TSE diagnostics: recent advances in immunoassaying prions.
1.1. Prions. Prion is by definition a "proteinaceous infectious particle," responsible for transmissibility of a group of fatal neurodegenerative diseases that affect humans and many other mammals. The so-called protein-only hypothesis, which postulated that the aberrantly folded protein is able to infect and replicate, made prion diseases (at that time quite heretically) distinct from infections caused by microorganisms .
Prion ([PrP.sup.Sc]) has an endogenous cellular counterpart, named prion protein ([PrP.sup.C]), which is expressed on the surface of various cell types, most abundantly in the central nervous system. [PrP.sup.Sc] and [PrP.sup.C] share the same amino acid sequence, but differ substantially in the secondary, tertiary, and quaternary structures. [PrP.sup.Sc] is believed to be acting like a mold for converting endogenous [PrP.sup.C] molecules into new prions. However, not only one, but several prion strains have been characterized so far, differing in their structure and biochemical characteristics [2, 3]. Moreover, [PrP.sup.C] as well as [PrP.sup.Sc] can be found in fragments of various lengths [4-6]. Therefore, considering the complex biochemical nature of the target, the difficulty of its detection is obvious.
1.2. Transmissible Spongiform Encephalopathies. In humans, the so-called transmissible spongiform encephalopathies (TSEs) or prion diseases have been known to either occur sporadically (sporadic Creutzfeldt-Jakob disease (sCJD)) or can be inherited (familial Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker syndrome). The transmissibility of these diseases was first demonstrated by Gajdusek et al., who successfully transmitted kuru to chimpanzees . However, TSEs came to public attention two decades later when the first case of bovine spongiform encephalopathy (BSE) was reported in United Kingdom, followed by an epidemic outburst of the disease in which more than 180 thousand animals have been diagnosed as BSE positive, and the estimation is that 1-3 million infected animals were slaughtered for human consumption before developing clinical signs . The source of the infection was found tobe the prion-infected meat and bone meal, produced from waste parts of sheep and cattle. About ten years after the appearance of BSE, the first cases of new variant CJD (vCJD) have been diagnosed in young patients in UK and later also in some other countries. vCJD was soon connected to the consumption of meat from BSE-infected cattle . Despite the fear, the vCJD cases did not reach the numbers of BSE epidemics (224 cases were described worldwide; http://www.cjd.ed.ac.uk/documents/worldfigs.pdf, 176 from which in UK; http://www.cjd.ed.ac.uk/documents/figs.pdf). However, it was shown that those young and active people, who have been carrying prions for years, before the outbreak of clinical signs, have transmitted them through blood donations. To date, four cases of probable and two cases of possible transmission of vCJD by blood transfusion have been described , but as blood or blood products of infected donors was given to many more (http://www.cjd.ed. ac.uk/TMER/summary.htm), thefearexiststhatmorewillfall ill. Apart from that, iatrogenic transmission of CJD has been connected to the use of infected surgical instrumentation, to the transplantation of cornea and dura mater grafting (228 reported cases), and to the application of cadaver-derived gonadotropin and human growth hormone (230 reported cases) .
1.3. TSE Diagnostics. Due to the remarkable biochemical diversity among prions on one hand and the disturbing presence of [PrP.sup.C] on the other, as well as due to the absence of specific nucleic acids, TSEs testing has remained one of the biggest challenges of diagnostics until now.
One way to assess the problem is to search for surrogate markers. For antemortem diagnosis of CJD, different liquor proteins, such as 14-3-3, Tau, phospho-Tau, amyloid-[beta] 1-42 and some others (for review, see ), have been employed in tests that reach considerably high sensitivity and are often used for CJD diagnostics complementary to neurological signs. However, it is important to stress that the presence of none of them is 100% specific for prion diseases and so far, their use in diagnostics has been limited to the advanced stages of the disease. With the development of new biomarkers and methods that would enable their detection at preclinical stages, liquor diagnostics is expected to take an even more important part in antemortem diagnostics of prion diseases in the near future.
Another way to approach TSE diagnostics is to exploit the physicochemical differences between [PrP.sup.C] and [PrP.sup.Sc]. Namely, [PrP.sup.Sc], being richer in beta sheet content, was found to be much more resistant to denaturation and proteolytic degradation than [PrP.sup.C]. Ever since, [PrP.sup.Sc] has been detected either by immunohistochemistry (IHC) after special pretreatments of tissue slices, which destroyed relevant [PrP.sup.C] epitopes, or by western blotting of brain homogenates after degradation of [PrP.sup.C] by proteinase K (PK). Many other commercially available diagnostic immunoassays that have been developed still relay on PK digestion of [PrP.sup.C]. Contemporary options of discrimination between [PrP.sup.C] and [PrP.sup.Sc] exploit the aggregation-prone nature of [PrP.sup.Sc] molecules in confrontation with to the monomeric [PrP.sup.C].
[PrP.sup.Sc]-specific monoclonal antibodies (mAbs) have always represented an ideal approach for prion diagnostics development. However, with the knowledge of various infectious prion strains and fragments, the idea of producing one mAb that would detect them all appears less credible.
In the present paper we have reviewed immunoassays designed to detect pathological form of prion protein as a diagnostic or research tool, discussing their evolution, their advantages, and their weaknesses. Because of the abundance of [PrP.sup.Sc], brain tissue is the most common and reliable diagnostic material. Routine testing of brain tissue is a good way to identify and remove diseased animals from the food chain, and many important advances have been achieved in this area in recent years. Nevertheless, detection of prions at presymptomatic levels of the disease in samples other than brain is the ultimate goal for which researchers still strive.
2. Immunoassaying Prions
2.1. Detection of Prions in Brain. Several types of ELISA or similar immunoassays have been developed for detection of [PrP.sup.Sc] in brain tissue (Table 1 and Figure 1). [PrP.sup.C] degradation by PK is still the most frequently used sample treatment prior to detection and analysis of [PrP.sup.Sc] and can successfully be transferred from western blot (WB) to ELISA format . ELISA enables simultaneous analysis of larger number of samples than WB, which represents a major advantage. After elimination of PK-sensitive PrP, the remaining resistant forms ([PrP.sup.res]) can be detected. PK digestion has in the recent years become somehow controversial. A number of studies have identified PK-sensitive [PrP.sup.Sc] strains, and it is believed that as much as 80% of [PrP.sup.Sc] is PKsensitive [14-19]. Complete [PrP.sup.C] removal and preservation of the whole [PrP.sup.Sc] at the same time is, therefore, hard or in some cases impossible to achieve. The determination of the existence of PK-sensitive [PrP.sup.Sc] strains raised the fear of resurgence of BSE due to the false negative results of routine testing as a consequence of using PK-based tests. Besides, when dealing with tests that rely on enzymes, the adequacy of storage conditions is of considerable importance, as the loss of the enzymatic activity may cause deceptive results. These issues are not to be overlooked since many routine diagnostic methods, especially for BSE, are still based on detection of [PrP.sup.res]. Differential resistance of prion strains to PK digestion, which usually poses a problem, can also be exploited for their distinction. Classical scrapie strain, for example, can be distinguished from more sensitive atypical scrapie strain based on the difference in resistance to low and high concentrations of PK . After mild PK digestion both classical and more sensitive atypical strains appear PK resistant. PK in higher concentrations further degrades [PrP.sup.Sc] in atypical strain, destroying relevant epitopes, while epitopes on [PrP.sup.Sc] in classical strain are preserved. The ratio of the signal after mild and harsh digestion is the measurement of sensitivity of certain strain .
During the transition from [PrP.sup.C] to [PrP.sup.Sc], and more importantly during the aggregation of [PrP.sup.Sc] molecules, certain epitopes become inaccessible. Upon denaturation of [PrP.sup.Sc], immunoreactivity is greatly enhanced presumably because the structure of the aggregates loosens and buried epitopesbecome accessible again . Conformation-dependent immunoassay (CDI) exploits this fact for analyzing different prion strains . The method is based on denaturation of different prion strains with rising denaturant concentration gradually revealing hidden [PrP.sup.Sc] epitopes. Denaturation profiles obtained for each strain differ from one another, namely, more stable (and less infectious) strains require higher denaturant concentration for dissociation. The measured optical density (OD) increases significantly for infected material after the denaturation and is, therefore, a measure for the amount of [PrP.sup.Sc] in the individual sample. Based on the difference between OD of denatured and nondenatured samples, infected samples can readily be distinguished from non-infected .
Denaturation has been used in numerous studies, in most cases with an important simplification of the original CDI method, although the main principle and the name of the method were retained [22-25]. Instead of measuring the denaturation profile of different prion strains, only one concentration of denaturant, was used for revealing hidden [PrP.sup.Sc] epitopes. Because different prion strains react differently to the same concentration of denaturant some strains, especially less stable, might be overlooked this way. However, according to the authors of these reports, the approach was successfully applied to bovine, ovine, elk, and deer tissues [22, 23]. Additional changes were applied to the first suggested CDI . In all assays sensitivity was increased by introduction of the sandwich immunoassay instead of the direct one. The other common modification was the change of denaturation conditions [22, 24, 25]. It was shown that precipitation step with sodium phosphotungstic acid (NaPTA), that was originally present in the protocol, can be omitted, shortening and simplifying the sample processing [22, 25]. Although the use of denaturation for [PrP.sup.Sc] epitope revealing eliminates the need for PK digestion, it can be applied for more efficient elimination of [PrP.sup.C] and therefore for improved discrimination between TSE-positive and TSE-negative samples [23, 25].
In a different set of assays, denaturation step was employed for differential extraction of PrP [26-28]. Samples were subjected first to low and subsequently to high concentrations of denaturant. [PrP.sup.Sc] aggregates were only soluble when the concentration of denaturant was high enough. Comparison of the two fractions in ELISA (enzyme-linked immunosorbent assay) or DELFIA (dissociation-enhanced lanthanide fluoroimmunoassay) enabled the discrimination between infected and noninfected bovine, murine, and human tissues.
An important issue of immunoassaying brains is the fact that different parts of brain may vary greatly in the abundance of [PrP.sup.Sc], which was shown for animal and also for human brain [15, 23]. Some parts of infected brain may therefore contain only very low amounts of [PrP.sup.Sc]. This issue can to some extent be managed by the knowledge of prion distribution patterns that are present in certain TSEs. Low quality of samples can also be the reason for low amounts of [PrP.sup.C] and [PrP.sup.Sc]. In such cases, a low OD is misleading. To avoid misinterpretation of results, normalization of detected [PrP.sup.Sc] against detected [PrP.sup.C] in the same sample can be very useful. A ratio between denatured and nondenatured sample (D/N) can be applied for this purpose [3, 29].
In sandwich ELISA capture, mAb is adsorbed to the bottom of the well and detector mAb is used to detect antigen bound to the capture Ab. This format requires two mAbs directed against two different epitopes on one antigen molecule. But in a case of aggregated proteins such as [PrP.sup.Sc], it is reasonable to assume that certain epitopes are represented more than once. This assumption is the basis of the so-called aggregation-specific ELISA (AS-ELISA) that detects only PrP aggregates in brain samples . Using the same mAb for capturing and detecting, it is possible to avoid the detection of [PrP.sup.C], which is usually present as a monomer, and observe only aggregates.
Ligands other than Abs can be used for the purpose of capturing PrP. Glycosaminoglycans (GAGs) that have been found to bind PrP in the cell [31, 32] can be immobilized onto the solid phase in the ELISA test instead of capturing Ab. Higher affinity of GAGs for [PrP.sup.Sc] in comparison to [PrP.sup.C] enables discrimination between normal and scrapie tissue . A protocol for glycotyping of PrP (which can be non-, mono-, or diglycosylated) was also developed based on the binding of different lectins to specific sugar moieties on PrP . This approach provides yet another advantage, since in comparison to WB in common ELISA the information about glycosylation is lost. Apart from GAGs, other polymeric compounds may bind [PrP.sup.Sc] selectively under defined conditions. This principle was successfully exploited in one of the commercially available BSE tests .
The above-mentioned methods all rely on frozen tissues that are sometimes not available. As IHC is still the golden standard for definite diagnosis of TSE, much of the tissue taken for analysis is paraffin embedded. Because IHC is not a high-throughput method, protocols for detection of [PrP.sup.Sc] from paraffin-embedded tissue by WB have been developed [35, 36]. They can readilybe transferred to ELISA test format , enabling the analysis of larger number of samples compared to IHC. In the first step, the tissue is separated from the paraffin bysubjecting the tissue sections to boil and freeze cycles. In the second step, the collected tissue is disrupted by sonication. Following tissue disruption, samples are analyzed with the chosen method.
Sensitivity of an immunoassay depends not only on the sample preparation and treatment, but largely also on the detection system. The simplest and most easily accessible is the ELISA format where detection of PrP is achieved via anti-PrP mAb coupled directly or indirectly to an enzyme which produces visible signal after the addition of the substrate. In more sensitive DELFIA, anti-PrP antibody is labeled with lanthanide chelates, most commonly Europium, that emit stable fluorescent signal. DELFIA was used in a number of studies described in this review [23, 24, 26, 28, 38]. To lower the detection limit even further, a surrounding optical fiber immunoassay (SOFIA) was developed . It is based on sandwich ELISA, but instead of an enzyme conjugate, Rhodamine Red X is coupled to streptavidin. Specially designed hardware that enables maximum light collection and very high sensitivity of the method, at the same time, makes SOFIA less accessible for the widespread use.
Yet another method that was proved to be more sensitive than WB and IHC is immuno-polymerase chain reaction (IPCR). Original protocol exploits the benefits of both specific antigen recognition in ELISA and exponential amplification of DNA in polymerase chain reaction (PCR) . Antigen is captured as in ELISA followed by the addition of biotinilated DNA instead of en enzyme for obtaining the signal. Bound DNA is amplificated by PCR for enhancing the sensitivity of the protocol [41, 42]. The IPCR was applied to classical ELISA for detection of [PrP.sup.res] in hamster and human brain tissues. Because of the afore mentioned concerns about PK-sensitive strains of prions, a need for PK digestion of the samples is a substantial drawback of the method. To our knowledge, IPCR was never applied to denaturation-based [PrP.sup.Sc] immunoassay.
The reports of the development of [PrP.sup.Sc]-specific mAb based immunoassay are very limited. Despite of the use of [PrP.sup.Sc]--or aggregate-specific mAb--, these immunoassays are still based on denaturation or PK digestion of samples. The V5B2 mAb, first described by our group in 2004 , was later discovered to be specific for a truncated PrP, which ends with the residue Y226 of the human PrP . Although this fragment, named PrP226*, can be present in minute quantities also in normal human brain, it accumulates abundantly in aggregates together with the whole [PrP.sup.Sc] in CJD infected brain . Because it is packed into aggregates and is therefore unavailable for V5B2 mAb, denaturation of samples is necessary for efficient discrimination between infected and noninfected tissues. Nevertheless, greater dissociation compared to the use of non-[PrP.sup.Sc]-specific anti-PrP mAb between [PrP.sup.Sc]-positive and-negative samples has been achieved in a simple, PK-independent immunoassay. A rationale that a [PrP.sup.Sc]-specific mAb-based immunoassay would not need any specific preparation of samples therefore does not seem so plausible anymore, at least for immunoassays using brain samples, where [PrP.sup.Sc] is known to be aggregated.
2.2. Detection of Prions in Blood. All the above-mentioned methods were developed for analysis of human and animal brain tissues, and can thus be applied only for postmortem diagnostics. For an ante mortem test, the use of blood and other body fluids needs to be applied (for summary of the blood tests for prions, see Table 2 and Figure 1). As [PrP.sup.Sc] is supposed to be present in extremely low amounts in these samples, immunoassays need to include an additional step of [PrP.sup.Sc] enrichment [44-47]. In an effort to reach high sensitivity, the specificity of the method should not be neglected. Even a small percentage of false positive results in blood donor testing would result in an extremely large number of misinterpreted asymptomatic carriers of the disease. Besides the obvious moral concern, there is also a financial aspect of the issue because all possible carriers should undergo additional diagnostic procedures and should stay under constant medical supervision .
As stated before, the main problem of detecting prions in blood or plasma is the extremely small quantity of prions and a high background of other proteins and [PrP.sup.C]; therefore, extreme sensitivity and specificity is a necessity for a blood test. Apart from that, samples of prion infected blood are rare, limited, and only accessible to few laboratories. To overcome that problem, many test developers make use of spiking brain homogenates or [PrP.sup.Sc] isolated from brain into the blood of healthy persons. This might not be the optimal solution of the problem since pathological PrP, if present in blood, not necessarily possesses the same characteristics as that of brain derived. Nevertheless, such studies are important as they represent an insight into the detection limits we are currently able to reach [38, 46, 47]. Besides the sensitivity issue, the susceptibility to PK digestion also represents a problem because a big portion of [PrP.sup.Sc] in blood may be PK sensitive. Tattum et al. addressed both of these problems in their research . They developed a sandwich ELISA for the detection of [PrP.sup.Sc] in samples of whole human blood spiked with vCJD brain homogenate. The sensitivity was enhanced by immunoprecipitation (IP), reaching the pg level . PK was replaced by a metalloproteinase thermolysin, which was shown to readily digest [PrP.sup.C] into small fragments while leaving [PrP.sup.Sc] intact [48, 49]. Whether the thermolysin is appropriate replacement of PK is a matter of discussion. Only a few studies have addressed this question so far, so it could turn out that certain strains of [PrP.sup.Sc] are sensitive to thermolysin digestion, as was shown for PK.
Instead of the immunoprecipitation, a precipitation on solid-state capture matrix can be performed 47] taking into account that prions readily bind to stainless steel [50, 51]. The precipitation of [PrP.sup.Sc] from blood on stainless steel particles  was more efficient than immunoprecipitation with anti-PrP Ab . [PrP.sup.Sc] was also detected in blood from symptomatic vCJD patients, which is a huge step forward in the antemortem diagnostics of prion diseases . However, tests for screening of blood donors represent a separate issue as their sensitivity should be even higher, as well as they should provide an excellent specificity and a low background. Although more efficient, the use of precipitation on solid-state matrix has a drawback in comparison to immunoprecipitation with anti-PrP Ab. Whereas the selection of Ab enables the specificity of immunoprecipitation, precipitation on solid-state matrix is nonspecific.
Besides PrP precipitation, another way to approach to the sensitivity issue is in vitro amplification of [PrP.sup.Sc]. The most widely used method for the in vitro amplification of [PrP.sup.Sc] is protein-misfolding cyclic amplification (PMCA) . PMCA exploits the fact that [PrP.sup.C] is converted in the presence of [PrP.sup.Sc]. [PrP.sup.C] that serves as a substrate and minute amount of [PrP.sup.Sc] in the sample that serves as a template are incubated together to form new aggregates which are then dissociated by sonication. New [PrP.sup.C] is added and incubation and sonication are repeated. Multiple repetitions of aggregation and sonication cycles enable multiplification of [PrP.sup.Sc] to the level, detectable in WB. The drawback of PMCA is the repetition of many cycles, which is time consuming and increases the possibility of arising of false positive results .
Two recent reports have shown the use of in vitro amplification of [PrP.sup.Sc] in combination with immunoassays more sensitive than WB. Chang et al. described a method that combines the in vitro [PrP.sup.Sc] amplification similar to PMCA with AS-ELISA and fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT), named Am-A-FACCT . In the first step, plasma is mixed with healthy brain homogenate and subjected to amplification. Subsequently, newly formed [PrP.sup.Sc] aggregates are captured by an aggregate-specific mAb in AS-ELISA in combination with FACTT  where detection is performed via biotin-conjugated DNA template. The transcription of DNA template into RNA is followed by the addition of the RNA-intercalating dye, and the intensity of the emitted light is measured. Incorporating more steps into the procedure may prolong the duration of an experiment and also increases the possibility of experimental mistake, but the sensitivity can be greatly enhanced. According to Chang et al. , Am-A-FACTT can detect [PrP.sup.Sc] aggregates in the blood of scrapie-infected mice and chronic wasting disease-(CWD-) infected mule deer in asymptomatic phase. Combination of limited PMCA, IP, and a very sensitive detection system SOFIA conserves the high sensitivity of the method despite the low number of cycles. This approach enabled the detection of [PrP.sup.Sc] in the blood of scrapie-infected sheep and CWD-infected white-tailed deer in the preclinical phase .
The knowledge about prions that has accumulated in the last three decades and the use of routine testing of bovine brain for BSE had great impact on reducing the risk of prion transmission. However, for complete prevention on prion transmission through food, drugs, and blood-derived products, the sensitivity of the methods for prion detection must be greatly improved and designed for analyzing low-content prion material.
The latest advances in [PrP.sup.Sc] immunoassaying set the course of development of testing in different directions, all headed for the same goal--the maximal sensitivity and specificity of the method.
Accumulating reports on PK-sensitive strains of prions have reflected unfavorably on the use of PK-based diagnostics and therefore in novel prion immunoassays, PK is being avoided.
For routine antemortem testing of potential TSE transmitters, a blood test would be most appropriate. In an effort to develop such a test, different obstacles need to be overcome. Firstly, testing systems, developed for brain tissue, cannot be transferred directly to blood because quantities of [PrP.sup.Sc] in blood are much lower than in brain. Secondly, little is known about biophysical properties of [PrP.sup.Sc] in blood which may differ from [PrP.sup.Sc] in brain. Moreover, samples of infected human blood are limited in number and availability, which is an important drawback. However, a recent study by Edgeworth et al. shows that it is possible to detect prions in the blood of symptomatic vCJD patients . Two other studies on experimentally infected animals demonstrated the detection of blood prions also in the asymptomatic phase of the disease [44, 45], reaching a long-expected milestone. However, both methods are quite complex and therefore do not seem to be applicable to large-scale screening blood tests. The question concerning artificial production of prions by in vitro amplification in medical institutions also needs to be taken into consideration.
A simple, inexpensive, high-throughput, and at the same time highly sensitive blood test for prions does not seem to be available in the near future. A more likely solution seems to be large-scale screening for TSE surrogate markers in combination with an extremely sensitive prion test applied only to the identified risk samples.
A. Lukan and T. Vranac contributed equally to this work.
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Anja Lukan, Tanja Vranac, and Vladka Curin Serbec
Department for Production of Diagnostic Reagents and Research, Blood Transfusion Centre of Slovenia, Slajmerjeva 6, 1000 Ljubljana, Slovenia
Correspondence should be addressed to Vladka Curin Serbec; email@example.com
Received 15 March 2013; Revised 27 May 2013; Accepted 2 July 2013
Academic Editor: Benaissa El Moualij
TABLE 1: Summary of the methods for detection of [PrP.sup.Sc] in brain tissue. Reference PrP source PK Denaturation  rMoPrP, rOvPrP, - - rBoPrP, rHuPrP, and mice brain  BSE bovine brain + - and scrapie ovine brain  Scrapie sheep + - brain and tonsils, BSE bovine brain, and scrapie hamster brain  sCJD human brain + -  Scrapie hamster + - brain  BSE bovine brain - 0.1M GdnSCN  BSE bovine brain - 1M GdnHCl 6 M GdnHCl  BSE bovine brain, + 4 M GdnHCl CWD white-tailed deer, mule deer, and elk brains  Scrapie mouse and - 8 M GdnHCl hamster brain 6 M GdnHCl  Scrapie sheep brain - 6 M GdnHCl  vCJD human spleen - 2 M GdnHCl and brain 6M GdnHCL  BSE ovine brain and + Heath scrapie ovine brain  Scrapie hamster and - 1% SDS sheep brain, CWD-infected white-tailed deer brain  TME hamster brain + 3 M GdnSCN  Paraffin-embedded - Denaturation scrapie ship, buffer CWD white-tailed (not deer and TME specified) cattle brains  CJD human brain - 3 M GdnSCN Detection Reference PrP source Antibodies method  rMoPrP, rOvPrP, C: 11G5 D: Sandwich rBoPrP, rHuPrP, 11G5-biotin ELISA and mice brain  BSE bovine brain C: 6H4 D: n.r. Sandwich and scrapie ELISA ovine brain  Scrapie sheep D: SAF70 ELISA brain and tonsils, Secondary BSE bovine brain, AB conjugated and scrapie with peroxidase hamster brain  sCJD human brain C: 1E5 D: IPCR 4F7-biotin Streptavidin- biotin-DNA  Scrapie hamster C: 8b4 or 7A12 IPCR brain D: 3F4-biotin Streptavidin- biotin-DNA  BSE bovine brain C: 6H4 D: Sandwich rabbit ELISA antiserum C15S Swine anti-rabbit Ab-HRP  BSE bovine brain C: FH11 D: DELFIA 3F4-Eu  BSE bovine brain, C: Fab D18 D: DELFIA CWD white-tailed recFab deer, mule deer, HuM-P-Eu and elk brains  Scrapie mouse and C: 11G5 D: Sandwich hamster brain 7A12, 2F8, ELISA 8F9, and 8B4-biotin  Scrapie sheep brain C: FH11 D: DELFIA 8H4-Eu  vCJD human spleen C: FH11 D: DELFIA and brain 3F4-Eu  BSE ovine brain and C: SAF34 D: Sandwich scrapie ovine brain Bar224-enzyme ELISA  Scrapie hamster and C: 11F12 D: SOFIA sheep brain, 5D6-biotin CWD-infected Streptavidin- white-tailed Rhodamine Red X deer brain  TME hamster brain D: 3F4 Goat ELISA anti-mouse-HRP  Paraffin-embedded HerdChek BSE- ELISA scrapie ship, Scrapie Ag CWD white-tailed Test C: deer and TME polyanionic cattle brains ligand D: anti-PrP-HRP  CJD human brain C: V5B2 D: DELFIA EM20 Reference PrP source Sensitivity *  rMoPrP, rOvPrP, 6 ng of rBoPrP, rHuPrP, aggregated and mice brain PrP  BSE bovine brain 6pg rPrP/well and scrapie 30 pg/mL ovine brain  Scrapie sheep 3 ng rBoPrP brain and tonsils, BSE bovine brain, and scrapie hamster brain  sCJD human brain n.r.  Scrapie hamster 1 x 104 brain [Pr.sup.PSc] molecules/mL or 19 fg/mL  BSE bovine brain 1 ug [PrP.sup.Sc]/mL  BSE bovine brain 36 pg PrP/well  BSE bovine brain, 1 ng rec CWD white-tailed [beta]- deer, mule deer, MBo2M PrP/mL and elk brains  Scrapie mouse and 0.05-5 ng hamster brain rHuPrP  Scrapie sheep brain 200 pg rOvPrP/ well  vCJD human spleen 10 pg and brain rHuPrP/mL  BSE ovine brain and n.r. scrapie ovine brain  Scrapie hamster and 10 ag sheep brain, rHaPrP, CWD-infected rMoPrP, white-tailed rOvPrP, deer brain and rDePr  TME hamster brain n.r.  Paraffin-embedded n.r. scrapie ship, CWD white-tailed deer and TME cattle brains  CJD human brain n.r. * We report the sensitivity as provided by the authors because of the lack of sufficient data for converting the results to the united form. rHuPrP: recombinant human prion protein, rMoPrP: recombinant mouse prion protein, rOvPrP: recombinant ovine prion protein, rBoPrP: recombinant bovine prion protein, rDePrP: recombinant deer prion protein, rHaPrP: recombinant hamster prion protein. ICSM is not an acronym but a name of two anti-prion antibodies (ICSM 35, ICSM 18). TABLE 2: Summary of the methods for detection of [PrP.sup.Sc] in blood. Reference PrP source PK Denaturation  Scrapie mice - - blood and CWD deer and elk blood  CJD human blood + -  Scrapie sheep - 1% SDS blood and CWD white-tailed deer blood  Healthy human Thermolysin 4 M GdnHCl blood spiked with vCJD brain  vCJD human blood - Heat and healthy human blood spiked with vCJD brain Detection Reference PrP source Antibodies method  Scrapie mice n.r. facct blood and CWD deer and elk blood  CJD human blood C: 6H4 D: DELFIA 3F4-biotin Streptavidin-Eu  Scrapie sheep C: 11F12 D: SOFIA blood and CWD 5D6-biotin white-tailed deer blood  Healthy human C: ICSM 10 D: Sandwich blood spiked ICSM 35-biotin ELISA with vCJD brain  vCJD human blood D: ICSM ELISA and healthy 18-biotin human blood spiked with vCJD brain Reference PrP source Sensitivity *  Scrapie mice n.r. blood and CWD deer and elk blood  CJD human blood 50 ul recPrP/mL PK-digested plasma 10 pg recPrP/well  Scrapie sheep n.r. blood and CWD white-tailed deer blood  Healthy human 2.8 pg [PrP.sup.Sc]/ blood spiked well 150000-fold with vCJD brain dilution (105-17)  vCJD human blood [10.sup.10]-fold and healthy dilution of human blood vCJD brain spiked with homogenate in vCJD brain whole blood * We report the sensitivity as provided by the authors because of the lack of sufficient data for converting the results to the united form. rHuPrP: recombinant human prion protein, rMoPrP: recombinant mouse prion protein, rOvPrP: recombinant ovine prion protein, rBoPrP: recombinant bovine prion protein, rDePrP: recombinant deer prion protein, rHaPrP: recombinant hamster prion protein. ICSM is not an acronym but a name of two anti-prion antibodies (ICSM 35, ICSM 18).
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|Author:||Lukan, Anja; Vranac, Tanja; Serbec, Vladka Curin|
|Publication:||Journal of Immunology Research|
|Date:||Jan 1, 2013|
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