Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets

A definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (C JD) relies on the detection of pathological prion protein (PrPSc). However, no test for PrPSc in cerebrospinal fluid (CSF) has been available thus far . Based on a setup for confocal dual-color fluorescence correlation spectro scopy, a technique suitable for single molecule detection, we developed a h ighly sensitive detection method for PrPSc. Pathological prion protein aggr egates were labeled by specific antibody probes tagged with fluorescent dye s, resulting in intensely fluorescent targets, which were measured by dual- color fluorescence intensity distribution analysis in a confocal scanning s etup. In a diagnostic model system, PrPSc aggregates were detected down to a concentration of 2 pM PrPSc, corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sens itive than Western blot analysis. A PrPSc-specific signal could also be det ected in a number of CSF samples from patients with CJD but not in control samples, providing the basis for a rapid and specific test for CJD and othe r prion diseases. Furthermore, this method could be adapted to the sensitiv e detection of other disease-associated amyloid aggregates such as in Alzhe imer's disease.