Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR.
Pathologic prion protein (PrP(Sc)), 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 PrP(Sc). 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 PrP(C) 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 PrP(Sc) in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrP(C) in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.
Barletta
Janet M
JM
Food and Drug Administration CDER/OPS/Microbiology, Rockville, MD, USA. barlettaj@cder.fda.gov
Edelman
Daniel C
DC
Highsmith
W E
WE
Constantine
Niel T
NT
D000818:Animals; D001921:Brain; D001923:Brain Chemistry; D006224:Cricetinae; D018620:PrPSc Proteins / Q000032:analysis* / Q000276:immunology; D017096:Prion Diseases / Q000175:diagnosis / Q000209:etiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012608:Scrapie / Q000378:metabolism
D018620:PrPSc Proteins