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

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

^†Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttingen, Germany; and Departments of ^‡Neuropathology and ^§Neurology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany

Contributed by M. Eigen

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.

Footnotes

↵ * J.B. and A.G. contributed equally to this work.
↵ ¶ To whom reprint requests should be addressed. E-mail: Haus.Kretzschmar{at}inp.med.uni-muenchen.de.
↵ ‖ Present address: Department of Neuropathology, University of Munich, Marchioninistrasse 17, 81377 München, Germany.
Abbreviations:

CJD,

Creutzfeldt–Jakob disease;

CSF,

cerebrospinal fluid;

FCS,

fluorescence correlation spectroscopy;

SIFT,

scanning for intensely fluorescent targets