Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy

Trieschmann, Lothar; Santos, Anne Navarrete; Kaschig, Katja; Torkler, Sandra; Maas, Elke; Schätzl, Hermann; Böhm, Gerald

doi:10.1186/1472-6750-5-26

Cited by 27 publications

(10 citation statements)

References 17 publications

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“…However, plasma components strongly inhibited both assays (data not shown), consistent with previously reported inhibition of protein misfolding cyclic amplification (PMCA) (38) and flow cytometric assays (23). Accordingly, we sought methods to capture and concentrate prions in a detectable form from plasma.…”

Section: Resultssupporting

confidence: 90%

Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion

Orrú

Wilham

Raymond

et al. 2011

mBio

138

153

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A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 1014-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call “enhanced QuIC” (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples.

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Section: Resultssupporting

confidence: 90%

Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion

Orrú

Wilham

Raymond

et al. 2011

mBio

138

153

View full text Add to dashboard Cite

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“…In BSE-afflicted cattle infectivity is absent from the lymphatic system and has never been reported in blood [35], [36]. However, seeding activity was demonstrated in a small number of BSE serum samples [37]. Considerable effort was spent not only in improving the sensitivity of the assay but also in optimizing the preparation of PrP aggregates from blood plasma.…”

Section: Discussionmentioning

confidence: 99%

Detection of Prion Protein Particles in Blood Plasma of Scrapie Infected Sheep

et al. 2012

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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.

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“…Based on those observations and suspecting that lack of infectivity detection in many experimental transmission experiments was due to the low sensitivity of the available techniques, several sophisticated methods for PrP Sc detection in blood were developed using animal models of prion infection or blood spiked with brain material from TSE-affected individuals [208][209][210][211][212][213][214][215][216][217][218]. The report of the first cases of vCJD transmission through contaminated blood transfusion in 2004 [219][220][221] entailed a point of inflexion, since a theoretical hazard became real, causing a significant increase in researchers dedicated to investigate infectivity and PrP Sc presence in blood as well as improved detection methods.…”

Section: Prp Sc In Blood According To Transmission Studiesmentioning

confidence: 99%

Detection of Pathognomonic Biomarker PrPSc and the Contribution of Cell Free-Amplification Techniques to the Diagnosis of Prion Diseases

et al. 2020

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Transmissible spongiform encephalopathies or prion diseases are rapidly progressive neurodegenerative diseases, the clinical manifestation of which can resemble other promptly evolving neurological maladies. Therefore, the unequivocal ante-mortem diagnosis is highly challenging and was only possible by histopathological and immunohistochemical analysis of the brain at necropsy. Although surrogate biomarkers of neurological damage have become invaluable to complement clinical data and provide more accurate diagnostics at early stages, other neurodegenerative diseases show similar alterations hindering the differential diagnosis. To solve that, the detection of the pathognomonic biomarker of disease, PrP Sc , the aberrantly folded isoform of the prion protein, could be used. However, the amounts in easily accessible tissues or body fluids at pre-clinical or early clinical stages are extremely low for the standard detection methods. The solution comes from the recent development of in vitro prion propagation techniques, such as Protein Misfolding Cyclic Amplification (PMCA) and Real Time-Quaking Induced Conversion (RT-QuIC), which have been already applied to detect minute amounts of PrP Sc in different matrixes and make early diagnosis of prion diseases feasible in a near future. Herein, the most relevant tissues and body fluids in which PrP Sc has been detected in animals and humans are being reviewed, especially those in which cell-free prion propagation systems have been used with diagnostic purposes.

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Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy

Cited by 27 publications

References 17 publications

Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion

Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion

Detection of Prion Protein Particles in Blood Plasma of Scrapie Infected Sheep

Detection of Pathognomonic Biomarker PrPSc and the Contribution of Cell Free-Amplification Techniques to the Diagnosis of Prion Diseases

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