Byron
通讯作者：
Caughey, Byron