title: Detection of prion protein particles in blood plasma of scrapie infected sheep.
abstract: Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.
authors.affiliation: Institute of Physical Biology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany. bannacho@hhu.de
authors.forename: Oliver
authors.initials: O
authors.lastname: Bannach
authors.forename: Eva
authors.initials: E
authors.lastname: Birkmann
authors.forename: Elke
authors.initials: E
authors.lastname: Reinartz
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.forename: Jan P M
authors.initials: JP
authors.lastname: Langeveld
authors.forename: Robert G
authors.initials: RG
authors.lastname: Rohwer
authors.forename: Luisa
authors.initials: L
authors.lastname: Gregori
authors.forename: Linda A
authors.initials: LA
authors.lastname: Terry
authors.forename: Dieter
authors.initials: D
authors.lastname: Willbold
authors.forename: Detlev
authors.initials: D
authors.lastname: Riesner
mesh_terms: D000818:Animals; D011328:Prions / Q000097:blood*; D012608:Scrapie / Q000097:blood*; D012756:Sheep
chemical_list: D011328:Prions