Summary
Prion diseases are characterized by the cerebral deposition of an aggregated pathological isoform of the prion protein (PrPSc) which constitutes the principal component of the transmissible agent termed prion. In order to develop a highly sensitive method for the detection of PrPSc aggregates in biological samples such as cerebrospinal fluid (CSF), we used a method based on Fluorescence Correlation Spectroscopy (FCS), a technique which allows detection of single fluorescently labeled molecules in solution. Within the FCS setup, fluorescent photons emitted by molecules passing an open volume element defined by the beam of an excitation laser focussed into a diffraction-limited spot are imaged confocally onto a single photon counting detector. Aggregates of PrPSc could be labeled by co-aggregation of probe molecules such as monomeric recombinant PrP or PrP-specific antibodies tagged with a fluorescent dye. In addition to slow diffusion, labeled aggregates are characterized by high fluorescence intensity, which allows detection and quantification by analysis of fluorescence intensity distribution. To improve detection of rare target particles, the accessible volume element was increased by scanning for intensely fluorescent targets (SIFT). To further improve sensitivity and specificity, two different probes were used simultaneously in a two-color setup. In a diagnostic model system of CSF spiked with purified prion rods, dual-color SIFT was more sensitive than Western blot analysis. In addition, a PrPSc-specific signal was also detected in a number of CSF samples derived from CJD patients but not in controls.
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Giese, A., Bieschke, J., Eigen, M., Kretzschmar, H.A. (2000). Putting prions into focus: application of single molecule detection to the diagnosis of prion diseases. In: Groschup, M.H., Kretzschmar, H.A. (eds) Prion Diseases. Archives of Virology. Supplementa, vol 16. Springer, Vienna. https://doi.org/10.1007/978-3-7091-6308-5_15
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DOI: https://doi.org/10.1007/978-3-7091-6308-5_15
Publisher Name: Springer, Vienna
Print ISBN: 978-3-211-83529-6
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