Dabaghian Reza H, Barnard Geoff, McConnell Ian, Clewley Jonathan P
Virus Reference Department, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK.
J Virol Methods. 2006 Mar;132(1-2):85-91. doi: 10.1016/j.jviromet.2005.09.002. Epub 2005 Oct 10.
Concern about the possible secondary spread of variant Creutzfeldt-Jakob disease (vCJD) through blood transfusion and blood products has increased the need for a sensitive and rapid test for the identification of PrP(Sc) in specimens collected non-invasively from living persons. Furthermore, an accurate estimate of the prevalence of pre-clinical vCJD in the British population would be possible if there were such a test that could be applied to specimens available readily (e.g. blood and urine). As a first step towards that goal, we have developed a simple and sensitive test for the detection of PrP(Sc) in peripheral tissues and brain of vCJD patients, based on the differential extraction of PrP(Sc) with guanidine hydrochloride. The prion protein (PrP) isoforms are extracted sequentially from homogenized tissue by applying two different concentrations of this chaotropic agent. Each extraction yields a fraction of the PrP isoforms with different solubilities in guanidine hydrochloride. Quantitation of the two fractions (relatively insoluble or relatively soluble) using time resolved fluorescence (DELFIA) as a reporter system allows differentiation between PrP(Sc) infected and non-infected tissues. The assay has a detection limit of 10 pg PrP, is robust and could be automated.
对变异型克雅氏病（vCJD）可能通过输血和血液制品发生二次传播的担忧，增加了对一种灵敏且快速的检测方法的需求，该方法用于鉴定从活人身上非侵入性采集的标本中的朊病毒蛋白（PrP）（Sc）。此外，如果有这样一种能够应用于容易获取的标本（如血液和尿液）的检测方法，就有可能准确估计英国人群中临床前vCJD的患病率。作为朝着该目标迈出的第一步，我们基于用盐酸胍对PrP（Sc）进行差异提取，开发了一种简单且灵敏的检测方法，用于检测vCJD患者外周组织和大脑中的PrP（Sc）。通过应用两种不同浓度的这种离液剂，从匀浆组织中依次提取朊病毒蛋白（PrP）异构体。每次提取都会得到在盐酸胍中具有不同溶解度的PrP异构体部分。使用时间分辨荧光（DELFIA）作为报告系统对这两个部分（相对不溶或相对可溶）进行定量，能够区分感染PrP（Sc）的组织和未感染的组织。该检测方法的检测限为10 pg PrP，稳定性好且可实现自动化。
