Deduction of the evaluation limit and termination timing ofmulti-round protein misfolding cyclic amplification from a titration curve
Deduction of the evaluation limit and termination timing ofmulti-round protein misfolding cyclic amplification from a titration curve
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从滴定曲线推导多轮蛋白质错误折叠循环扩增的评价限和终止时机
DOI：
10.1111/j.1348-0421.2011.00340.x
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发表时间：
2011
影响因子：
2.6
通讯作者：
Morita M
中科院分区：
文献类型：
--
作者：
Takeuchi A;Komiya M;Kitamoto T;Morita M
在本研究中，通过多轮蛋白错误折叠循环扩增（PMCA）技术评估了消毒剂降低部分蛋白酶抗性朊病毒异构体的效果。感染痒病衍生菌株263K的仓鼠大脑均质化，在灭活或模拟条件下处理，并进行多轮PMCA。分析了四组连续10倍稀释的模拟处理样品。尽管在信号模式中观察到相当大的变异性，但在第二轮和第六轮之间，PMCA轮的数量与模拟处理的感染大脑的平均稀释系数呈线性相关，对应于3.8-7.3 log的对数减少因子（LRF）。在正常仓鼠脑匀浆扩增的PMCA产物中未观察到信号。在1 M和2 M NaOH处理的样品中，信号首次出现时的平均轮数分别为4.33和4。用线性回归线作为滴定曲线，各消毒剂的lrf分别为6.1和5.8 log；这些数值无显著差异。碱性清洁剂和十二烷基硫酸钠的平均循环次数分别为9次和10.33次，均不在线性回归线和评价限的范围内。lrf≥7.3 log，认为消毒剂非常有效。这些估计与先前基于生物测定的报告一致。因此，评价限在消毒剂评价和工艺验证等多轮PMCA的一些应用中似乎是有价值的。
In this study, the efficacy of disinfectants in reducing the partially protease‐resistant isoform of prion protein was evaluated by a multi‐round protein misfolding cyclic amplification (PMCA) technique. Hamster brains infected with scrapie‐derived strain 263K were homogenized, treated under inactivating or mock conditions, and subjected to multi‐round PMCA. Four sets of serial 10‐fold dilutions of mock‐treated samples were analyzed. Although considerable variability was observed in the signal patterns, between the second and sixth rounds the number of the PMCA round correlated in a linear fashion with the mean dilution factor of mock‐treated, infected brains, corresponding to a log reduction factor (LRF) of 3.8–7.3 log. No signals were observed in the PMCA products amplified from normal hamster brain homogenates. The mean numbers of rounds at the first appearance of the signal for 1 M and 2 M NaOH‐treated samples were 4.33 and 4, respectively. Using the linear regression line as the titration curve, the LRFs of these disinfectants were found to be 6.1 and 5.8 log, respectively; these values were not significantly different. The mean number of rounds for the alkaline cleaner and sodium dodecyl sulfate were 9 and 10.33, respectively, and were outside the range of both the linear regression line and evaluation limit. The disinfectants were considered very effective because their LRFs were ≥7.3 log. These estimations were concordant with previous bioassay‐based reports. Thus, the evaluation limit seems to be valuable in some applications of multi‐round PMCA, such as disinfectant assessment and process validation.