Prion protein detection via direct immuno-quantitative real-time PCR.
We describe a simple and robust assay for the quantitative detection of prions using immuno-quantitative real-time PCR (iQ-RT-PCR) made possible by a direct conjugate of a prion-specific antibody (ICSM35) and a synthetic 99-bp DNA tail. The DNA tail was engineered to include a single ScrFI restriction site, which enabled subsequent quantification of restricted DNA tails using real-time PCR. The assay was tested with scrapie prions bound to polyvinylidene difluoride membranes and to 96-well plates coated with a capturing antibody from a commercially available immuno-based assay (TeSeE). The iQ-RT-PCR assay had a detection limit corresponding to 2.32x10(2) prion epitopes, which represented a 1000-fold increase in detection sensitivity over the commercial assay. Detection of prions from diluted scrapie-positive brain homogenate bound to membranes was linear over a range of 1.06x10(4) to 3.24x10(2) epitopes (R(2)=0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies.
Reuter
Tim
T
Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, Canada T1J 4B1.
Gilroyed
Brandon H
BH
Alexander
Trevor W
TW
Mitchell
Gordon
G
Balachandran
Aru
A
Czub
Stefanie
S
McAllister
Tim A
TA
D000818:Animals; D001923:Brain Chemistry; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D009698:Nucleoproteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D011328:Prions / Q000032:analysis*; D012680:Sensitivity and Specificity
D009698:Nucleoproteins; D011328:Prions