Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR

Janet M. Barletta, Daniel C. Edelman, W Edward Jr. Highsmith, Niel T. Constantine

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)154-164
Number of pages11
JournalJournal of Virological Methods
Volume127
Issue number2
DOIs
StatePublished - Aug 2005

Fingerprint

Scrapie
Cricetinae
Real-Time Polymerase Chain Reaction
Polymerase Chain Reaction
Brain
Endopeptidase K
Prion Diseases
Enzyme-Linked Immunosorbent Assay
Western Blotting
Antibodies
Biological Assay
Brain Stem
Digestion
Prion Proteins
Infection
Proteins

Keywords

  • BSE
  • Immuno-PCR
  • IPCR
  • Prion
  • PrP
  • PrP
  • Real-time IPCR
  • Scrapie
  • TSE

ASJC Scopus subject areas

  • Virology

Cite this

Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR. / Barletta, Janet M.; Edelman, Daniel C.; Highsmith, W Edward Jr.; Constantine, Niel T.

In: Journal of Virological Methods, Vol. 127, No. 2, 08.2005, p. 154-164.

Research output: Contribution to journalArticle

Barletta, Janet M. ; Edelman, Daniel C. ; Highsmith, W Edward Jr. ; Constantine, Niel T. / Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR. In: Journal of Virological Methods. 2005 ; Vol. 127, No. 2. pp. 154-164.
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