Lee D C, Stenland C J, Hartwell R C, Ford E K, Cai K, Miller J L, Gilligan K J, Rubenstein R, Fournel M, Petteway S R
Department of Pathogen Safety Research/Biological Products, Bayer Corp., Research Triangle Park, NC 27709, USA.
J Virol Methods. 2000 Jan;84(1):77-89. doi: 10.1016/s0166-0934(99)00135-4.
Determining the risk of transmissible spongiform encephalopathy (TSE) transmission by blood or plasma-derived products requires sensitive and specific assays for the detection of either infectivity or a reliable marker for infectivity. To this end, a Western blot assay that is both sensitive and reproducible for the detection of PrP(RES), a marker for TSE infectivity, was developed. Using the 263K strain of TSE as a model system, the Western blot assay proved to be sensitive, specific and quantitative over a 3-4 log dynamic range. Compared to the rodent bioassay, the assay was shown to detect PrP(RES) down to approximately 10(3.4) IU/ml which is approximately 5-10 pg of PrP or approximately 10-20 ng brain equivalents. The Western blot was applied to monitor the partitioning of spiked PrP(Sc) through three plasma fractionation steps, cryoprecipitation, fraction I and fraction III, that are common to the purification of several human plasma-derived therapeutic products including albumin and immunoglobulins. The results from these studies demonstrated 1 log, 1 log and 4 logs of PrP(Sc) partitioning away from the effluent fraction for the cryoprecipitation, fraction I and fraction III steps, respectively.
确定血液或血浆衍生产品传播可传播性海绵状脑病（TSE）的风险，需要用于检测传染性或传染性可靠标志物的灵敏且特异的检测方法。为此，开发了一种对检测TSE传染性标志物PrP(RES)灵敏且可重复的蛋白质印迹检测法。以TSE的263K毒株作为模型系统，蛋白质印迹检测法在3至4个对数动态范围内被证明具有灵敏性、特异性和定量性。与啮齿动物生物测定法相比，该检测法能检测到低至约10(3.4) IU/ml的PrP(RES)，这大约是5至10 pg的PrP或约10至20 ng脑当量。蛋白质印迹法用于监测掺入的PrP(Sc)在三个血浆分级分离步骤（冷沉淀、I组分和III组分）中的分配情况，这三个步骤是包括白蛋白和免疫球蛋白在内的几种人血浆衍生治疗产品纯化过程中常见的。这些研究结果表明，在冷沉淀、I组分和III组分步骤中，PrP(Sc)分别从流出组分中分配出1个对数、1个对数和4个对数。
