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 (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.
Bieschke
J
J
Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen, Germany.
Giese
A
A
Schulz-Schaeffer
W
W
Zerr
I
I
Poser
S
S
Eigen
M
M
Kretzschmar
H
H
D000818:Animals; D007562:Creutzfeldt-Jakob Syndrome / Q000134:cerebrospinal fluid* / Q000175:diagnosis*; D006224:Cricetinae; D005456:Fluorescent Dyes; D006801:Humans; D010865:Pilot Projects; D018620:PrPSc Proteins / Q000134:cerebrospinal fluid*; D011328:Prions / Q000032:analysis; D015203:Reproducibility of Results; D012608:Scrapie; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; D018620:PrPSc Proteins; D011328:Prions