Counting of single prion particles bound to a capture-antibody surface (surface-FIDA)

Heinrich-Heine University Düsseldorf, Institute of Physical Biology, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
Veterinary Microbiology (Impact Factor: 2.51). 09/2007; 123(4):294-304. DOI: 10.1016/j.vetmic.2007.04.001
Source: PubMed


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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    • "The method discriminates aggregated PrP forms from monomeric PrP without the use of the proteinase K (PK) digestion step and therefore recognizes both PK-resistant and PK-sensitive PrPTSE. Surface-FIDA enabled the counting of bovine and hamster PrP aggregates in brain homogenates and in bovine cerebrospinal fluid [47]. PrP aggregates were also blind-detected in blood of scrapie-infected sheep (n = 15) with high specificity and sensitivity [46], although it remains unsettle whether the detection of PrP aggregates correlates with infectivity. "
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    ABSTRACT: Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal rare neurodegenerative disorders affecting man and animals and caused by a transmissible infectious agent. TSE diseases are characterized by spongiform brain lesions with neuronal loss and the abnormal deposition in the CNS, and to less extent in other tissues, of an insoluble and protease resistant form of the cellular prion protein (PrP(C)), named PrP(TSE). In man, TSE diseases affect usually people over 60 years of age with no evident disease-associated risk factors. In some cases, however, TSE diseases are unequivocally linked to infectious episodes related to the use of prion-contaminated medicines, medical devices, or meat products as in the variant Creutzfeldt-Jakob disease (CJD). Clinical signs occur months or years after infection, and during this silent period PrP(TSE), the only reliable marker of infection, is not easily measurable in blood or other accessible tissues or body fluids causing public health concerns. To overcome the limit of PrP(TSE) detection, several highly sensitive assays have been developed, but attempts to apply these techniques to blood of infected hosts have been unsuccessful or not yet validated. An update on the latest advances for the detection of misfolded prion protein in body fluids is provided.
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    • "In an earlier study, we described the detection of PrP aggregates with high sensitivity in brain homogenate of BSE cattle, and in a small number of cerebrospinal fluid samples from BSE cattle [26]. According to the literature, infectivity in blood - even in symptomatic experimental hamsters - is as low as 10 infectious units per ml [33], [34]. "
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    • "A scheme of the measurement principle can be found in the figure. At least, three detection antibodies are used in the assay procedure, providing high specificity [48]. Additionally, detection of monomers can be excluded by the application of the same antibody as capture and as detection probe. "
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