Putting prions into focus: application of single molecule detection to thediagnosis of prion diseases

Prion diseases are characterized by the cerebral deposition of an aggregate d pathological isoform of the prion protein (PrPSc) which constitutes the p rincipal component of the transmissible agent termed prion. In order to dev elop a highly sensitive method for the detection of PrPSc aggregates in bio logical samples such as cerebrospinal fluid (CSF), we used a method based o n Fluorescence Correlation Spectroscopy (FCS), a technique which allows det ection of single fluorescently labeled molecules in solution. Within the FC S setup, fluorescent photons emitted by molecules passing: an open volume e lement defined by the beam of an excitation laser focussed into a diffracti on-limited spot are imaged confocally onto a single photon counting detecto r. Aggregates of PrPSc could be labeled by coaggregation 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 ch aracterized by high fluorescence intensity, which allows detection and quan tification by analysis of fluorescence intensity distribution. To improve d etection of rare target particles, the accessible volume element was increa sed by scanning for intensely fluorescent targets (SIFT). To further improv e sensitivity and specificity, two different probes were used simultaneousl y in a two-color setup. In a diagnostic model system of CSF spiked with pur ified prion rods, dual-color SIFT was more sensitive than Western blot anal ysis. In addition, a PrPSc-specific signal was also detected in a number of CSF samples derived from CJD patients but not in controls.