USDA Agricultural Research Service, Foodborne Contaminants Research Unit, Albany, CA, USA.
Prion. 2010 Apr-Jun;4(2):94-104. doi: 10.4161/pri.4.2.12622. Epub 2010 Apr 9.
Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrP(C) into alternately folded PrP(Sc). Immunoassays toward pre-clinical detection of infectious PrP(Sc) have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrP(Sc) selective antibodies. We report a method to purify infectious PrP(Sc) from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrP(Sc) was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM <20-fold and purified DRM immunogen <40-fold) relative to 1% crude brain homogenate. Purification of PrP(Sc) from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrP(Sc) antigen was performed using wild-type (wt) and Prnp(0/0) mice, both on Balb/cJ background. A robust immune response against PrP(Sc) was observed in all inoculated Prnp(0/0) mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrP(C) and PrP(Sc), while binding to other brain-derived protein was not observed. In contrast, the PrP(Sc) inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.
朊病毒病是由朊病毒蛋白（PrP）引起的致命神经退行性疾病。传染性朊病毒通过内源性 PrP(C) 模板介导的构象转换积累在大脑中，转化为交替折叠的 PrP(Sc)。针对传染性 PrP(Sc) 的临床前检测的免疫测定受到非神经元组织中低水平朊病毒积累和缺乏 PrP(Sc) 选择性抗体的困扰。我们报告了一种从生物组织中纯化传染性 PrP(Sc) 的方法，可将其用作免疫原和样品富集物，以提高免疫测定的灵敏度。通过蔗糖梯度离心感染组织并从脂质筏（DRM）中的去污剂抗性膜中分离，可以实现朊病毒的显著富集。与粗脑组织相比，直接 ELISA 观察到 DRM 分数中可检测到的 PrP(Sc) 增加了 50 倍，而蛋白质浓度相同。与 1%的粗脑组织匀浆相比，连续的纯化步骤导致特异性感染性增加（DRM <20 倍，纯化的 DRM 免疫原<40 倍）。在用蛋白酶 K (PK) 消化后，用磷钨酸蛋白沉淀法从 DRM 中纯化 PrP(Sc)，然后进行凝胶过滤层析，以将 PK 和残留的蛋白质片段与较大的朊病毒聚集体分离。用纯化的 PrP(Sc) 抗原免疫野生型（wt）和 Prnp(0/0) 小鼠，均在 Balb/cJ 背景下进行。在所有接种 Prnp(0/0) 小鼠中观察到针对 PrP(Sc) 的强烈免疫反应，导致抗血清中含有针对朊病毒蛋白的高滴度抗体。来自这些小鼠的抗血清识别 PrP(C) 和 PrP(Sc)，而未观察到与其他脑源性蛋白的结合。相比之下，wt 小鼠中的 PrP(Sc) 接种物是无免疫原性的，抗血清与 PrP 或任何其他蛋白均无反应性。
