NR AXDP
AU Birkmann,E.; Henke,F.; Weinmann,N.; Dumpitak,C.; Groschup,M.H.; Funke,A.; Willbold,D.; Riesner,D.
TI Counting of single prion particles bound to a capture-antibody surface (surface-FIDA)
QU Veterinary Microbiology 2007 Aug 31; 123(4): 294-304
PT journal article; research support, non-u.s. gov't
AB Hitherto accredited prion tests use the PK resistance of PrPsc, the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrPsc. Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrPsc was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.
MH Animals; Blotting, Western/veterinary; Cattle; Cricetinae; Electrophoresis, Polyacrylamide Gel/veterinary; Encephalopathy, Bovine Spongiform/diagnosis; Endopeptidase K/chemistry/metabolism; PrPsc Proteins/*cerebrospinal fluid/*isolation & purification; Prion Diseases/diagnosis/*veterinary; Prions/isolation & purification; Scrapie/diagnosis; Sensitivity and Specificity; Spectrometry, Fluorescence/methods/*veterinary
AD Heinrich-Heine University Düsseldorf, Institute of Physical Biology, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
SP englisch
PO Niederlande