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This 96-well-plate ‘real-time quaking-induced conversion’ assay allows the detection of abnormal prion protein in human brain and CSF samples. It can be applied to study many protein misfolding diseases, as well as for drug screening and prion strain discrimination. The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrP Sc ) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrP Sc seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays—such as protein misfolding cyclic amplification (PMCA)—which are based on sonication, the RT-QuIC technique is based on prion seed–induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes ∼87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimer's and Parkinson's disease.

We report a case of preclinical variant Creutzfeldt-Jakob disease (vCJD) in a patient who died from a non-neurological disorder 5 years after receiving a blood transfusion from a donor who subsequently developed vCJD. Protease-resistant prion protein (PrP res) was detected by western blot, paraffin-embedded tissue blot, and immunohistochemistry in the spleen, but not in the brain. Immunohistochemistry for prion protein was also positive in a cervical lymph node. The patient was a heterozygote at codon 129 of PRNP, suggesting that susceptibility to vCJD infection is not confined to the methionine homozygous PRNP genotype. These findings have major implications for future estimates and surveillance of vCJD in the UK.

Chronic wasting disease is a transmissible spongiform encephalopathy of cervids. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein (PrPC) to pathogenic conformers (PrPSc), and the pathogenic forms accumulate in the brain and other tissues. Real-time Quaking Induced Conversion (RT-QuIC) can be used for the detection of prions and for prion strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how either PrPSc from cervids of different genotypes or PrPSc from different sources of CWD influence the fibril formation of recombinant bank vole (BV) or human prion proteins using RT-QuIC. We found that reaction mixtures seeded with PrPSc from different genotypes of white-tailed deer or reindeer brains have similar conversion efficiency with both substrates. Also, we observed similar results when assays were seeded with different sources of CWD. Thus, we conclude that the genotypes of all sources of CWD used in this study do not influence the level of conversion of PrPC to PrPSc.

Prion diseases are caused by an unconventional infectious agent termed prion, composed mainly of the misfolded prion protein (PrP Sc ) 1 . The development of highly sensitive assays for biochemical detection of PrP Sc in blood is a top priority for minimizing the spread of the disease 2 . Here we show that the protein misfolding cyclic amplification (PMCA) technology 3 can be automated and optimized for high-efficiency amplification of PrP Sc . We show that 140 PMCA cycles leads to a 6,600-fold increase in sensitivity over standard detection methods. Two successive rounds of PMCA cycles resulted in a 10 million–fold increase in sensitivity and a capability to detect as little as 8,000 equivalent molecules of PrP Sc . Notably, serial PMCA enables detection of PrP Sc in blood samples of scrapie-afflicted hamsters with 89% sensitivity and 100% specificity. These findings represent the first time that PrP Sc has been detected biochemically in blood, offering promise for developing a noninvasive method for early diagnosis of prion diseases.

Prions are thought to be the proteinaceous infectious agents responsible for transmissible spongiform encephalopathies (TSEs). PrPSc, the main component of the infectious agent, is also the only validated surrogate marker for the disease, and its sensitive detection is critical for minimizing the spread of the disease. We detected PrPSc biochemically in the blood of hamsters infected with scrapie during most of the presymptomatic phase of the disease. At early stages of the incubation period, PrPSc detected in blood was likely to be from the peripheral replication of prions, whereas at the symptomatic phase, PrPSc in blood was more likely to have leaked from the brain. The ability to detect prions biochemically in the blood of infected but not clinically sick animals offers a great promise for the noninvasive early diagnosis of TSEs.

The misfolded form of the prion protein, PrP Sc , can be quantified in a variety of tissues and fluids using a quantitative version of the popular protein misfolding cyclic amplification (PMCA) assay. Prions, the proteinaceous infectious agent responsible for prion diseases, can be detected with high sensitivity by protein misfolding cyclic amplification (PMCA) technology. Here we describe a quantitative PMCA procedure to calculate the concentration of very low levels of prions in biological samples. Using this procedure, we determined the quantities of misfolded prion protein (PrP Sc ) in brain, spleen, blood and urine of scrapie-affected hamsters.

It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 10³ID₅₀ as measured by intracerebral inoculation of tg338 mice (ID₅₀ IC in tg338). This was consistent with a whole blood titer greater than 10³·⁶ID₅₀ IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation.

The most widely recognized human prion disease, or transmissible spongiform encephalopathy is variant Creutzfeldt-Jakob disease (vCJD)2. vCJD is related to consumption of beef from cattle infected with bovine spongiform encephalopathy (BSE) colloquially referred to as mad cow disease. The mature PrPSc aggregates are characterized by their resistance to proteinase K digestion, apple-green birefringence when stained with Congo red, and their ability to enhance the fluorescence emission of the dye thioflavin T. In vitro, conversion of PrPC to PrPSc can be driven by high concentrations of PrP, low pH conditions, chemical denaturants, and the presence of PrPSc to seed polymerization. For comparison, the amount of PrPSc in whole blood of symptomatic individuals with vCJD was estimated to be equivalent to a 10 9 dilution of vCJD brain homogenate on the basis of the number of PMCA cycles required for detection of a majority of the vCJD blood samples tested. Previously published online at DOI: 10.1373/clinchem.2017.272229 ©2017 American Association for Clinical Chemistry 2 Nonstandard abbreviations: vCJD, variant Creutzfeldt-Jakob Disease; sCJD, sporadic CJD; PrP, prion protein; CSF, cerebrospinal fluid; PMCA, protein misfolding cyclic amplification; RT-QuIC, real-time quaking-induced conversion.

On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein (PrPSc), we have been interested in how these peptides interact with PrPSc. After screening peptides from the entire human PrP sequence, we found two peptides (PrP₁₉₋₃₀ and PrP₁₀₀₋₁₁₁) capable of binding full-length PrPSc in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein (PrPC). The limit of detection for captured PrPSc was calculated to be 8 amol from a [almost equal to]10⁵-fold dilution of 10% (wt/vol) human variant Creutzfeldt-Jakob disease brain homogenate, with >3,800-fold binding specificity to PrPSc over PrPC. Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and PrPSc. Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide-PrPSc interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in PrPSc, is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of PrPC may play a role in the recruitment of PrPC to PrPSc.

The disease-associated water-soluble form of hamster prion protein (ws-PrPSc) has recently been found to be less stable than classical PrPSc. Since the stability of PrP to degradation correlates with its glycosylation level, the aim of this study was to investigate whether there are differences between the glycosylation of ws-PrPSc and classical PrPSc of hamster which might account for the ws-PrPSc minor stability compared with that of the classical PrPSc. Thus, ws-PrP and classical PrP were captured from noninfected or scrapie-infected hamster brain homogenate [high-speed supernatant (SHS) and high-speed pellet (PHS)] and blood plasma by anti-PrP antibodies (3F4 and 6H4) and subjected to screening for glycans by lectins under denaturing or nondenaturing procedures in a sandwich lectin-ELISA. Glycans have been found in minor quantities and differently exposed on ws-PrPSc from SHS and plasma compared with classical PrPSc from PHS. These differences have been shown to be potentially responsible for the instability of ws-PrPSc. Treatment of infected blood with GdnHCl significantly (P<0.01) increased the detection of ws-PrPSc in ELISA, reflecting an increase in its stability, and showed efficacy in removing high-abundance proteins in silver-stained gels. This increase in ws-PrPSc stability is due to an interaction of GdnHCl not only with high-abundance proteins but also with the ws-PrPSc glycosylation with particular regard to the mannose sugar. Analysis of lectins immunoreactivity toward total proteins from plasma collected before and at different time points after infection revealed that mannose might exert a stabilizing effect toward all of hamster blood glycoproteins, regardless of scrapie infection. Since low levels of ws-PrPSc/soluble-infectivity have been estimated both in blood and brain of hamster, this glycosylation-related instability may have negatively influenced the propensity of ws-PrPC to convert to ws-PrPSc both in blood and the brain. Therefore, PrPC glycosylation characteristics may provide a tool for the determination risk of prion transmissibility.