Establishing quantitative real-time quaking-induced conversion (qRT-QuIC) for highly sensitive detection and quantification of PrPSc in prion-infected tissues
© Shi et al.; licensee BioMed Central Ltd. 2013
Received: 20 July 2013
Accepted: 20 July 2013
Published: 2 August 2013
PrPSc, the only known constituent of prions, the infectious agents causing prion diseases, can be detected by real-time quaking-induced conversion (RT-QuIC). However, there is no efficient method to quantify the amount of PrPSc by RT-QuIC.
Here we introduce quantitative RT-QuIC (qRT-QuIC) to quantify with high accuracy minute amounts of PrPSc in the brain and various peripheral tissues at levels far below detection by in vivo transmission. PrPSc is relatively resistant to treatment with proteinase K (PK). However, as there can also be a fraction of pathological PrP that is digested by PK, we use the term PrP27-30 to denote to the amount of PrPSc that can be detected by immunoblot after PK treatment. qRT-QuIC is based upon the quantitative correlation between the seeded amount of PrP27-30 and the lag time to the start of the conversion reaction detected by RT-QuIC. By seeding known amounts of PrP27-30 quantified by immunoblot into qRT-QuIC a standard calibration curve can be obtained. Based on this calibration curve, seeded undetermined amounts of PrP27-30 can be directly calculated. qRT-QuIC allowed to quantify PrP27-30 concentrations at extremely low levels as low as 10-15.5 g PrP27-30, which corresponds to 0.001 LD50 units obtained by in vivo i.c. transmission studies. We find that PrP27-30 concentration increases steadily in the brain after inoculation and can be detected at various time points during the incubation period in peripheral organs (spleen, heart, muscle, liver, kidney) in two experimental scrapie strains (RML, ME7) in the mouse.
We suggest that an automatic quantitative system to measure disease progression as well as prion contamination of organs, blood and food product is feasible. Moreover, the concept of qRT-QuIC should be applicable to measure other disease-associated proteins rich in β-pleated structures (amyloid) that bind ThT and that show seeded aggregation.
Prion diseases are a group of transmissible neurodegenerative lethal disorders in humans, cattle, sheep, elk, mink and experimental animals. These diseases are characterized by neuronal death and the accumulation of pathological disease-associated prion protein (PrPSc) in the central nervous system . PrPSc is thought to be the essential, if not the exclusive, component of the transmissible agent, or prion. Prion propagation seems to rely on autocatalytic amplification of PrPSc by converting the host-encoded cellular prion protein (PrPC) to the pathogenic PrPSc form without the participation of nucleic acids [2, 3]. The conversion of PrPC to PrPSc is a post-translational event and involves a conformational change of the protein [4, 5]. To distinguish between PrPSc which is isolated from infectious tissue and is per definitionem associated with the TSE agent on the one hand and structurally altered PrP, which has been converted into a ProteinaseK-resistant form in vitro, on the other, we refer to the latter as ‘PrPres’. The most commonly used approach to distinguish PrPSc from PrPC when analyzing infected tissues is based on pre-treatment with proteinase K as PrPSc is relatively PK-resistant. However, as there can also be a fraction of pathological PrP that is digested by PK treatment , we use the term PrP27-30  in this manuscript whenever we refer to the amount of PK-resistant PrPSc that can be detected by immunoblotting.
Although protease-resistant PrP27-30 is often used as a definitive biological marker for TSE infection, the titer of infectivity measured by inoculation in experimental animals does not always fully correlate with the amount of PrP27-30 detected by immunoblot. The correlation of the seeding activity on rPrPsen conversion into rPrPres measured by RT-QuIC with the amount of PrP27-30 is also a complex issue. For example, it has been shown that vCJD prions have less seeding activity than sCJD prions despite the relatively high PrP27-30 concentration , and that prions from brains of 263 K-affected mice with little immunoblot-detectable PrP27-30 have a seeding activity comparable to that associated with the high-PrPSc strain, 139A . In previous studies, we showed that the complex correlation between amounts of PrP27-30 and infectivity in vivo and seeding activity in vitro can be explained at least in part by differences in size distribution of PrP aggregates . Consequently, RT-QuIC seeding activity may correlate more closely with prion infectivity than with PrP27-30 levels, which can be considered an advantage in regard to the development of assays for prion detection.
We observed that in the RT-QuIC reaction, adding small amounts of PrPSc resulted in a delayed initiation of conversion detected by ThT fluorescence (Figure 1b). This delay and the shape of the resulting ThT fluorescence curve appeared similar to the kinetics of amplification of DNA that is used for quantitative RT-PCR analysis [14, 15]. Thus we investigated whether real-time protein amplification was quantitative and established a highly sensitive assay for the quantification of prion infectivity in a high-throughput system based on measuring lag time of detectable conversion. We termed this approach qRT-QuIC and show that it allows quantitation of prions in various tissues with a detection limit corresponding to 0.001 LD50 units.
Results and discussion
Establishing quantitative RT-QuIC
Measuring PrP27-30 concentrations in brains and peripheral organs at different dpi of prion infection
To demonstrate the feasibility of qRT-QuIC in the assessment of bio-hazard risks, the concentration of PrP27-30/g tissue (g/g) in various tissues measured by qRT-QuIC was compared to the reported half-lethal doses (LD50) of both prion strains. The known LD50 of RML and ME7 are 10-8.8 and 10-8.3 g of terminal brains, respectively . Using the PrP27-30 concentration measured by qRT-QuIC, we can roughly extrapolate that 1 LD50 unit contained 10-12.93 g of PrP27-30 for RML and 10-12.72 g for ME7. One LD50 unit is shown as the gray dotted lines in Figure 5 to indicate the extent of prion concentration in each gram of tissue (see also Additional file 1: Tables S2 and S3), suggesting that qRT-QuIC can be used for estimating prion contamination in biological materials. As our protocol uses an extraction method for PrP27-30 prior to qRT-QuIC that removes potentially interfering factors present in peripheral tissues, the calibration curve obtained for brain-derived PrP27-30 should also provide meaningful results for other tissues from the same species. Moreover, our findings for RML and ME7 indicate that similar assay conditions can be efficient for different strains. However, to ensure optimum sensitivity, the exact conditions of the qRT-QuIC assay need to be established for different species and strains.
In a recent study, Wilham and colleagues  used SD50 (50% seeding dose) to quantify seeding activity in the RT-QuIC. SD50 was defined as minimum seeded amount causing 50% of RT-QuIC reactions to be positive. Using end-point titration in a dilution series for quantification, SD50 was shown to correlate with the infectivity of 263 K strains in hamster. To obtain SD50 values for one prion strain in RT-QuIC, serially diluted standard sample are required, e.g., serially diluted prion-infected reference brain homogenate and serially diluted undetermined materials. Thus, this approach requires multiple repeats to yield the percentage of positive reactions for every dilution. In contrast, qRT-QuIC is a lag time-based assay and the amount of PrP27-30 equivalents in suspected materials can be directly calculated and quantified with much fewer repeats based on the calibration curve. The respective advantages and disadvantages of end-point titration and lag time assays are well known from assays of prion infection in vivo. End-point titration provides an accurate measure of infectious units. However, end-point in vivo assay also requires multiple repeats for each dilution of seeds to obtain the titration of one prion strain, whereas the incubation time in vivo assay is more commonly used for prion quantification as much less animals are required. Hence, we conclude that the qRT-QuIC assay which uses lag time is more suitable than SD50 measurements for quantitative detection and high-throughput assay for prion diagnosis.
In conclusion, qRT-QuIC is a new advancement of the RT-QuIC system, which enables us to quantify PrP27-30 concentrations at extremely low levels as low as 10-15.5 g PrP27-30, which corresponds to 0.001 LD50 units. qRT-QuIC is based upon the quantitative correlation between the seeded amount of PrP27-30 and the lag time to the start of the conversion reaction detected by RT-QuIC. The principle of generating standard curves by monitoring the fluorescence and setting threshold levels makes qRT-QuIC applicable to the development of an automatic system similar to quantitative RT-PCR. qRT-QuIC can be used as a rapid and reliable novel research tool, increase the precision of diagnosis, and help to follow the effects of therapy of prion diseases. Moreover, this concept should be applicable to measure other proteins rich in β-pleated structures (amyloids) that bind ThT and that show seeded aggregation.
rPrPsen expression and purification
BL21 (DE3) E.coli and pET41a system (Merk, Germany) were used to express mouse rPrPsen. Bacteria were cultured in LB medium at 37°C with 220 rpm shaking and were added 1 mM of IPTG (final concentration) when OD reached 0.7 to 0.9. The cell pellet was harvested by 10,000 g centrifugation at 4°C for 10 min after 4 hours induction. Thereafter, cells were resuspended by BugBuster Master Mix (Novagen, Germany) containing rLysozyme and Benzonase for extracting inclusion bodies (manual for BugBuster, Novagen, Germany). The extracted inclusion bodies were denatured with 8 M Guanidine hydrochloride at 25°C for 1 hour. The denatured protein solution was centrifuged at 16,000 g at 4°C for 20 min to remove the debris.
We chose Ni2+-NTA superflow (Qiagen, Germany) resin for protein purification. The NTA resin was prepared by following the manufacturer’s manual. The denatured protein was loaded onto prepared resin followed by binding with inversion on the rotor at 25°C for 1 hour. After that, the resin was loaded into the column (GE healthcare, USA) followed by connecting to the AKTA prime (GE healthcare, USA). The followed purification procedures including refolding and elution were performed as described .
After elution, the rPrPsen solution was loaded into 6 KDa Cellu Sep dialysis tubing (Interchim, France) followed by immersion in pre-chilled dialysis buffer (9 mM NaH2PO4, 1 mM Na2HPO4, pH 5.9) at 4°C. The dialysis was done in 2 successive steps, which were 2 hours and 18 hours, respectively. 100 volumes of dialysis buffer were used for each step. The dialyzed solutions were sterilized with a 0.22 μm filter (Millipore, USA) and the absorption was measured at 280 nm for calculating the concentration of rPrPsen. The concentration of each fraction was adjusted to 0.5 mg/ml by adding sterilized and chilled dialysis buffer. The rPrPsen solution was aliquoted and frozen in liquid nitrogen, followed by transfer to a freezer (-80°C) for long-term storage.
6-week old C57BL/6 mice were inoculated with mouse-adapted RML and ME7 scrapie (i.c). 10 μl of 10% brain homogenate in 1 × PBS (pH 7.2) was used for each inoculum. 7 time-points (30, 60, 90, 120, 135, 150 and 170 dpi, days post inoculation) were set for RML-infection and 6 time-points (30, 60, 90, 120, 135 and 150 dpi) were for ME7-infection. At each dpi, five mice were sacrificed with CO2. To harvest the tissues (brain, heart, liver, spleen, lung, kidney and hindlimb muscle) they were washed with chilled 1 × PBS containing 5% sodium citrate to remove the blood. Then tissues were weighed and stored in liquid nitrogen. The tissues of age-related control C57BL/6 mice were prepared following the same procedure.
We followed a published protocol  to purify PrP27-30. The RML- and ME7-infected mouse tissue was prepared to 10% homogenate (w/v) with lysis buffer (pH 7.2) containing 130 mM NaCl, 10 mM NaH2PO4, 10 mM Na2HPO4, 0.5% Triton X-100, 0.5% sodium deoxycholate, 2 mM MgCl2, 2.5 U/ml of Benzonase (Merck, Germany) and EDTA-free protease inhibitor cocktail (Roche, Switzerland). Homogenate was incubated at 25°C for 30 min for digesting nucleic acids followed by 1,000 g centrifugation at 4°C for 5 min for removing debris. Thereafter, 100 μl of supernatant were doubly diluted with lysis buffer to reach 200 μl of total volume followed by 20 μg/ml of PK-digestion at 37°C for 1 h. The digestion was stopped by adding 5 mM PMSF (Sigma-Aldrich, Switzerland) and then the PK-treated supernatants were transferred into 300 μl of 1 × QuIC buffer (130 mM NaCl, 5 mM NaH2PO4, 5 mM Na2HPO4 and 1 mM EDTA, pH 7.4). The total volume reached 500 μl.
The preparations were then brought to equal volume of buffer containing 20% NaCl and 0.1% sarkosyl. These solutions were vortexed vigorously followed by incubating on ice with gentle shaking for 10 min. After centrifugation at 16,000 g at 4°C for 10 min, the pellets were washed by 500 μl of 20 mM Tris–HCl containing 0.05% sarkosyl followed by 16,000 g of centrifugation at 4°C for 10 min. This washing step was repeated twice. The pellet was stored at -80°C till being used as the seed.
For preparing the prion seeds, the frozen pellets were thawed at 4°C followed by washing with 500 μl of ddH2O. The resuspended solutions were precipitated by centrifugation at16,000 g for 10 min at 4°C. This step was repeated twice. The last pellets were resxuspended thoroughly by 50 μl of ddH2O followed by 1,000 g of centrifugation at 4°C for 1 min. 45 μl of supernatant from peripheral tissues (tissue extract) was brought to qRT-QuIC to be both the seed and required water. 10 μl of PrP27-30 with known concentration quantified by semi-quantitative immunoblotting  was used as the seed for obtaining standard a calibration curve.
We referred to a published protocol  to purify mouse PrPC. The brain from a healthy 20-week old C57BL/6 mouse was homogenized in chilled 1 × PBS (pH 7.2) containing EDTA-free protease inhibitor cocktail (Roche, Switzerland) to make a 10% homogenate (w/v). After centrifugation at 3,000 g at 4°C for 30 min, the pellet was resuspended with an equal volume of chilled buffer (pH 7.2) containing 130 mM NaCl, 10 mM NaH2PO4, 10 mM Na2HPO4, 2% NP-40, 1% sodium deoxycholate and EDTA-free protease inhibitor cocktail (Roche, Switzerland). After incubation on ice for 30 min, the homogenate was subjected to 100,000 g centrifugation at 4°C for 30 min to remove the debris. The supernatant was filtrated with a 0.22 μm filter (Millipore, USA) followed by pouring over the ImmunoPure Immobilized Protein A column (Pierce, USA) to remove the endogenous immunoglobulins. The filtrated solution was incubated with 1 ml of Protein A resin cross-link to mouse-PrP specific monoclonal 4H11 antibody at 4°C over night. Then the resin was washed with 10 volumes of washing buffer (20 mM Tris–HCl, 500 mM NaCl and 5 mM EDTA, pH 8.0) followed by washing with 10 volumes of 1 × PBS containing 0.5% NP-40. PrPC was eluted with 5 ml of 200 mM glycine. The solution was loaded into 10 kDa filter centrifugal tubes (Millipore, USA) to centrifuge at 3,000 g at 4°C for 1 h for desalting and buffer exchanging. Then the protein was resuspended with 5 ml of 1 × QuIC buffer and subjected to gel filtration by passing over the Superdex 75 column (GE lifescience, USA). The harvested protein in the peak was concentrated with a 10 kDa filter centrifugal tube (Millipore, USA) followed by estimating protein concentration with quantitative immunoblot. The yield of mouse PrPC was approx. 3 μg/brain.
The ingredients of qRT-QuIC preparation
10 x QuIC buffer
(100 mM NaH2PO4, 100 mM Na2HPO4, 10 mM EDTA, 1.3 M NaCl, pH 6.9)
35 μl for standard curve, not for testing tissue extract
3 M NaCl
rPrPsen (0.3 mg/ml)
34 μl (final 0.1 mg/ml)
1 mM thioflavin T
10 μl for standard curve, 45 μl for testing tissue extract
total volume of each RT-QuIC
PK-digestion and immunoblot
The 10% brain homogenates of RML- and ME7-infected mice and purified RML- and ME7-PrP27-30 were digested with 100 μg/ml of proteinase-K (Roche, Switzerland) at 37°C for 1 h. The digestions were stopped by heating at 100°C for 10 min with 2 × loading buffer. The proteins were separated in a 15% SDS-PAGE followed by transfer to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% non-fat milk for 1 h at room temperature. The proteins were detected with mouse-PrP specific 4H11 monoclonal antibody (a gift from E. Kremmer, National Research Center for Environment and Health, Munich, Germany). Signals were measured on a Diana III luminescence imaging system (Raytest, Germany).
We thank Dr. B. Caughey, Dr. A.G. Hughson, G.J. Raymond (These three researchers are from NIAID, NIH, USA), Dr. L.I. McGuire and Dr. A.J. Green (both two researchers are from NCJDRSU, University of Edinburgh, UK) for nicely sharing their experiences on RT-QuIC technique. We thank Dr. E. Kremmer (National Research Center for Environment and Health, Munich, Germany) for providing 4H11 monoclonal antibody. We thank Janina Mielke for technique supports. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 596).
- Prusiner SB: Novel proteinaceous infectious particles cause scrapie. Science 1982, 216: 136–144. 10.1126/science.6801762View ArticlePubMedGoogle Scholar
- Prusiner SB: Prions. Proc Natl Acad Sci USA 1998, 95: 13363–13383. 10.1073/pnas.95.23.13363PubMed CentralView ArticlePubMedGoogle Scholar
- Bieschke J, Weber P, Sarafoff N, Beekes M, Giese A, Kretzschmar H: Autocatalytic self-propagation of misfolded prion protein. Proc Natl Acad Sci USA 2004, 101: 12207–12211. 10.1073/pnas.0404650101PubMed CentralView ArticlePubMedGoogle Scholar
- Caughey B, Raymond GJ, Ernst D, Race RE: N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. J Virol 1997, 65: 6597–6603.Google Scholar
- Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE: Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA 1993, 90: 10962–10966. 10.1073/pnas.90.23.10962PubMed CentralView ArticlePubMedGoogle Scholar
- Safar J, Wille H, Itri V, Groth D, Serban H, Torchia M, Cohen FE, Prusiner SB: Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med 1998, 4: 1157–1165. 10.1038/2654View ArticlePubMedGoogle Scholar
- Wilham JM, Orrú CD, Bessen RA, Atarashi R, Sano K, Race B, Meade-White KD, Taubner LM, Timmes A, Caughey B: Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays. PLoS Pathog 2010, 6: e1001217. 10.1371/journal.ppat.1001217PubMed CentralView ArticlePubMedGoogle Scholar
- Atarashi R, Satoh K, Sano K, Fuse T, Yamaguchi N, Ishibashi D, Matsubara T, Nakagaki T, Yamanaka H, Shirabe S, Yamada M, Mizusawa H, Kitamoto T, Klug G, McGlade A, Collins SJ, Nishida N: Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion. Nat Med 2011, 17: 175–178. 10.1038/nm.2294View ArticlePubMedGoogle Scholar
- McGuire LI, Peden AH, Orrú CD, Wilham JM, Appleford NE, Mallinson G, Andrews M, Head MW, Caughey B, Will RG, Knight RS, Green AJ: Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease. Ann Neurol 2012, 72: 278–285. 10.1002/ana.23589PubMed CentralView ArticlePubMedGoogle Scholar
- Peden AH, McGuire LI, Appleford NE, Mallinson G, Wilham JM, Orrú CD, Caughey B, Ironside JW, Knight RS, Will RG, Green AJ, Head MW: Sensitive and specific detection of sporadic Creutzfeldt-Jakob disease brain prion protein using real-time quaking-induced conversion. J Gen Virol 2012, 93: 438–449. 10.1099/vir.0.033365-0PubMed CentralView ArticlePubMedGoogle Scholar
- Orrù CD, Hughson AG, Race B, Raymond GJ, Caughey B: Time course of prion seeding activity in cerebrospinal fluid of scrapie-infected hamsters after intratongue and intracerebral inoculations. J Clin Microbiol 2012, 50: 1464–1466. 10.1128/JCM.06099-11PubMed CentralView ArticlePubMedGoogle Scholar
- Vascellari S, Orrù CD, Hughson AG, King D, Barron R, Wilham JM, Baron GS, Race B, Pani A, Caughey B: Prion seeding activities of mouse scrapie strains with divergent PrPSc protease sensitivities and amyloid plaque content using RT-QuIC and eQuIC. PLoS One 2012, 7: e48969. 10.1371/journal.pone.0048969PubMed CentralView ArticlePubMedGoogle Scholar
- Weber P, Reznicek L, Mitteregger G, Kretzschmar H, Giese A: Differential effects of prion particle size on infectivity in vivo and in vitro. Biochem Biophys Res Commun 2008, 369: 924–928. 10.1016/j.bbrc.2008.02.115View ArticlePubMedGoogle Scholar
- Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW: Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal Biochem 2000, 285: 194–204. 10.1006/abio.2000.4753View ArticlePubMedGoogle Scholar
- Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A: Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 2004, 313: 856–862. 10.1016/j.bbrc.2003.11.177View ArticlePubMedGoogle Scholar
- Polymenidou M, Verghese-Nikolakaki S, Groschup M, Chaplin MJ, Stack MJ, Plaitakis A, Sklaviadis T: A short purification process for quantitative isolation of PrP Sc from naturally occurring and experimental transmissible spongiform encephalopathies. BMC Infect Dis 2002, 2: 23. 10.1186/1471-2334-2-23PubMed CentralView ArticlePubMedGoogle Scholar
- Saá P, Castilla J, Soto C: Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J Biol Chem 2006, 281: 35245–35252. 10.1074/jbc.M603964200View ArticlePubMedGoogle Scholar
- Deleault NR, Harris BT, Rees JR, Supattapone S: Formation of native prions from minimal components in vitro. Proc Natl Acad Sci USA 2007, 104: 9741–9746. 10.1073/pnas.0702662104PubMed CentralView ArticlePubMedGoogle Scholar
- Browning S, Baker CA, Smith E, Mahal SP, Herva ME, Demczyk CA, Li J, Weissmann C: Abrogation of complex glycosylation by swainsonine results in strain- and cell-specific inhibition of prion replication. J Biol Chem 2011, 286: 40962–40973. 10.1074/jbc.M111.283978PubMed CentralView ArticlePubMedGoogle Scholar
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