Berardi Vito Angelo, Cardone Franco, Valanzano Angelina, Lu Mei, Pocchiari Maurizio
Degenerative and Inflammatory Neurological Diseases Unit, Department of Cell Biology and Neurosciences, Italian National Institute of Health, Rome.
Transfusion. 2006 Apr;46(4):652-8. doi: 10.1111/j.1537-2995.2006.00763.x.
Concern about the safety of blood, blood components, and plasma-derived products with respect to prions has increased since the report of two blood-related infections of variant Creutzfeldt-Jakob disease in the United Kingdom. Efforts were directed toward the development of procedures able to remove or inactivate prions from blood components or plasma-derived products with brain fractions of transmissible spongiform encephalopathy (TSE)-infected rodents as spiking materials. These spiking materials, however, are loaded with pathological prion protein (PrP(TSE)) aggregates that are likely not associated to blood infectivity. The presence of these aggregates may invalidate these studies.
Brains from 263K scrapie-infected hamsters were suspended in 10 percent phosphate-buffered saline. After low-speed centrifugation, the supernatant was collected and ultracentrifuged at 220,000 x g at 25 degrees C for 30 minutes. The high-speed supernatants (S(HS)) and pellets were collected; the proteinase-resistant PrP(TSE) was measured by Western blot and infectivity by intracerebral inoculation into weanling hamsters.
A substantial amount of prion infectivity (more than 10(5) LD(50) per mL of a 10% suspension of brain tissues) is present in the S(HS) fraction of 263K scrapie-infected hamster brains. Concomitantly, this fraction contains none or only traces of PrP(TSE) in its aggregate form.
This study describes a simple and fast protocol to prepare infectious material from 263K scrapie-infected brains that is not contaminated with PrP(TSE) aggregates. This S(HS) fraction is likely to be the most relevant material for endogenous spiking of human blood in validation experiments aimed at demonstrating procedures to remove or inactivate TSE infectious agents.
自英国报告两例与血液相关的变异型克雅氏病感染病例以来，人们对血液、血液成分和血浆衍生产品在朊病毒方面的安全性的关注度有所增加。人们致力于开发能够从血液成分或血浆衍生产品中去除或灭活朊病毒的程序，以感染可传播性海绵状脑病（TSE）的啮齿动物的脑部分作为加标材料。然而，这些加标材料中含有病理性朊病毒蛋白（PrP(TSE)）聚集体，而这些聚集体可能与血液传染性无关。这些聚集体的存在可能使这些研究无效。
将263K株羊瘙痒病感染仓鼠的大脑悬浮于10%磷酸盐缓冲盐水中。低速离心后，收集上清液，并在25℃下以220,000×g超速离心30分钟。收集高速上清液（S(HS)）和沉淀；通过蛋白质印迹法测量抗蛋白酶PrP(TSE)，并通过脑内接种断奶仓鼠来检测感染性。
263K株羊瘙痒病感染仓鼠大脑的S(HS)部分存在大量朊病毒感染性（每毫升10%脑组织悬液中超过10(5) LD(50)）。与此同时，该部分聚集体形式的PrP(TSE)含量为零或仅有微量。
本研究描述了一种简单快速的方案，用于从263K株羊瘙痒病感染的大脑中制备未被PrP(TSE)聚集体污染的感染性材料。在旨在证明去除或灭活TSE感染因子程序的验证实验中，该S(HS)部分可能是用于人体血液内源性加标的最相关材料。
