Prion protein detection via direct immuno-quantitative real-time PCR
Reuter, T., Gilroyed, B.H., Alexander, T.W., Mitchell, G., Balachandran, A., Czub, S., McAllister, T.A. (2009). Prion protein detection via direct immuno-quantitative real-time PCR, 78(3), 307-311. http://dx.doi.org/10.1016/j.mimet.2009.07.001
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 2.32 × 102 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 1.06 × 104 to 3.24 × 102 epitopes (R2 = 0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies. Crown Copyright © 2009.
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