NR ATBU
AU Bastian,F.O.; McDermott,M.E.; Perry,A.S.; Carver,L.A.; Dash,S.; Garry,R.F.
TI Safe method for isolation of prion protein and diagnosis of Creutzfeldt-Jakob disease
QU Journal of Virological Methods 2005 Dec; 130(1-2): 133-9
PT journal article
AB Creutzfeldt-Jakob disease (CJD) is a fatal progressive infectious encephalopathy of humans characterized by spongiform degeneration of the brain. Detection of protease-resistant low molecular weight proteins, referred to as 'prions', in the brain is essential for diagnosis. Protease-based methods for prion detection are problematic due to variable susceptibility of prion proteins to proteinase-K digestion. Since CJD brain samples are infectious at all stages of the prion extraction process, we set out to develop a laboratory safe method for prion purification. We lysed the tissues with guanidine thiocyanate followed by phenol extraction of the proteins. Western blotting using prion-specific MAB 3F4 revealed primarily low molecular weight unglycosylated prion (UGP) bands in all CJD cases (19) while the predominant banding in all normal brains (14) represented glycosylated prion (GP). Density readings of the blots revealed the UGP/GP ratio to be significantly different in CJD versus normal brains, with an inverse UGP/GP ratio in CJD. Using this method, we discovered one previously undiagnosed CJD case when we screened 19 brains from the Louisiana State University Alzheimer disease brain bank. Our method is a safe and reliable way of detecting abnormal prion proteins (p<0.0001) and is adaptable to both diagnostic and research laboratories.
IN Weil manchmal auch PrPsc nicht über die typische Proteinase-K-Resistenz verfügt, entwickelten die Autoren eine alternative Methode zur Prionprotein-Isolierung. Sie lysieren Gewebe mit Guanidinthiocyanat und extrahieren anschließend Proteine mit Phenol. Die Unterscheidung zwischen PrPc und PrPsc erfolgt nicht durch eine Proteinase-K-Behandlung, sondern durch das Bandenmuster im Western blot. Als typisch für Fälle von Creutzfeldt-Jakob-Krankheit fanden sie eine Bande mit nichtglykolisiertem Prionprotein mit niedrigem Molekulargewicht. In normalem Gehirn hingegen fanden sie hauptsächlich glykosiliertes Prionprotein. Zusätzlich zu der Bedeutung für den Nachweis atypischer TSE-Fälle betonen die Autoren die Sicherheit ihrer Methode.
MH Blotting, Western/*methods; Brain Chemistry; Creutzfeldt-Jakob Syndrome/*diagnosis; Glycosylation; Guanidines; Humans; Phenol; Prions/*analysis; Research Support, N.I.H., Extramural; Safety; Thiocyanates
AD Department of Pathology and Laboratory Medicine, Tulane University Medical Center, 1430 Tulane avenue, New Orleans, LA 70112, USA. fbastian@tulane.edu
SP englisch
PO Niederlande