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

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Abstract
A definite diagnosis of prion diseases such as Creutzfeldt–Jakob disease (CJD) relies on the detection of pathological prion protein (PrP Sc ). However, no test for PrP Sc in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule detection, we developed a highly sensitive detection method for PrP Sc . Pathological prion protein aggregates were labeled by specific antibody probes tagged with fluorescent dyes, resulting in intensely fluorescent targets, which were measured by dual-color fluorescence intensity distribution analysis in a confocal scanning setup. In a diagnostic model system, PrP Sc aggregates were detected down to a concentration of 2 pM PrP Sc , corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sensitive than Western blot analysis. A PrP Sc -specific signal could also be detected 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 other prion diseases. Furthermore, this method could be adapted to the sensitive detection of other disease-associated amyloid aggregates such as in Alzheimer's disease.