Prion disease blood test using immunoprecipitation and improved quaking-induced conversion.
Prion disease blood test using immunoprecipitation and improved quaking-induced conversion.
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作者：
Orrú CD;Wilham JM;Raymond LD;Kuhn F;Schroeder B;Raeber AJ;Caughey B
在医学、农业和野生动物生物学中管理传染性海绵状脑病（TSE）或朊病毒疾病的一个关键挑战是开发处于或低于感染水平的朊病毒的实用测试。特别感兴趣的是能够检测血液成分如血浆中朊病毒的测试，但血液通常具有极低的朊病毒浓度，并含有最敏感的朊病毒测试的抑制剂。后者的测试之一是震动诱导转化（QuIC），它可以像体内生物测定一样灵敏，但更快，更高的通量，更便宜。现在，我们已经将基于抗体15 B3的免疫沉淀与QuIC反应相结合，以提高灵敏度，并将朊病毒与血浆样品中的抑制剂分离。免疫沉淀和改进的实时QuIC反应的耦合显著增强了稀释到人血浆中的变异型克雅氏病（vCJD）脑组织的检测。稀释1014倍，每毫升含有约2阿托克（ag）的蛋白酶K-抗性朊病毒蛋白，很容易检测到，这表明vCJD脑的灵敏度比以前报道的高约10，000倍。我们还区分了羊瘙痒病感染和未感染仓鼠的血浆和血清样本，即使在早期临床前阶段。我们称之为“增强型QuIC”（eQuIC）的这种联合检测显著改善了组织、体液或环境样本中低水平朊病毒的常规检测前景。传染性海绵状脑病（TSEs）基本上无法治疗，并且在不可逆转的临床衰退或死亡之前很难明确诊断。TSE在物种内和物种间的传播性突出了即使是最小量的感染性也需要进行实际测试。已经报道了一些足够敏感的体外方法，但大多数方法都有很大的局限性，这将妨碍它们在常规诊断或筛查应用中的使用。我们的新检测方法改善了此类关键应用的前景。我们最初专注于血浆，因为朊病毒的实用血液检测对TSE诊断和降低风险特别有价值。特别是变异型克雅氏病（vCJD）通过输血在人类之间传播。增强的实时震动诱导转换（eQuIC）提供了迄今为止最灵敏的vCJD检测。15 B3抗体结合多个物种的朊病毒，这表明我们的测定可用于人类和动物的各种TSE的临床和基础研究。
A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 1014-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call “enhanced QuIC” (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples. Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eQuIC) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals.