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      Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays


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          A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP C to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD 50). Brain tissue from 263K scrapie-affected hamsters gave SD 50 values of 10 11-10 12/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD 50 values of 10 3.5–10 5.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD 50 values of 10 2.0–10 2.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.

          Author Summary

          Prion diseases are deadly infectious neurodegenerative disorders of mammals which involve the misfolding of host prion protein. To better manage these diseases, we need to be able to detect and quantify the infectious particles, or prions, in biological samples. However, current tests lack the sensitivity, speed and/or quantitative capabilities required for many important applications in medicine, agriculture, wildlife biology and research. To address this problem, we have developed a new prion assay that is highly sensitive, rapid, and quantitative. This assay takes advantage of the ability of miniscule amounts of infectious prions to seed the misfolding of large excesses of normal prion protein in test tube reactions. Quantitation is achieved by testing a range of sample dilutions and determining loss of seeding activity, i.e. the end-point dilution. Similar analyses have long been used to quantify prions by inoculation into animals; however, such bioassays take months or years to perform and are both animal-intensive and expensive. Our new method provides a more practical means of detecting and quantifying prions. So far, we have applied this assay to prions from sheep, deer, and hamsters, and have found surprisingly high levels of prions in the nasal and cerebral spinal fluids of infected hamsters.

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          Most cited references31

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          In vitro generation of infectious scrapie prions.

          Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathy (TSE) diseases. They are thought to be composed exclusively of the protease-resistant prion protein (PrPres) that replicates in the body by inducing the misfolding of the cellular prion protein (PrPC). Although compelling evidence supports this hypothesis, generation of infectious prion particles in vitro has not been convincingly demonstrated. Here we show that PrPC --> PrPres conversion can be mimicked in vitro by cyclic amplification of protein misfolding, resulting in indefinite amplification of PrPres. The in vitro-generated forms of PrPres share similar biochemical and structural properties with PrPres derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrPres led to a scrapie disease identical to the illness produced by brain infectious material. These findings demonstrate that prions can be generated in vitro and provide strong evidence in support of the protein-only hypothesis of prion transmission.
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            Formation of native prions from minimal components in vitro.

            The conformational change of a host protein, PrP(C), into a disease-associated isoform, PrP(Sc), appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt-Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrP(C) and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrP(Sc) molecules in a purified system requires accessory polyanion molecules. In addition, we found that PrP(Sc) molecules could be formed de novo from these defined components in the absence of preexisting prions. Inoculation of samples containing either prion-seeded or spontaneously generated PrP(Sc) molecules into hamsters caused scrapie, which was transmissible on second passage. These results show that prions able to infect wild-type hamsters can be formed from a minimal set of components including native PrP(C) molecules, copurified lipid molecules, and a synthetic polyanion.
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              Quantification of beta-sheet amyloid fibril structures with thioflavin T.


                Author and article information

                Role: Editor
                PLoS Pathog
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                December 2010
                December 2010
                2 December 2010
                : 6
                : 12
                : e1001217
                [1 ]Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, Hamilton, Montana, United States of America
                [2 ]Department of Biomedical Sciences and Technologies, University of Cagliari, Monserrato, Italy
                [3 ]Veterinary Molecular Biology, Montana State University, Bozeman, Montana, United States of America
                [4 ]Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Kyushu, Japan
                University of Alberta, Canada
                Author notes

                Conceived and designed the experiments: JMW CDO RAB RA KS BC. Performed the experiments: JMW CDO RAB BR KDMW LMT AT. Analyzed the data: JMW RAB. Contributed reagents/materials/analysis tools: JMW RAB. Wrote the paper: JMW BC.

                This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
                : 4 May 2010
                : 3 November 2010
                Page count
                Pages: 15
                Research Article
                Biochemistry/Protein Chemistry
                Infectious Diseases
                Infectious Diseases/Prion Diseases
                Neurological Disorders/Prion Diseases

                Infectious disease & Microbiology
                Infectious disease & Microbiology


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