Efficient propagation of variant Creutzfeldt-Jakob disease prion protein using the cell-protein misfolding cyclic amplification technique with samples containing plasma and heparin.
Efficient propagation of variant Creutzfeldt-Jakob disease prion protein using the cell-protein misfolding cyclic amplification technique with samples containing plasma and heparin.
复制标题
使用细胞蛋白错误折叠循环扩增技术，使用含有血浆和肝素的样品，有效繁殖克雅氏病朊病毒蛋白。
DOI：
10.1111/trf.13279
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Morita M.
中科院分区：
文献类型：
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作者：
Oshita M;Yokoyama T;Takei Y;Takeuchi A;Ironside JW;Kitamoto T;Morita M.
为了防止变异型克雅氏病（vCJD）通过血液制品或输血在人类之间的医源性传播，高度敏感的体外筛查试验是必要的。蛋白质错误折叠循环扩增（PMCA）就是这样一种候选测试。然而，据报道，血浆可抑制PMCA反应。因此，我们研究了在血浆存在下允许vCJD朊病毒扩增的细胞-PMCA条件。研究设计和方法通过加入不同终浓度的混合血浆、柠檬酸盐-磷酸盐-葡萄糖（CPD）、白蛋白、球蛋白或用离子交换剂处理的混合血浆，进行vCJD样品的细胞-PMCA。当肝素化细胞-PMCA中加入1%-50%的混合血浆时，扩增效率在低于1%和40%的最终血浆浓度下显示出双峰曲线，表明血浆中不仅含有PMCA抑制剂，而且还含有启动子。静脉注射球蛋白不抑制细胞-PMCA，但蛋白G结合部分。CPD、去白蛋白血浆和阴离子交换色谱的未结合部分抑制细胞-PMCA，但白蛋白和阳离子交换色谱的未结合部分不抑制。异常朊病毒蛋白在多轮细胞-PMCA的检测限，当维持在40%的最终血浆浓度在每一轮，是10 - 10稀释的vCJD脑samples. CONCLUSION我们已经建立了一个新的细胞-PMCA格式在血浆中没有任何预处理的存在下，其中vCJD朊病毒蛋白的扩增水平相当，发现没有血浆。我们的数据表明cell-PMCA作为vCJD朊病毒的实用血液检测的可行性。
BACKGROUNDTo prevent the iatrogenic spread of variant Creutzfeldt‐Jakob disease (vCJD) between humans via blood products or transfusion, highly sensitive in vitro screening tests are necessary. Protein misfolding cyclic amplification (PMCA) is one such candidate test. However, plasma has been reported to inhibit the PMCA reaction. Therefore, we investigated the cell‐PMCA conditions that permit vCJD prion amplification in the presence of plasma.STUDY DESIGN AND METHODSCell‐PMCA of vCJD samples was performed by adding various final concentrations of pooled plasma, citrate‐phosphate‐dextrose (CPD), albumin, globulin, or pooled plasma treated with ion exchangers. After heparin and plasma concentrations were optimized, multiround cell‐PMCA was performed.RESULTSWhen 1% to 50% of pooled plasma was added to heparinized cell‐PMCA, amplification efficiency showed a double‐peaked profile at less than 1% and 40% final plasma concentrations, indicating that plasma contains not only PMCA inhibitors but also promoters. Intravenous globulin did not inhibit cell‐PMCA, but the protein G–bound fraction did. CPD, albumin‐depleted plasma, and the unbound fraction of anion‐exchange chromatography inhibited cell‐PMCA, but albumin and the unbound fraction of the cation‐exchange chromatography did not. The detection limit of abnormal prion protein in multiround cell‐PMCA, when maintaining the final plasma concentration at 40% at each round, was 10−10dilutions of a vCJD brain specimen.CONCLUSIONWe have established a novel cell‐PMCA format in the presence of plasma without any pretreatment, where vCJD prion protein was amplified at comparable levels to that found without plasma. Our data suggest the feasibility of cell‐PMCA as a practical blood test for vCJD prions.