Pre‐symptomatic detection of prions by cyclic amplification of protein misfolding

Soto, Claudio; Anderes, Laurence; Suardi, Silvia; Cardone, Franco; Castilla, Joaquı́n; Frossard, Marie Jose; Peano, Sergio; Saá, Paula; Limido, Lucia; Carbonatto, Michaela; Ironside, James W.; Torres, Juan María; Pocchiari, Maurizio; Tagliavini, Fabrizio

doi:10.1016/j.febslet.2004.12.035

Cited by 132 publications

(102 citation statements)

References 30 publications

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“…However, high-molecular-mass bands were observed in the amplified samples and may represent PrP aggregates or incomplete PK digestion. This was only observed when using PolyA to assist the PMCA reaction in this study but has been reported by others following PMCA without the addition of PolyA (Soto et al, 2005). No amplification of PrP Sc was observed in reaction mixtures containing only the CBH (Fig.…”

Section: Resultsmentioning

confidence: 50%

“…PMCA has been reported previously to increase the sensitivity of the detection of PrP Sc from brains of experimentally scrapie-infected rodents (Saborio et al, 2001;Bieschke et al, 2004;Deleault et al, 2003), cattle and sheep naturally infected with bovine spongiform encephalopathy and scrapie, respectively (Soto et al, 2005), and more recently from humans with Creutzfeldt-Jakob disease (Jones et al, 2007) and deer with chronic wasting disease (Kurt et al, 2007). Furthermore, the application of this technology for the detection of PrP Sc in blood, both at terminal stages of disease and in pre-symptomatic animals Fig.…”

Section: Discussionmentioning

confidence: 99%

“…We have demonstrated in this study, using PMCA, the efficient amplification of PrP Sc from brain and blood from sheep naturally infected with scrapie. Previously Soto et al (2005) indicated that PMCA could be applied successfully to sheep with natural scrapie with low levels of amplification (86) after 10 cycles. Here, we show increases of sensitivity up to 650 000-fold after 192 cycles of PMCA, which has facilitated the observation of PrP Sc present at very low concentrations.…”

Section: Discussionmentioning

confidence: 99%

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In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie

Thorne

Terry

2008

Journal of General Virology

101

116

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Scrapie is a fatal, naturally transmissible, neurodegenerative prion disease that affects sheep and goats and is characterized by the accumulation of a misfolded protein, PrPSc, converted from host-encoded PrPc, in the central nervous system of affected animals. Highly efficient in vitro conversion of host PrPc to PrPSc has been achieved in models of scrapie and in natural prion diseases by protein misfolding cyclic amplification (PMCA). Here, we demonstrate amplification, by serial PMCA, of PrPSc from individual sources of scrapie-infected sheep. Efficiency of amplification was affected by the pairing of the source of PrPSc with the control brain substrate of different genotypes of PrP. In line with previous studies, efficiency of amplification was greatly enhanced with the addition of a synthetic polyanion, polyadenylic acid (PolyA), facilitating rapid detection of low levels of PrPSc from body fluids such as blood. To this end PrPSc was amplified, in a 3 day PMCA assay, from blood leukocyte preparations from VRQ/VRQ scrapie-affected sheep at clinical end point. While PolyA-assisted PMCA resulted in spontaneous conversion of PrPc, we were able to distinguish blood samples from unaffected and affected sheep under controlled conditions. This study demonstrates that highly efficient amplification of PrPSc can be achieved for ovine scrapie from both brain and blood from naturally infected sheep and shows potential applications for improvements in current diagnostics and pre-mortem testing.

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Section: Resultsmentioning

confidence: 50%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie

Thorne

Terry

2008

Journal of General Virology

101

116

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“…PMCA allows an undetectable amount of PrP Sc to convert exogenously provided PrP C to PrP Sc after multiple cycles of sonication (11,33,40). Another study suggested that simple incubation without sonication is sufficient (41).…”

mentioning

confidence: 99%

Test for Detection of Disease-Associated Prion Aggregate in the Blood of Infected but Asymptomatic Animals

Chang

Cheng

Yin

et al. 2007

Clin Vaccine Immunol

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We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrP Sc . However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.

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“…In this method (Am-A-FACTT), an in vitro PrP Sc amplification step (Am) is included to further improve the detection limit. This methodology is based on the Protein Misfolding Cyclic Amplification (PMCA) which allows an undetectable amount of PrP Sc to convert exogenously provided PrP C to PrP Sc with [41][42][43] or without [44] multiple cycles of sonication. Similiarly, in the Am-A-FACTT, plasma or tissue homogenate from normal animals is incubated with infected samples to amplify PrP Sc in the sample.…”

Section: Prion Diseasementioning

confidence: 99%

Non-invasive, ultra-sensitive, high-throughput assays to quantify rare biomarkers in the blood

Freudenberg

Bembas

Greene

et al. 2008

Methods

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Many diseases are easier to treat and control when detected at an early stage of disease progression. Often, disease-related antigens or biomarkers are shed from the primary site and present in the blood. Unfortunately, there are very few tests capable of detecting these rare biomarkers in the blood. A blood test would be very useful to diagnose the disease earlier, monitor effectiveness of treatments, predict recurrence, and monitor recurrence. There is certainly a need to develop assays that are ultrasensitive, non-invasive and high-throughput. Here we describe several highly sensitive immunological assays we have developed to detect rare serum antigens. Initially we created an assay named immunodetection amplified by T7 RNA polymerase (IDAT). To enhance the effectiveness and streamline the procedure, this assay was amended to the facile amplification system termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). These assays have been used to analyze the tumor antigen HER2 and the prion protein PrP Sc . They can also be applied to other tumor markers or antigens from a variety of diseases such as cardiovascular disease, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, and hepatitis. These tests are not limited to testing only serum, but may also be applicable to detecting biomarkers in tissue, saliva, urine, cerebrospinal fluid, etc. Clearly, the FACTT-based technology represents an important step in the detection of rare molecules in fluids or tissues for a variety of diseases.

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Pre‐symptomatic detection of prions by cyclic amplification of protein misfolding

Cited by 132 publications

References 30 publications

In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie

In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie

Test for Detection of Disease-Associated Prion Aggregate in the Blood of Infected but Asymptomatic Animals

Non-invasive, ultra-sensitive, high-throughput assays to quantify rare biomarkers in the blood

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