Bauman P A, Lawrence L A, Biesert L, Dichtelmüller H, Fabbrizzi F, Gajardo R, Gröner A, Jorquera J I, Kempf C, Kreil T R, von Hoegen I, Pifat D Y, Petteway S R, Cai K
Talecris Biotherapeutics, Research Triangle Park, NC, USA.
Vox Sang. 2006 Jul;91(1):34-40. doi: 10.1111/j.1423-0410.2006.00790.x.
Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by aberrantly folded cellular proteins (PrP(Sc); prions) that are generally resistant to conventional pathogen-inactivation techniques. To ensure effective decontamination and inactivation of prions that could be present in source material, we investigated critical factors that influence prion inactivation by NaOH.
A decrease in prion infectivity correlates with the disappearance of the protease-resistant core of PrPSc (PrPRES) observed in biochemical assays. To model prion inactivation, hamster scrapie (strain 263K) brain homogenate (SBH) was incubated for specific periods of time in 0.1 m NaOH at 4 or 18 degrees C, with or without detergent. Neutralized samples were subjected to limited digestion with proteinase K (PK) and then analysed using an endpoint dilution western blot assay and antibody 3F4. Structural changes in prions exposed to NaOH were examined using differential immunoprecipitation.
Treatment of SBH with 0.1 m NaOH for 15 min, in the absence of detergent, at 4 and 18 degrees C caused a reduction in the PrP(RES) signal of 3.5 and 4.0 log10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60-min incubation in NaOH further enhanced PrPRES reduction to > or = 4.5 log10 units (i.e. below the limit of detection). NaOH treatment induced conformational changes in PrP that resulted in the exposure of a hidden epitope and enabled prion immunoprecipitation by antibody 3F4.
The use of NaOH can effectively reduce prion levels in an in vitro inactivation assay. After pretreatment of SBH with detergent, NaOH completely eliminates the PrPRES signal. Detergent may liberate lipid membrane-protected PrPSc to improve access to NaOH, which can then inactivate PrPSc by altering its structure. In cases of unidentified exposure to PrPSc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.
传染性海绵状脑病（TSEs）是由异常折叠的细胞蛋白（PrP(Sc)；朊病毒）引起的致命性神经退行性疾病，这些蛋白通常对传统的病原体灭活技术具有抗性。为确保对源材料中可能存在的朊病毒进行有效去污和灭活，我们研究了影响氢氧化钠对朊病毒灭活的关键因素。
在生化检测中，朊病毒感染性的降低与PrPSc蛋白酶抗性核心（PrPRES）的消失相关。为模拟朊病毒灭活，将仓鼠瘙痒病（263K株）脑匀浆（SBH）在4℃或18℃下于0.1 m氢氧化钠中孵育特定时间，有无去污剂均可。中和后的样品用蛋白酶K（PK）进行有限消化，然后使用终点稀释免疫印迹法和3F4抗体进行分析。使用差异免疫沉淀法检测暴露于氢氧化钠的朊病毒的结构变化。
在4℃和18℃下，在无去污剂的情况下，用0.1 m氢氧化钠处理SBH 15分钟，分别使PrP(RES)信号降低3.5和4.0 log10单位，仍有一些残留信号。在氢氧化钠中孵育60分钟期间去污剂十二烷基肌氨酸钠的存在进一步增强了PrPRES的降低至≥4.5 log10单位（即低于检测限）。氢氧化钠处理诱导了PrP的构象变化，导致一个隐藏表位的暴露，并使朊病毒能被3F4抗体免疫沉淀。
在体外灭活试验中，使用氢氧化钠可有效降低朊病毒水平。用去污剂对SBH进行预处理后，氢氧化钠可完全消除PrPRES信号。去污剂可能会释放脂质膜保护的PrPSc，以改善氢氧化钠的作用，然后氢氧化钠可通过改变其结构使PrPSc失活。在生产过程中发生未识别的PrPSc暴露的情况下，结合使用去污剂和氢氧化钠的消毒程序可能有助于确保产品中污染残留量降至最低。
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