Defining and Assessing Analytical Performance Criteria for Transmissible Spongiform Encephalopathy–Detecting Amyloid Seeding Assays
2016; Elsevier BV; Volume: 18; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2016.01.005
ISSN1943-7811
AutoresJohn G. Gray, Catherine Graham, Sandor Dudas, Eric Paxman, Ben Vuong, Stefanie Czub,
Tópico(s)Amyotrophic Lateral Sclerosis Research
ResumoTransmissible spongiform encephalopathies (TSEs) are infectious, fatal neurodegenerative diseases that affect production animal health, and thus human food safety. Enhanced TSE detection methods mimic the conjectured basis for prion replication, in vitro; biological matrices can be tested for prion activity via their ability to convert recombinant cellular prion protein (PrP) into amyloid fibrils; fluorescent spectra changes of amyloid-binding fluorophores in the reaction vessel detect fibril formation. In vitro PrP conversion techniques have high analytical sensitivity for prions, comparable with that of bioassays, yet no such protocol has gained regulatory approval for use in animal TSE surveillance programs. This study describes a timed in vitro PrP conversion protocol with accurate, well-defined analytical criteria based on probability density and mass functions of TSE+ and TSE− associated thioflavin T signal times, a new approach within this field. The prion detection model used is elk chronic wasting disease (CWD) in brain tissues. The protocol and analytical criteria proved as sensitive for elk CWD as two bioassay models, and upward of approximately 1.2 log10 more sensitive than the most sensitive TSE rapid test we assessed. Furthermore, we substantiate that timing in vitro PrP conversion may be used to titrate TSE infectivity, and, as a result, provide a comprehensive extrapolation of analytical sensitivity differences between bioassay, TSE rapid tests, and in vitro PrP conversion for elk CWD. Transmissible spongiform encephalopathies (TSEs) are infectious, fatal neurodegenerative diseases that affect production animal health, and thus human food safety. Enhanced TSE detection methods mimic the conjectured basis for prion replication, in vitro; biological matrices can be tested for prion activity via their ability to convert recombinant cellular prion protein (PrP) into amyloid fibrils; fluorescent spectra changes of amyloid-binding fluorophores in the reaction vessel detect fibril formation. In vitro PrP conversion techniques have high analytical sensitivity for prions, comparable with that of bioassays, yet no such protocol has gained regulatory approval for use in animal TSE surveillance programs. This study describes a timed in vitro PrP conversion protocol with accurate, well-defined analytical criteria based on probability density and mass functions of TSE+ and TSE− associated thioflavin T signal times, a new approach within this field. The prion detection model used is elk chronic wasting disease (CWD) in brain tissues. The protocol and analytical criteria proved as sensitive for elk CWD as two bioassay models, and upward of approximately 1.2 log10 more sensitive than the most sensitive TSE rapid test we assessed. Furthermore, we substantiate that timing in vitro PrP conversion may be used to titrate TSE infectivity, and, as a result, provide a comprehensive extrapolation of analytical sensitivity differences between bioassay, TSE rapid tests, and in vitro PrP conversion for elk CWD. CME Accreditation Statement: This activity ("JMD 2016 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2016 CME Program in Molecular Diagnostics") for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2016 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2016 CME Program in Molecular Diagnostics") for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Transmissible spongiform encephalopathies (TSEs) are invariably fatal neurodegenerative diseases, involving infectious proteinacious particles known as prions.1Prusiner S.B. Novel proteinaceous infectious particles cause scrapie.Science. 1982; 216: 136-144Crossref PubMed Scopus (4097) Google Scholar The prion-related protein (PrP) is host-encoded within the PRNP gene and is the principle component of prions.2Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Prusiner S.B. Weissmann C. A cellular gene encodes scrapie PrP 27-30 protein.Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1251) Google Scholar PrP is understood to exist in either the cellular isoform (PrPc), which is regarded as normal/nonpathogenic, or, as the prion/disease-associated isoform, PrPd; PrPd is conformationally different from PrPc, has amyloidogenic properties, and is associated with TSE pathogenicity. Mediated by an uncertain process, PrPc changes from a mainly α-fold into a β-fold, which is characteristic of PrPd. This event is recognized to occur sporadically, genetically (due to predisposed genetic susceptibilities in the PRNP gene), or via exposure to pre-existing prions. The latter is the basis for concern about the hazard TSEs pose on human/animal food safety.3Belay E.D. Schonberger L.B. The public health impact of prion diseases.Annu Rev Public Health. 2005; 26: 191-212Crossref PubMed Scopus (90) Google Scholar Once introduced into the host, PrPd ostensibly coaxes endogenous PrPc to convert into additional PrPd, which becomes a self-perpetuating process.4Cobb N.J. Surewicz W.K. Prion diseases and their biochemical mechanisms.Biochemistry. 2009; 48: 2574-2585Crossref PubMed Scopus (156) Google Scholar The significance of TSEs on human health was not entirely realized until cases of variant Creutzfeldt-Jakob disease in humans was discovered in the years after the bovine spongiform encephalopathy (BSE) outbreak in the United Kingdom.5Smith P.G. Bradley R. Bovine spongiform encephalopathy (BSE) and its epidemiology.Br Med Bull. 2003; 66: 185-198Crossref PubMed Scopus (98) Google Scholar, 6Heim D. Mumford E. The future of BSE from the global perspective.Meat Sci. 2005; 70: 555-562Crossref PubMed Scopus (10) Google Scholar These cases of variant Creutzfeldt-Jakob disease were associated with consuming meat products contaminated with BSE prions.7Anderson R.M. Donnelly C.A. Ferguson N.M. Woolhouse M.E. Watt C.J. Udy H.J. MaWhinney S. Dunstan S.P. Southwood T.R. Wilesmith J.W. Ryan J.B. Hoinville L.J. Hillerton J.E. Austin A.R. Wells G.A. Transmission dynamics and epidemiology of BSE in British cattle.Nature. 1996; 382: 779-788Crossref PubMed Scopus (531) Google Scholar, 8Prusiner S.B. Prion diseases and the BSE crisis.Science. 1997; 278: 245-251Crossref PubMed Scopus (857) Google Scholar Since then, the zoonotic potential of all TSEs has been an integral food safety concern. Other notable animal TSEs include Scrapie (affecting ovines and caprines) and chronic wasting disease (CWD) that affects cervids. Unlike BSE, the zoonotic hazard of CWD and Scrapie are debatable,9Kong Q. Huang S. Zou W. Vanegas D. Wang M. Wu D. Yuan J. Zheng M. Bai H. Deng H. Chen K. Jenny A.L. O'Rourke K. Belay E.D. Schonberger L.B. Petersen R.B. Sy M.S. Chen S.G. Gambetti P. Chronic wasting disease of elk: transmissibility to humans examined by transgenic mouse models.J Neurosci. 2005; 25: 7944-7949Crossref PubMed Scopus (203) Google Scholar, 10Sandberg M.K. Al-Doujaily H. Sigurdson C.J. Glatzel M. O'Malley C. Powell C. Asante E.A. Linehan J.M. Brandner S. Wadsworth J.D. Collinge J. Chronic wasting disease prions are not transmissible to transgenic mice overexpressing human prion protein.J Gen Virol. 2010; 91: 2651-2657Crossref PubMed Scopus (91) Google Scholar, 11Race B. Meade-White K.D. Phillips K. Striebel J. Race R. Chesebro B. Chronic wasting disease agents in nonhuman primates.Emerg Infect Dis. 2014; 20: 833-837Crossref PubMed Scopus (45) Google Scholar, 12Kurt T.D. Jiang L. Fernández-Borges N. Bett C. Liu J. Yang T. Spraker T.R. Castilla J. Eisenberg D. Kong Q. Sigurdson C.J. Human prion protein sequence elements impede cross-species chronic wasting disease transmission.J Clin Invest. 2015; 125: 1485-1496Crossref PubMed Scopus (47) Google Scholar although new evidence suggests it is conceivable.13Cassard H. Torres J.-M. Lacroux C. Douet J.-Y. Benestad S.L. Lantier F. Lugan S. Lantier I. Costes P. Aron N. Reine F. Herzog L. Espinosa J.-C. Beringue V. Andréoletti O. Evidence for zoonotic potential of ovine scrapie prions.Nat Commun. 2014; 5: 5821Crossref PubMed Scopus (99) Google Scholar Many countries have enacted TSE surveillance programs, aiming to eradicate livestock-related TSEs.14Regulation (EC) No. 999/2001 of The European Parliament and of the Council; laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies.Off J Eur Communities. 2001; L147: 1-40Google Scholar A focal point of eradication strategies has been the development of highly sensitive TSE diagnostic methods, capable of detecting infections in their earliest stages. This is of particular importance for CWD and Scrapie, because the prion agent is shed into the environment during preclinical phases of disease.15Gough K.C. Maddison B.C. Prion transmission: prion excretion and occurrence in the environment.Prion. 2010; 4: 275-282Crossref PubMed Scopus (96) Google Scholar TSE surveillance programs use streamlined biochemical screening tests known as TSE rapid tests. TSE rapid tests use PrP-specific antibodies to capture and/or probe for PrPd via formats such as enzyme-linked immunosorbent assay (ELISA) or Western blot analysis. Tissues that express the highest abundance of PrPc are reservoirs for PrPd replication and thus are targeted for TSE diagnosis; this primarily involves nervous and lymphatic tissues. Generally, brainstem is the most accurate sampling location; however, lymphatic tissues can also be an acceptable antemortem testing option for CWD and Scrapie.16Race R. Jenny A. Sutton D. Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis.J Infect Dis. 1998; 178: 949-953Crossref PubMed Scopus (172) Google Scholar, 17Williams E.S. Scrapie and chronic wasting disease.Clin Lab Med. 2003; 23: 139-159Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 18Williams E.S. Miller M.W. Kreeger T.J. Kahn R.H. Thorne E.T. Chronic wasting disease of deer and elk: a review with recommendations for management.J Wild Management. 2002; 66: 551-563Crossref Scopus (235) Google Scholar Although accurate tests, TSE rapid tests are not optimized for the detection of TSE infectivity that exists in blood, saliva, and urine, as reported in CWD and Scrapie.16Race R. Jenny A. Sutton D. Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis.J Infect Dis. 1998; 178: 949-953Crossref PubMed Scopus (172) Google Scholar, 19Haley N.J. Seelig D.M. Zabel M.D. Telling G.C. Hoover E.A. Detection of CWD prions in urine and saliva of deer by transgenic mouse bioassay.PLoS One. 2009; 4: e4848Crossref PubMed Scopus (178) Google Scholar, 20Mathiason C.K. Powers J.G. Dahmes S.J. Osborn D.A. Miller K.V. Warren R.J. Mason G.L. Hays S.A. Hayes-Klug J. Seelig D.M. Wild M.A. Wolfe L.L. Spraker T.R. Miller M.W. Sigurdson C.J. Telling G.C. Hoover E.A. Infectious prions in the saliva and blood of deer with chronic wasting disease.Science. 2006; 314: 133-136Crossref PubMed Scopus (387) Google Scholar, 21Tamgüney G. Richt J.A. Hamir A.N. Greenlee J.J. Miller M.W. Wolfe L.L. Sirochman T.M. Young A.J. Glidden D.V. Johnson N.L. Giles K. DeArmond S.J. Prusiner S.B. Salivary prions in sheep and deer.Prion. 2012; 6: 52-61Crossref PubMed Scopus (43) Google Scholar Such infectivity has only been detected via bioassay models or via in vitro methods that aim to mimic the conjectured basis of in vivo prion propagation. As such, the consensus is that a highly sensitive TSE detection platform based on in vitro prion amplification will further enhance TSE detection in currently targeted tissues and prospectively offer less-invasive sampling options (eg, blood), the latter offering high-throughput antemortem testing possibilities. The theory behind these in vitro techniques is that the presence of a minute quantity of PrPd can be detected via the misfolding (or conversion) activity it will exert on a source of PrPc; the PrPc source acts as conversion substrate. Protein misfolding cyclic amplification was among the first in vitro techniques to prove this concept22Soto C. Anderes L. Suardi S. Cardone F. Castilla J. Frossard M.-J. Peano S. Saa P. Limido L. Carbonatto M. Ironside J. Torres J.-M. Pocchiari M. Tagliavini F. Pre-symptomatic detection of prions by cyclic amplification of protein misfolding.FEBS Lett. 2005; 579: 638-642Crossref PubMed Scopus (130) Google Scholar: TSE− brain homogenate could be converted to a TSE+ brain homogenate by spiking-in a minute quantity of TSE+-infected material, followed by cyclical rounds of sonication and rest periods. Initially TSE− brain homogenates would react as TSE+ via Western blot analysis detection protocols. Various protocols/approaches have since been created.23Orru C.D. Wilham J.M. Vascellari S. Hughson A.G. Caughey B. New generation QuIC assays for prion seeding activity.Prion. 2012; 6: 147-152Crossref PubMed Scopus (83) Google Scholar, 24Ryou C. Mays C.E. Prion propagation in vitro: are we there yet?.Int J Med Sci. 2008; 5: 347-353Crossref PubMed Scopus (14) Google Scholar The amyloid seeding assay (ASA) and real-time quaking induced conversion (real-time QuIC) assay both use recombinant PrPc as a conversion substrate.25Colby D.W. Zhang Q. Wang S. Groth D. Legname G. Riesner D. Prusiner S.B. Prion detection by an amyloid seeding assay.Proc Natl Acad Sci U S A. 2007; 104: 20914-20919Crossref PubMed Scopus (188) Google Scholar, 26Orrú C.D. Wilham J.M. Hughson A.G. Raymond L.D. McNally K.L. Bossers A. Ligios C. Caughey B. Human variant Creutzfeldt-Jakob disease and sheep scrapie PrPres detection using seeded conversion of recombinant prion protein.Protein Eng Des Sel. 2009; 22: 515-521Crossref PubMed Scopus (55) Google Scholar, 27Wilham J.M. Orru C.D. Bessen R.A. Atarashi R. Sano K. Race B. Meade-White K.D. Taubner L.M. Timmes A. Caughey B. Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.PLoS Pathog. 2010; 6: e1001217Crossref PubMed Scopus (319) Google Scholar A solution of PrPc is seeded with a minute volume of tissue homogenate or bodily fluid (seed material). Should the seed material be TSE+, conversion activity causes the PrPc substrate to form PrP amyloid fibrils; amyloid-binding fluorophores, such as thioflavin T (ThT), signal the formation of fibrils via changes in their fluorescence spectra.25Colby D.W. Zhang Q. Wang S. Groth D. Legname G. Riesner D. Prusiner S.B. Prion detection by an amyloid seeding assay.Proc Natl Acad Sci U S A. 2007; 104: 20914-20919Crossref PubMed Scopus (188) Google Scholar, 27Wilham J.M. Orru C.D. Bessen R.A. Atarashi R. Sano K. Race B. Meade-White K.D. Taubner L.M. Timmes A. Caughey B. Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.PLoS Pathog. 2010; 6: e1001217Crossref PubMed Scopus (319) Google Scholar, 28Ban T. Hamada D. Hasegawa K. Naiki H. Goto Y. Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence.J Biol Chem. 2003; 278: 16462-16465Crossref PubMed Scopus (305) Google Scholar Ideally, the assay readout is strictly a binary outcome. Only TSE+ seed material will cause PrP conversion, thus producing ThT signals; TSE− seed material will not cause PrP conversion, thus no ThT signals are produced, although this is not always the case. ASA and real-time QuIC are fundamentally derived from earlier autocatalytic conversion studies of PrPc.29Baskakov I.V. Autocatalytic conversion of recombinant prion proteins displays a species barrier.J Biol Chem. 2004; 279: 7671-7677Crossref PubMed Scopus (73) Google Scholar, 30Baskakov I.V. Bocharova O.V. In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features.Biochemistry. 2005; 44: 2339-2348Crossref PubMed Scopus (74) Google Scholar In short, autocatalytic conversion showed that PrPc has an inherent tendency to spontaneously convert into amyloid fibrils/aggregates; however, the introduction of preformed PrP fibrils to monomeric PrPc bypasses the necessary monomer nucleation phase to initiate amyloid assembly, which is not the case for spontaneous PrPc conversion.29Baskakov I.V. Autocatalytic conversion of recombinant prion proteins displays a species barrier.J Biol Chem. 2004; 279: 7671-7677Crossref PubMed Scopus (73) Google Scholar, 31Bocharova O.V. Breydo L. Parfenov A.S. Salnikov V.V. Baskakov I.V. In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP(Sc).J Mol Biol. 2005; 346: 645-659Crossref PubMed Scopus (231) Google Scholar Therefore, spontaneous PrP conversion events have a longer lag-phase before amyloid formation than those seeded by pre-existing fibrils. On this premise, the ASA showed PrPc conversion can be seeded by semipurified PrPd fibrils from TSE+ tissues.25Colby D.W. Zhang Q. Wang S. Groth D. Legname G. Riesner D. Prusiner S.B. Prion detection by an amyloid seeding assay.Proc Natl Acad Sci U S A. 2007; 104: 20914-20919Crossref PubMed Scopus (188) Google Scholar However, the high incidence of spontaneous PrP conversion/ThT signals from TSE− and nonseeded ASA reactions was viewed as problematic to a preferred dichotomous present/absent (ThT+ = TSE+ versus ThT− = TSE−) diagnostic interpretation of signals.23Orru C.D. Wilham J.M. Vascellari S. Hughson A.G. Caughey B. New generation QuIC assays for prion seeding activity.Prion. 2012; 6: 147-152Crossref PubMed Scopus (83) Google Scholar, 27Wilham J.M. Orru C.D. Bessen R.A. Atarashi R. Sano K. Race B. Meade-White K.D. Taubner L.M. Timmes A. Caughey B. Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.PLoS Pathog. 2010; 6: e1001217Crossref PubMed Scopus (319) Google Scholar Various modifications to the ASA protocol led to the real-time QuIC assay, which had reduced spontaneous PrP conversion events within the allotted assay duration.27Wilham J.M. Orru C.D. Bessen R.A. Atarashi R. Sano K. Race B. Meade-White K.D. Taubner L.M. Timmes A. Caughey B. Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays.PLoS Pathog. 2010; 6: e1001217Crossref PubMed Scopus (319) Google Scholar, 32Atarashi R. Wilham J.M. Christensen L. Hughson A.G. Moore R.A. Johnson L.M. Onwubiko H.A. Priola S.A. Caughey B. Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking.Nat Methods. 2008; 5: 211-212Crossref PubMed Scopus (234) Google Scholar Efforts to eliminate the occurrence of spontaneous PrP conversion show that the phenomenon depends on the combination of assay environment/buffer conditions, PrPc substrate (ie, host species primary amino acid sequence), and/or seed material matrix, and can sometimes be resolved with protocol revisions.33Atarashi R. Sano K. Satoh K. Nishida N. Real-time quaking-induced conversion: a highly sensitive assay for prion detection.Prion. 2011; 5: 150-153Crossref PubMed Scopus (114) Google Scholar, 34McGuire L.I. Peden A.H. Orrú C.D. Wilham J.M. Appleford N.E. Mallinson G. Andrews M. Head M.W. Caughey B. Will R.G. Knight R.S.G. Green A.J.E. Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt–Jakob disease.Ann Neurol. 2012; 72: 278-285Crossref PubMed Scopus (266) Google Scholar, 35Vascellari S. Orru C.D. Hughson A.G. King D. Barron R. Wilham J.M. Baron G.S. 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: e48969Crossref PubMed Scopus (48) Google Scholar When spontaneous PrP conversion does occur, reference to the ThT signal's excessive lag-phase duration has been used to identify these events and to justify their irrelevance.35Vascellari S. Orru C.D. Hughson A.G. King D. Barron R. Wilham J.M. Baron G.S. 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: e48969Crossref PubMed Scopus (48) Google Scholar, 36John T.R. Schatzl H.M. Gilch S. Early detection of chronic wasting disease prions in urine of pre-symptomatic deer by real-time quaking-induced conversion assay.Prion. 2013; 7: 253-258Crossref PubMed Scopus (55) Google Scholar, 37Shi S. Mitteregger-Kretzschmar G. Giese A. Kretzschmar H.A. Establishing quantitative real-time quaking-induced conversion (qRT-QuIC) for highly sensitive detection and quantification of PrPSc in prion-infected tissues.Acta Neuropathol Commun. 2013; 1: 44Crossref PubMed Scopus (25) Google Scholar As such, to avoid witnessing false-positive ThT signals (type I errors), an assay termination time is used as a binary classifier for TSE+ versus TSE− diagnostic interpretation. For example, a 55-hour cutoff time is used for the detection of BSE with the use of hamster and human PrPc types, because no spontaneous PrP conversion was seen to occur before that time.38Orru C.D. Favole A. Corona C. Mazza M. Manca M. Groveman B.R. Hughson A.G. Acutis P.L. Caramelli M. Zanusso G. Casalone C. Caughey B. Detection and discrimination of classical and atypical L-type bovine spongiform encephalopathy by real-time quaking-induced conversion.J Clin Microbiol. 2015; 53: 1115-1120Crossref PubMed Scopus (42) Google Scholar Other real-time QuIC protocols appear to use assay cessation times between 24 and 60 hours when detecting CWD in deer tissues via a hamster PrPc substrate.39Haley N.J. Carver S. Hoon-Hanks L.L. Henderson D.M. Davenport K.A. Bunting E. Gray S. Trindle B. Galeota J. LeVan I. Dubovos T. Shelton P. Hoover E.A. Detection of chronic wasting disease in the lymph nodes of free-ranging cervids by real-time quaking-induced conversion.J Clin Microbiol. 2014; 52: 3237-3243Crossref PubMed Scopus (36) Google Scholar, 40Elder A.M. Henderson D.M. Nalls A.V. Wilham J.M. Caughey B.W. Hoover E.A. Kincaid A.E. Bartz J.C. Mathiason C.K. In vitro detection of prionemia in TSE-infected cervids and hamsters.PLoS One. 2013; 8: e80203Crossref PubMed Scopus (71) Google Scholar Ultimately, it is the time component of ThT signals which is used to substantiate the specificity of the result, much like the ASA. Therefore, the principle on which these cutoff times are set can be more accurately defined; associating a ThT signal with TSE+ seed material (as opposed to being a spontaneous PrP conversion event) is subject to a probability density function over assay duration. No related study has formally applied cumulative distribution principles to substantiate the precision of the reported TSE+ classifier criteria for ThT signals. Furthermore, cutoff times only mitigate the risk of witnessing false-positive ThT signals. Many related studies test individual samples via numerous replicates to avoid type II errors (false negatives), which essentially allows weak TSE+ samples a chance to generate a ThT signal before the assay cessation time or classifying criteria. No study has formally quantified how the number of replicates conducted per sample affects the statistical power of the result. Here, we use a combination of statistical methods to address the analytical quandaries surrounding ASA-based TSE-detecting assays. Subsequently, we have developed a well-defined, substantiated timed ASA protocol for the accurate detection of elk CWD in brain tissues. ThT signal time distributions were created by seeding PrP conversion reactions with CWD− and CWD+ brain material (varying in infectivity/PrPd content), such that receiver-operating characteristic (ROC) curve analyses could be used to set specific CWD+ classifying cutoff times. We also describe how the probability of committing type I and/or type II errors for any given PrP conversion reaction can be quantified, based on the established time-dependent criteria. The analytical sensitivity of the ASA was then evaluated versus two transgenic (Tg) mouse bioassay models [Tg(CerPrP-M132)1536+/− Tg(CerPrP-E226)5037+/−)],41Bian J. Napier D. Khaychuck V. Angers R. Graham C. Telling G. Cell-based quantification of chronic wasting disease prions.J Virol. 2010; 84: 8322-8326Crossref PubMed Scopus (64) Google Scholar and three regulatory-approved TSE rapid test platforms: the Prionics CheckWESTERN, the Bio-Rad TeSeE ELISA, and the IDEXX HerdChek CWD enzyme-linked immunoassay (EIA). Furthermore, because timing in vitro PrP conversion has the potential to quantify PrPd within tissue samples and potentially to titrate CWD infectivity in elk tissues (more recently shown for deer tissues as well),37Shi S. Mitteregger-Kretzschmar G. Giese A. Kretzschmar H.A. Establishing quantitative real-time quaking-induced conversion (qRT-QuIC) for highly sensitive detection and quantification of PrPSc in prion-infected tissues.Acta Neuropathol Commun. 2013; 1: 44Crossref PubMed Scopus (25) Google Scholar, 42Gray J.G. Timing recombinant prion protein conversion as a measure of prion activity in chronic wasting disease. University of Calgary, Calgary, AB, Canada2014Google Scholar, 43Henderson D.M. Davenport K.A. Haley N.J. Denkers N.D. Mathiason C.K. Hoover E.A. Quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion.J Gen Virol. 2015; 96: 210-219Crossref PubMed Scopus (73) Google Scholar we could also extrapolate the analytical sensitivity differences between TSE rapid tests and Tg mouse bioassay. CWD+ and CWD− brain tissue samples were obtained from a tissue bank, created from a previous parallel-group elk CWD pathogenesis study (CFIA project: L04-003; CFIA Lethbridge/Burnaby Animal Care Animal Use Protocol: 04002). The clinical history of the elk was known; elk had been obtained as calves from a certified CWD-free facility, in a nonendemic area. Elk selected for CWD+ challenge had received 1 g (perorally) of CWD+ material of elk origin. Sham-challenged elk served as controls. Animals were housed in a level-3 containment facility, where CWD+ and sham-challenged groups were kept separate for the duration of the study. Disease outcomes were confirmed on postmortem immunohistochemical analysis of the obex and retropharyngeal lymph nodes.44Accredited Veterinarian's Manual Chronic Wasting Disease Voluntary Herd Certification Program. Chapter 13.6 Appendix 1B: Lymph Node Sampling Procedures. Canadian Food Inspection Agency.Date: 2012Google Scholar All brain tissue used in this study was from elk that had been sacrificed at least 1.5 years after challenge. Tissues were sampled in a class II biosafety cabinet; measures to prevent cross-contamination were strictly observed. CWD− tissues were always sampled before CWD+ tissues, and disposable scalpels (single use) were used on each individual tissue sample, regardless of CWD+ or CWD− status. Elk brain samples tested on the ASA were processed to a final 10% (w/v) stock homogenate in the following homogenization buffer: 1% (w/v) 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) detergent, 1 mol/L 3-(1-Pyridinio)-1-propanesulfonate (NDSB-201), PBS. [10 mmol/L Na2HPO4, 2 mmol/L KH2PO4, 137 mmol/L NaCl, 2 mmol/L KCl, pH 7.4], 1 mmol/L EDTA, Complete Protease Inhibitors (Roche Applied Science, Mannheim, Germany). NDSB-201 was incorporated in buffers with CHAPS detergent to enhance protein solubility and possibly to preserve protein-fold integrity during processes such as homogenization.45Vuillard L. Braun-Breton C. Rabilloud T. Non-detergent sulphobetaines: a new class of mild solubilization agents for protein purification.Biochem J. 1995; 305: 337-343Crossref PubMed Scopus (75) Google Scholar, 46Willis M.S. Hogan J.K. Prabhakar P. Liu X. Tsai K. Wei Y. Fox T. Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions.Protein Sci. 2005; 14: 1818-1826Crossref PubMed Scopus (63) Google Scholar Homogenization was facilitated with a ribolyser (Precess 24; Bio-Rad Laboratories, Hercules, CA) with grinding bead tubes (1-mm beads). Homogenates were stored at −80°C in 100-μL aliquots until further use for seeding PrP conversion/ASA reactions. A 20% (w/v) homogenate was prepared by pooling brain tissues from three CWD+ elk, all in advanced stages of disease. The CWD infectivity of the homogenate was previously titrated
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