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By Jason M. Wilham1, Christina D. Orrú2, Richard A. Bessen3, Ryuichiro Atarashi4, Kazunori Sano5, Brent Race6, Kimberly D. Meade-White7, Lara M. Taubner8, Andrew Timmes9, Byron Caughey10
PLOS Pathogens, 2010
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 PrPC 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 (SD50). Brain tissue from 263K scrapie-affected hamsters gave SD50 values of 1011-1012/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 SD50 values of 103.5–105.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD50 values of 102.0–102.9/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.
Introduction
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The transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders that include human Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), sheep scrapie, cervid chronic wasting disease (CWD), and transmissible mink encephalopathy (TME). The infectious agent, or prion, of the TSEs appears to be composed primarily of an abnormal, misfolded, oligomeric form of prion protein (PrPSc). PrPSc is formed post-translationally from the normal cellular prion protein (PrPC) [1], [2]. PrPSc, which in purified form can resemble amyloid fibrils, induces the polymerization and conformational conversion of PrPC to infectious PrPSc [3]–[5] or to PrPSc-like partially protease-resistant forms (PrPres) in a variety of in vitro reactions [4], [6]–[8]. These studies demonstrate that PrPSc can self-propagate, and although the mechanism is not fully understood, it appears to be a seeded or templated polymerization [9]–[11].
The ability to detect prions rapidly and sensitively would be an important asset in managing TSEs. Early prion detection in individuals is critical to the prevention of spread and the initiation of potential treatments. Prions can be found in a wide variety of tissues and accessible bodily fluids from infected mammalian hosts, including blood [12]–[17], breast milk [18], [19], saliva [15], [20], urine [21], [22], feces [23], and nasal fluids [24]. In most cases, our ability to rapidly measure prion infectivity in these fluids is limited by the low amount of infectious agent. Knowledge of the prion titers in these fluids or tissues and their products is key for prion diagnosis and in assessing the public health exposure risks to those materials.
The most direct and reliable assay for the detection of TSE infectivity is animal bioassay. Quantitation of infectivity can be achieved by end-point [25] or limiting dilution bioassays [26]. For some combinations of prion agent and host species, strong correlations between infectivity titer and disease incubation period have been established in laboratory rodents, allowing the use of incubation period to measure infectivity levels [27], [28]. The disadvantage of these bioassays is that they are animal-intensive, time-consuming and expensive. For certain murine-adapted scrapie strains, the cell culture based standard scrapie cell assay (SSCA) can also be used to measure infectivity levels by end-point and limiting dilution methods [29]. The SSCA offers several advantages over animal bioassays but it still requires weeks to perform and has been limited to a few mouse-adapted scrapie strains. The limitations of the animal bioassay and SSCA have provided strong motivation to develop more practical assays for prion quantitation.
Several extremely sensitive in vitro methods for prion detection have been reported. Using the protein misfolding cyclic amplification (PMCA) reaction in multi-round sonicated reactions with brain-derived PrPC as a substrate, as little as 1 ag of PrPsc can be detected [30]. The speed and practicality of PMCA assays have been improved by the use of bacterially-expressed recombinant PrPC (rPrPc) [31] and by substituting shaking for the sonication step as described for quaking-induced conversion (QuIC) reactions [32], [33]. The QuIC assay can detect sub-femtogram amounts of PrPSc (less than one lethal intracerebral dose) in hamster brain homogenates (BH) within a single day. The effectiveness of the QuIC assay for prion detection was demonstrated by its ability to discriminate normal from prion-infected hamsters using 2 µl samples of cerebral spinal fluid (CSF) [32], [33] or nasal lavage [24]. The latest adaptations of QuIC reactions have led to the sensitive detection of variant CJD (vCJD) in human tissue and scrapie in sheep tissue [33]. The readout for QuIC and PMCA assays is the detection of specific protease-resistant prion-seeded products by immunoblotting, which is difficult to adapt to automated high-throughput formats. An alternative, and potentially higher-throughput approach was used for the amyloid seeding assay (ASA) in which the fluorescent dye thioflavin T (ThT) was used to detect prion seeding of rPrPC polymerization [34]. The ASA can also detect protease sensitive disease-causing prions and has a 98% correlation with neuropathological signs of prion disease [35]. However, a potentially confounding aspect of ASA is the frequent spontaneous formation of rPrP fibrils (without seeding by prions) soon after prion-seeded reactions [34].
Until very recently, a major limitation of the PMCA, QuIC and ASA methods was the lack of prion quantitation. While the present paper was under review, Chen and colleagues reported a method called quantitative PMCA (qPMCA) in which PrPSc content is estimated by the number of PMCA rounds necessary for a positive response [36]. Here we describe a distinct end-point dilution approach to relative prion quantitation with in vitro prion seeding assays which is analogous to the end-point dilution titrations classically used in animal bioassays. At the same time, we describe a new prion-seeded rPrPc polymerization assay, real-time (RT)-QuIC, which combines several aspects of the QuIC assay (intermittent shaking, rPrPC preparation, sample preparation, and a lack of chaotropic salts) with a fluorescent ThT readout, but with greatly reduced spontaneous rPrP fibril formation. Some elements of this new assay were first developed using human rPrPc and designated “real-time QUIC” by analogy with real-time PCR (R. Atarashi, K. Satoh, K. Sano, T. Fuse, N. Yamaguchi, D. Ishibashi, T. Matsubara, T. Nakagaki, H. Yamanaka, S. Shirabe, M. Yamada, H. Mizusawa, T. Kitamoto, G. Klug, A. McGlade, S. J. Collins, and N. Nishida, manuscript submitted). The latter manuscript describes the non-quantitative application of RT-QuIC to the detection of prions in CSF of human patients with multiple types of sporadic CJD. In the present study, we have applied RT-QuIC to prions of sheep, deer and hamsters, and measured prion seeding activity in the nasal fluids and CSF of prion-infected hamsters. In conjunction with the end-point dilution analysis that we describe here, the RT-QuIC assay can rapidly determine relative prion concentrations with a sensitivity that rivals that of animal bioassays, but with greatly reduced time and cost.
Discussion
The RT-QuIC is a rapid prion detection assay that is more amenable to high-throughput applications than the original QuIC and much less prone to generate spontaneous, unseeded positive reactions than the ASA assay. The sensitivity of the RT-QuIC is similar to the in vivo bioassay in hamsters, but is roughly 50–200 times faster and much less expensive.
Using this assay, we have been able to rapidly detect and quantify prion seeding activities in nasal lavages from clinically TME-affected hamsters. Considering that nasal lavages are likely to dilute endogenous nasal cavity fluids by at least 100-fold, these results confirm and extend a previous report of substantial prion infectivity in nasal secretions from hamsters in the clinical phase of HY TME infection [24]. In the previous report, we detected nasal fluid prions by bioassay and the original immunoblot-based QuIC assay. In the current study, our ability to rapidly detect and quantitate prion seeding activity in nasal lavages using the RT-QuIC raises the possibility that such testing of nasal lavages or swabs could help in diagnosing prion disease infections of humans and animals on a high-throughput basis.
Our detection and quantitation of prion seeding activity in the CSF of 263K scrapie-infected hamsters suggests that CSF, being a relatively accessible specimen, should be collected for prion disease diagnosis by RT-QuIC. Interestingly, the CSF SD50 levels (105.7 and 104.6/ml) (Fig. 8) were similar to the highest value obtained for nasal lavages (105.7/ml) (Table 2). However, the CSF should have at least 100-fold lower levels of prion seeding activity than the endogenous nasal fluids, given the considerable dilution that occurs when the nasal cavities are flushed with lavage buffer.
The origin of the rare positives that we observed in negative control RT-QuIC reactions (Fig. 3) is difficult to ascertain. Because we simultaneously tested both positive and negative controls on the same plates, there was some, but obviously very low, potential for prion seeds to be inadvertently transferred from prion-seeded wells to adjacent negative control reactions. Moreover, given the very high sensitivity of the assay, even a minute contamination could elicit a false-positive reaction. Yet another explanation could be a cross contamination due to a failure of our plate sealer tape during the course of the reaction incubation. Fortunately, whether due to contamination or spontaneous amyloidogenesis, such apparent false positives are extremely rare and can simply be retested for confirmation.
It is likely that the same multimeric particles of abnormal PrP that stimulate conversion of PrPC or rPrPc to an abnormally folded form in in vitro reactions also cause prion “infections” in vivo. Consistent with this idea, we found that positive RT-QuIC reactions were obtained only with seeds derived from TSE-infected animals (except for the rare exceptions described above). Moreover, we obtained similar end-point dilutions of scrapie BH with both the bioassay and the RT-QuIC (Fig. 3). These results gave the appearance of a direct quantitative correspondence between the activities measured in these assays. Indeed, we expect that for prions of a particular strain and tissue source, there will be a proportional relationship between the activities measured by end-point dilution analyses with the RT-QuIC and animal bioassay. However, the sensitivities of these distinct assays will likely be influenced by some fundamentally different factors in vitro and in vivo and should not be expected to coincide as closely as they have in Figure 3 with all types of prion samples or all permutations of the assays. Indeed, further studies will be required to determine whether RT-QuIC assays detect naturally occurring PrP aggregates that are associated with familial PrP mutations and disease, but are non-infectious in bioassays. This anticipated variability of the RT-QuIC and bioassay with different prion sample types does not diminish the utility of the RT-QuIC in assessing the relative amount of prion seeding activity in samples of similar nature. In further developments of RT-QuIC assays for certain purposes, e.g. diagnostic testing, the possibility that certain abnormal non-PrP amyloids could give false positive RT-QuIC reactions should also be considered.
The end-point dilution strategy for determining relative seed concentrations should be applicable to amyloid seeding assays for a variety of misfolded protein aggregates regardless of the means of detecting the amyloid product, e.g. by ThT fluorescence as in the ASA [34] and RT-QuIC assays, or immunoblotting as in PMCA [8], rPrP-PMCA [31] or original QuIC [32] assays. Like the RT-QuIC, many amyloid-seeded polymerization reactions progress rapidly to completion after a lag phase, providing an all-or-nothing response within appropriately selected time frames. This typical feature of seeded polymerization reactions should facilitate determinations of the proportion of positive reactions among replicates at a given sample dilution. Analyses of data from serial dilutions of various samples using the Spearman-Kärber [39] or Reed-Muench [42] algorithms can improve estimates of SD50 values per unit volume, which then indicate the relative concentrations of seeding activity in the samples.
As noted above, Chen and colleagues have recently described an alternative means of obtaining quantitative estimates of prion seeding activity using PMCA reactions, called qPMCA [36]. Rather than assaying serial dilutions of a sample and determining the end point dilution, as we demonstrate here, a single sample dilution is assayed in serial PMCA reactions and the relative seeding activity is estimated from the number of serial PMCA rounds that are required to detect a positive response. The accuracy of qPMCA therefore depends on the strength of the inverse correlation between the prion seed concentration and number of rounds required. Although these investigators have documented such a correlation, its biochemical/kinetic basis remains unclear. In contrast, end-point dilution analyses can simply be explained as a titration of the active species to the detection limit. Further studies will be required to determine which approach to estimating relative prion concentrations is more robust and practical for comparing specific sample types.
Within individual RT-QuIC experiments composed of multiple, simultaneous reactions, we observed a clear dependence of the lag phase on the concentration of seed, as illustrated in Figure 1. The lag phase might be considered analogous to the TSE incubation period between the inoculation and the near terminal stage of disease. In certain combinations of host and TSE strain, standard curves correlating bioassay incubation period with inoculated dose can be established and used to determine relative prion infectivity levels in unknown samples without resorting to more time-consuming and animal-intensive end-point dilution analyses. An analogous correlation between prion seed concentration and lag phase in the seeding assays like the RT-QuIC or ASA might also allow for seeding activity estimation without testing serial dilutions of each unknown. However, further work will be required to determine the efficacy, reproducibility, and validity of such an approach. In the mean time, the end-point dilution approach described in the current manuscript provides a clear means of quantitating prion seeding activity.
In summary, the end-point dilution RT-QuIC analysis provides quantitative comparisons of prion seeding activity. Although the extent to which prion seeding activity correlates quantitatively with infectivity in vivo under various other circumstances remains to be determined, we have shown that the RT-QuIC assay provides rapid and highly sensitive discrimination of prion-infected and uninfected brain tissues, nasal lavages, and CSF.
Read full study at PLOS pathogens.
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Citation
Wilham JM, Orrú CD, Bessen RA, Atarashi R, Sano K, et al. (2010) Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays. PLoS Pathog 6(12): e1001217. doi:10.1371/journal.ppat.1001217
This page created 16 Dec 2010 21:14
Last updated 10 May 2011 14:19
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