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| Home > Publications database > Counting of single prion particles bound to a capture-antibody surface (surface-FIDA) |
| Journal Article | PreJuSER-60108 |
; ; ; ; ; ; ;
2007
Elsevier Science
Amsterdam [u.a.]
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Please use a persistent id in citations: doi:10.1016/j.vetmic.2007.04.001
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.
Keyword(s): Animals (MeSH) ; Blotting, Western: veterinary (MeSH) ; Cattle (MeSH) ; Cricetinae (MeSH) ; Electrophoresis, Polyacrylamide Gel: veterinary (MeSH) ; Encephalopathy, Bovine Spongiform: diagnosis (MeSH) ; Endopeptidase K: chemistry (MeSH) ; Endopeptidase K: metabolism (MeSH) ; PrPSc Proteins: cerebrospinal fluid (MeSH) ; PrPSc Proteins: isolation & purification (MeSH) ; Prion Diseases: diagnosis (MeSH) ; Prion Diseases: veterinary (MeSH) ; Prions: isolation & purification (MeSH) ; Scrapie: diagnosis (MeSH) ; Sensitivity and Specificity (MeSH) ; Spectrometry, Fluorescence: methods (MeSH) ; Spectrometry, Fluorescence: veterinary (MeSH) ; PrPSc Proteins ; Prions ; Endopeptidase K ; J ; prion (auto) ; protemase K-free diagnosis (auto) ; TSE diagnosis (auto) ; single. particle detection (auto) ; Surface-FIDA (auto) ; BSE (auto) ; scrapie (auto) ; CSF (auto)
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