Jackman Roy, Everest David J, Schmerr Mary Jo, Khawaja Mohammed, Keep Pat, Docherty John
Immunochemistry Group, Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom.
J AOAC Int. 2006 May-Jun;89(3):720-7.
An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 7-12 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.
描述了一种用于检测感染羊瘙痒病的绵羊血液中血沉棕黄层部分内源性疾病相关朊病毒蛋白的分析方法。该方法已得到改进，并对其在绵羊传染性海绵状脑病临床前诊断中的性能进行了评估。该测试系统采用了包括提取和浓缩在内的样品制备方案以及使用朊病毒蛋白液相竞争免疫测定的测试方法。在测定中使用了针对朊病毒蛋白（PrP）C末端肽序列的抗体和荧光素标记的肽缀合物。使用带有激光诱导荧光检测的自由区毛细管电泳来分离抗体结合的荧光标记肽和游离标记肽。在该测定中，PrP与荧光标记肽竞争有限的抗体结合位点，这导致代表与荧光标记肽结合的抗体免疫复合物的峰减少。当分析来自7至12个月大的感染羊瘙痒病的绵羊以及瘙痒病易感PrP基因型VRQ/VRQ和VRQ/ARQ的血液样本时，在血液样本中发现了异常PrP。这些结果与瘙痒病的死后诊断相关。这些绵羊处于临床前阶段，在测试时看起来正常，但在测试后约12个月死于临床疾病。在年龄较大的动物以及有临床症状的动物中，检测呈阳性的动物比例较小。这项研究表明，该技术可作为一种灵敏、快速的临床前测试，用于检测感染羊瘙痒病的绵羊血液中的疾病相关PrP。提取方案和毛细管电泳条件的改进将提高该测试的稳健性。
