Counting of single prion particles bound to a capture-antibody surface (surface-FIDA).
Hitherto accredited prion tests use the PK resistance of PrP(Sc), 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 PrP(Sc). 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, PrP(Sc) 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.
Birkmann
Eva
E
Heinrich-Heine University Düsseldorf, Institute of Physical Biology, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
Henke
Franziska
F
Weinmann
Nicole
N
Dumpitak
Christian
C
Groschup
Martin
M
Funke
Aileen
A
Willbold
Dieter
D
Riesner
Detlev
D
D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002417:Cattle; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D016643:Encephalopathy, Bovine Spongiform / Q000175:diagnosis; D019286:Endopeptidase K / Q000737:chemistry / Q000378:metabolism; D018620:PrPSc Proteins / Q000134:cerebrospinal fluid* / Q000302:isolation & purification*; D017096:Prion Diseases / Q000175:diagnosis / Q000662:veterinary*; D011328:Prions / Q000302:isolation & purification; D012608:Scrapie / Q000175:diagnosis; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods / Q000662:veterinary*
D018620:PrPSc Proteins; D011328:Prions; D019286:Endopeptidase K