Mahal Sukhvir P, Demczyk Cheryl A, Smith Emery W, Klohn Peter-Christian, Weissmann Charles
Department of Infectology, Scripps Florida, Jupiter, FL, USA.
Methods Mol Biol. 2008;459:49-68. doi: 10.1007/978-1-59745-234-2_4.
Prions are usually quantified by bioassays based on intracerebral inoculation of animals, which are slow, imprecise, and costly. We have developed a cell-based prion assay that is based on the isolation of cell lines highly susceptible to certain strains (Rocky Mountain Laboratory and 22L) of mouse prions and a method for identifying individual, prion-infected cells and quantifying them. In the standard scrapie cell assay (SSCA), susceptible cells are exposed to prion-containing samples for 4 days, grown to confluence, passaged two or three times, and the proportion of rPrP(Sc)-containing cells is determined with automated counting equipment. The dose response is dynamic over 2 logs of prion concentrations. The SSCA has a standard error of +/-20-30%, is as sensitive as the mouse bioassay, 10 times faster, at least 2 orders of magnitude less expensive, and it is suitable for robotization. Assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar.
朊病毒通常通过基于动物脑内接种的生物测定法进行定量，这种方法缓慢、不精确且成本高昂。我们开发了一种基于细胞的朊病毒测定法，该方法基于分离对某些小鼠朊病毒株（落基山实验室株和22L株）高度敏感的细胞系，以及一种识别单个朊病毒感染细胞并对其进行定量的方法。在标准羊瘙痒病细胞测定法（SSCA）中，将敏感细胞暴露于含朊病毒的样品中4天，使其生长至汇合，传代两到三次，然后用自动计数设备测定含rPrP(Sc)细胞的比例。剂量反应在朊病毒浓度的2个对数范围内呈动态变化。SSCA的标准误差为±20 - 30%，与小鼠生物测定法一样灵敏，速度快10倍，成本至少低2个数量级，并且适合自动化操作。以更耗时的终点滴定形式进行的测定提高了灵敏度，结果表明在组织培养和小鼠中测得的感染性滴度相似。
