Prion detection by an amyloid seeding assay
Prion detection by an amyloid seeding assay
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DOI：
10.1073/pnas.0710152105
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发表时间：
2007-12-26
影响因子：
11.1
通讯作者：
Prusiner, Stanley B.
中科院分区：
文献类型：
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作者：
Colby, David W.;Zhang, Qiang;Prusiner, Stanley B.
重组朊病毒蛋白（recPrP），这是在细菌中产生的，聚合成淀粉样纤维，伴随着获得朊病毒感染性。我们在这里报告，部分纯化的朊病毒制剂种子聚合recPrP成淀粉样蛋白检测到的荧光位移的染料硫磺素T。我们的淀粉样蛋白播种试验（阿萨）检测PrP（sc），朊病毒的唯一组成部分，在大脑样本中，从人类散发性克雅氏病，以及在啮齿动物与实验朊病毒疾病。阿萨检测到了在小鼠和仓鼠体内传代的各种朊病毒株。阿萨的灵敏度随菌株类型而变化;对于仓鼠Sc237朊病毒，检测限接近1 fg。一些朊病毒菌株主要由蛋白酶敏感的PrPsc（sPrP（sc））组成，这些菌株很容易被阿萨检测到。我们的研究表明，阿萨提供了一种替代方法，用于检测sPrP（SC）和蛋白酶耐药PrP（SC），不依赖于蛋白酶消化或免疫检测。
Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrP(sc), the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt-Jakob disease, as well as in rodents with experimental prion disease. The ASA detected a variety of prion strains passaged in both mice and hamsters. The sensitivity of the ASA varied with strain type; for hamster Sc237 prions, the limit of detection was approximate to 1 fg. Some prion strains consist largely of protease-sensitive PrPsc (sPrP(sc)), and these strains were readily detected by ASA. Our studies show that the ASA provides an alternative methodology for detecting both sPrP(sc) and protease-resistant PrP(sc) that does not rely on protease digestion or immunodetection.