NR AOYV
AU Saunders,G.C.; Jones,V.; Tout,A.C.; Martin,T.C.
TI Development of a novel immuno-fret assay for the detection of PrPsc
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-41
PT Konferenz-Poster
AB
In this study we explore the feasibility of using FRET (fluorescence resonance energy transfer)-based technology as a diagnostic tool for TSEs. FRET is a distance-dependant interaction between two dye molecules where the excitation is transferred from one molecule (the donor) to another (the acceptor). This transfer only occurs when the 2 dye molecules are in very close proximity and when the absorption spectrum of the acceptor overlaps the emission spectrum of the donor.
The assay design consists of two commercial antibodies (abs) raised against different or overlapping PrP epitopes labeled with either a donor or acceptor fluorophore. The PrP containing sample is incubated with the labeled abs followed by excitation at the donor excitation wavelength. A FRET signal is detected by calculating the change in fluorescence of the donor and acceptor.
Proof of principle was established and the assay optimised using recPrP and PrPc before progressing to scrapie and BSE derived PrPsc; 25ng of recPrP (at 1ug/ml conc.) could be detected. We identified critical parameters such as ab ratio, incubation time and proteinase K inhibitors and explored the performance of the assay using crude and extracted samples and on a variety of tissue types (brain, CSF and mesenteric lymph node).
One potential advantage of the homogeneous FRET assay is that the change in signal only occurs when the labeled abs are bound to the specific target. Consequently, non-bound abs do not need to be removed from the assay, as in ELISA formats, thereby improving the rapidity, and safety of sample processing. Successful application of the technique to BSE and scrapie samples could lead to a screening assay and be useful in differentiating conformers of PrP.
This work was funded by DEFRA, UK
AD G.C. Saunders, V. Jones, A.C. Tout, T.C. Martin, TSE Molecular Biology Dept, Veterinary Laboratories Agency, UK
SP englisch
PO Deutschland