Counting of single prion particles bound to a capture-antibody surface (surface-FIDA) |
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Authors: | Birkmann Eva Henke Franziska Weinmann Nicole Dumpitak Christian Groschup Martin Funke Aileen Willbold Dieter Riesner Detlev |
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Institution: | 1. Heinrich-Heine University Düsseldorf, Institute of Physical Biology, Universitätsstrasse 1, 40225 Düsseldorf, Germany;2. Friedrich-Loeffler-Institut (FLI), Institute for Novel and Emerging Infectious Diseases, Boddenblick 5a, 17493 Greifswald/Insel Riems, Germany |
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Abstract: | 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. |
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Keywords: | Prion Proteinase K-free diagnosis TSE diagnosis Single particle detection Surface-FIDA BSE Scrapie CSF |
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