Curin Serbec Vladka, Bresjanac Mara, Popovic Mara, Pretnar Hartman Katrina, Galvani Vesna, Rupreht Ruth, Cernilec Maja, Vranac Tanja, Hafner Iva, Jerala Roman
Blood Transfusion Center of Slovenia, Slajmerjeva 6, Ljubljana, Slovenia.
J Biol Chem. 2004 Jan 30;279(5):3694-8. doi: 10.1074/jbc.M310868200. Epub 2003 Oct 29.
Current methods for diagnosing transmissible spongiform encephalopathies rely on the degradation of the cellular prion protein (PrP(C)) and the subsequent detection of the protease-resistant remnant of the pathological prion isoform PrP(Sc) by antibodies that react with all forms of PrP. We report on a monoclonal antibody, V5B2, raised against a peptide from the C-terminal part of PrP, which recognizes an epitope specific to PrP(Sc). In cryostat sections from Creutzfeldt-Jacob's disease (CJD) patients' brains, V5B2 selectively labels various deposits of PrP(Sc) without any pretreatment for removal of PrP(C). V5B2 does not bind to non-CJD brain samples or to recombinant PrP, either in its native or denatured form. Specificity for PrP is confirmed by a sandwich enzyme-linked immunosorbent assay utilizing V5B2, which discriminates between CJD and normal samples without proteinase K treatment, and by immunoprecipitation from CJD brain homogenate. The PrP(Sc)-specific epitope is disrupted by denaturation. We conclude that the C-terminal part of PrP in disease-associated PrP(Sc) aggregates forms a structural epitope whose conformation is distinct from that of PrP(C).
目前诊断传染性海绵状脑病的方法依赖于细胞朊蛋白(PrP(C))的降解,以及随后通过与所有形式的PrP发生反应的抗体来检测病理性朊病毒异构体PrP(Sc)的蛋白酶抗性残余物。我们报道了一种针对PrP C末端肽产生的单克隆抗体V5B2,它识别PrP(Sc)特有的一个表位。在克雅氏病(CJD)患者大脑的冷冻切片中,V5B2无需对PrP(C)进行任何预处理去除就能选择性地标记PrP(Sc)的各种沉积物。V5B2不与非CJD脑样本或重组PrP结合,无论是天然形式还是变性形式。利用V5B2的夹心酶联免疫吸附测定法证实了对PrP的特异性,该方法在不进行蛋白酶K处理的情况下就能区分CJD和正常样本,并且通过从CJD脑匀浆中进行免疫沉淀也得到了证实。疾病相关的PrP(Sc)聚集体中PrP的C末端部分的表位会因变性而被破坏。我们得出结论,疾病相关的PrP(Sc)聚集体中PrP的C末端部分形成了一个结构表位,其构象与PrP(C)不同。