NR ASSE
AU Barletta,J.M.; Edelman,D.C.; Highsmith,W.E.; Constantine,N.T.
TI Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR
QU Journal of Virological Methods 2005 Aug; 127(2): 154-64
PT journal article
AB Pathologic prion protein (PrPsc), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrPsc. The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrPc was consistently detected at 1 fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10(-8) (approximately 10-100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrPsc in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrPc in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.
IN Anscheinend lassen sich mit der sogenannten real-time immuno-PCR Prionproteine in Konzentrationen von nur 1 fg/ml und PrPsc nach Proteinase-K-Behandlung sogar noch nach 100-millionenfacher Verdünnung Scrapie-infizierten Hamsterhirns nachweisen, indem man an die Antikörper gebundene DNA-Fragmente mittels PCR amplifiziert. Damit soll sich die Empfindlichkeit von ELISA und Western-blot um den Faktor 1 Million steigern lassen und präklinische BSE-Nachweise ermöglichen. Allerdings verstärkt die gesteigerte Nachweisempfindlichkeit auch das Problem der falschpositiven Testergebnisse.
MH Animals; Brain; Brain Chemistry; Hamsters; PrPsc Proteins/*analysis/immunology; Prion Diseases/diagnosis/etiology; Research Support, U.S. Gov't, Non-P.H.S.; Reverse Transcriptase Polymerase Chain Reaction/*methods; Scrapie/metabolism
AD Janet M. Barletta (barlettaj@cder.fda.gov), Food and Drug Administration CDER/OPS/Microbiology, Rockville, MD, USA; Daniel C. Edelman, Niel T. Constantine, Department of Pathology, University of Maryland Baltimore, 725 W. Lombard St., Baltimore, MD 21201, USA; W.E. Highsmith, Department of Laboratory Medicine and Pathology, Division of Laboratory Genetics, Mayo Clinic, Rochester, MD, USA
SP englisch
PO Niederlande
SP englisch