Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment
Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment
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DOI：
10.1080/19336896.2016.1189053
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发表时间：
2016-01-01
期刊：
影响因子：
2.3
通讯作者：
Horiuchi, Motohiro
中科院分区：
文献类型：
--
作者：
Shan, Zhifu;Yamasaki, Takeshi;Horiuchi, Motohiro
朊病毒感染的细胞已被用于分析化合物对朊病毒蛋白异常同种型（PrPSc）形成的影响。PrPSc通常在通过蛋白酶K（PK）处理去除朊病毒蛋白（PrPC）的细胞同种型后使用抗朊病毒蛋白（PrP）抗体检测。然而，预期PK敏感性PrPSc（PrPSc-sen）（其具有比PK耐药PrPSc（PrPSc-res）更高的感染性和转化活性）也通过PK处理被消化。为了克服这一问题，我们建立了一种新的基于细胞的ELISA，其中PrPSc可以直接检测持续感染朊病毒的细胞，使用抗PrP单克隆抗体（mAb）132，识别由小鼠PrP氨基酸119-127组成的表位。新的基于细胞的ELISA可以区分朊病毒感染的细胞与朊病毒未感染的细胞，而无需细胞裂解和PK处理。MAb 132可同时检测PrPSc-sen和PrPSc-res，即使所有PrPSc分子均未被检测到。PrPSc检测的分析动态范围约为1 log。变异系数和信号背景比分别为7%-11%和2.5-3.3，证明了该测定法的重现性。在PrPSc检测前立即增加细胞毒性试验不影响随后的PrPSc检测。因此，包括细胞培养、细胞毒性测定和PrPSc检测在内的所有程序均在同一平板中完成。简单且不需要细胞裂解或PK处理是抗朊病毒化合物高通量筛选的优势。
Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrPSc). PrPSc is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrPC) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrPSc (PrPSc-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrPSc (PrPSc-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrPSc can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrPSc-sen and PrPSc-res even if all PrPSc molecules were not detected. The analytical dynamic range for PrPSc detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrPSc detection did not affect the following PrPSc detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrPSc detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.