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.

Factors intrinsic and extrinsic to blood hamper the development of a routine blood test for human prion diseases

“…Rather, it may be related to the variable presence of factors intrinsic and extrinsic to blood that differentially affect the concentration and/or detection of PrP TSE in blood as compared to other tissues. This supposition is supported by the repeated confirmation that blood PrP TSE probably differs from brain PrP TSE in its conformation and aggregation properties (Bannach et al, 2012;Lacroux et al, 2012; reviewed by Lukan et al, 2013). It is also supported by the fact that PrP TSE present in blood is both associated with cellular components and in a cellfree state (Table 1), implying that, due dynamic equilibrium, the ratio of free to associated PrP TSE is continuously subject to change during the disease period.…”

“…In this assay, PrP TSE was detected in the PBMCs of 55 % and 71 % of scrapie-and BSE-infected animals, respectively, but was not detected in the plasma (Terry et al, 2009). The same blood pool used for this experiment by Terry et al (2009) was also used for sFIDA, an approach developed by Bannach et al (2012). sFIDA exploits the strong tendency of PrP TSE to form large aggregates.…”

“…However, based on the fact that PrP TSE from vCJD-infected brain homogenate spiked into whole blood can be detected with good sensitivity at dilution of 10 210 , hypothetically, it should be possible to detect prions in the blood of vCJD patients at the preclinical stage of the disease (Edgeworth et al, 2011). Moreover, given that other blood tests based on different principles showed similar behaviour, but that not all blood samples obtained from clinically affected subjects tested positive for PrP TSE (Terry et al, 2009;Bannach et al, 2012;Jackson et al, 2014;Lacroux et al, 2014), the sensitivity of the tests is unlikely to be the only factor responsible for the failed detection of PrP TSE . Rather, it may be related to the variable presence of factors intrinsic and extrinsic to blood that differentially affect the concentration and/or detection of PrP TSE in blood as compared to other tissues.…”

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

“…can reliably identify preclinical cases to minimize the risk of iatrogenic disease transmission via blood-derived products. Experimental detection of PrP TSE in animal cerebrospinal fluid (CSF) (29), whole blood (21,22), plasma (27)(28)(29)(30)71), buffy coat (24 -26), and urine (74) and in human CSF (12,75), whole blood (6 -8), and urine (76,77) has been achieved by different methods that rely on the concentration or amplification techniques to bring PrP TSE levels to the detection threshold of biochemical assays. Although the presence of PrP TSE in blood exosomes has been suggested previously (48), biochemical detection has been complicated by the low levels of PrP TSE in blood and the concomitantly large volumes required for exosome isolation by standard methods.…”

“…PrP TSE has been successfully detected in the following: in whole blood of experimentally infected mice and hamsters, in sheep with natural scrapie and experimental BSE, and in white-tailed deer afflicted with CWD (21)(22)(23); in buffy coats of hamsters and sheep experimentally infected with scrapie (24 -26), as well as in plasma of mice and hamsters with scrapie; and in sheep and white-tailed deer naturally and experimentally infected with scrapie and CWD, respectively (27)(28)(29)(30). However, it is still not fully understood in what blood component TSE infectivity and/or PrP TSE reside and whether and how blood contributes to the spread of the disease from the periphery to the brain.…”