Myskiw Jennifer, Lamoureux Lise, Peterson Anne, Knox David, Jansen Gerard H, Coulthart Michael B, Booth Stephanie A
One Health Division, Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Manitoba, Canada; Department of Medical Microbiology and Infectious Diseases, Faculty of Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada.
One Health Division, Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Manitoba, Canada.
Lab Invest. 2023 Mar;103(3):100029. doi: 10.1016/j.labinv.2022.100029. Epub 2023 Jan 10.
Creutzfeldt-Jakob disease (CJD) comprises a group of transmissible neurodegenerative diseases with vast phenotypic diversity. Sporadic CJD heterogeneity is predominantly influenced by the genotype at codon 129 of the prion-encoding gene and the molecular weight of PrP fragments after protease digestion, resulting in a classification of 6 subtypes of CJD (MM1, MM2, MV1, MV2, VV1, and VV2). The majority of cases with CJD can be distinguished using this classification system. However, a number of reported CJD cases are phenotypically unique from others within their same subtype, such as variably protease-sensitive prionopathies, or exist as a mixture of subtypes within the same patient. Western blotting of brain tissue, along with the genotyping of codon 129 of the prion-encoding gene, is considered the "gold standard" for the biochemical characterization of CJD. Western blotting requires a significant amount of prion protein for detection, is labor-intensive, and is also associated with high interassay variability. In addition to these limitations, a growing body of research suggests that unique subtypes of CJD are often undetected or misdiagnosed using standard diagnostic western blotting protocols. Consequently, we successfully optimized and developed a capillary-based western assay using the JESS Simple Western (ProteinSimple) to detect and characterize prion proteins from patients with CJD. We found that this novel assay consistently differentiated CJD type 1 and type 2 cases with a limit of detection 10 to 100× higher than traditional western blotting. Cases with CJD in which type 1 and type 2 coexist within the same brain region can be detected using type 1-specific and type 2-specific antibodies, and we found that there was remarkable specificity for the detection of cases with variably protease-sensitive prionopathy. The assay presented displays outstanding sensitivity, allowing for the preservation of valuable samples and enhancing current detection methods.
克雅氏病（CJD）是一组具有广泛表型多样性的可传播性神经退行性疾病。散发性CJD的异质性主要受朊病毒编码基因第129密码子的基因型以及蛋白酶消化后PrP片段的分子量影响，由此将CJD分为6种亚型（MM1、MM2、MV1、MV2、VV1和VV2）。大多数CJD病例可通过该分类系统加以区分。然而，一些已报道的CJD病例在表型上与同一亚型中的其他病例不同，如可变蛋白酶敏感性朊病毒病，或在同一患者体内以亚型混合的形式存在。脑组织的蛋白质印迹分析，以及朊病毒编码基因第129密码子的基因分型，被认为是CJD生化特征鉴定的“金标准”。蛋白质印迹分析需要大量的朊病毒蛋白进行检测， labor-intensive（此处可能有误，推测为labor - intensive，意为劳动强度大），并且检测间的变异性也很高。除了这些局限性外，越来越多的研究表明，使用标准诊断性蛋白质印迹分析方案往往无法检测到或误诊CJD的独特亚型。因此，我们成功优化并开发了一种基于毛细管的蛋白质印迹分析方法，使用JESS Simple Western（ProteinSimple）来检测和鉴定CJD患者的朊病毒蛋白。我们发现，这种新型分析方法能够持续区分1型和2型CJD病例，其检测限比传统蛋白质印迹分析高10至100倍。同一脑区内1型和2型共存的CJD病例可使用1型特异性和2型特异性抗体进行检测，并且我们发现对可变蛋白酶敏感性朊病毒病病例的检测具有显著特异性。所展示的分析方法具有出色的灵敏度，能够保存珍贵样本并改进现有检测方法。
