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Short Communication |
1 National CJD Surveillance Unit, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
2 Ames Laboratory, USDOE, Iowa State University, Ames, IA 500011, USA
3 SNBTS Products and Components R&D Group, National Science Laboratory, 21 Ellen's Glen Road, Edinburgh EH17 7QT, UK
Correspondence
Mark W. Head
m.w.head{at}ed.ac.uk
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ABSTRACT |
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).
Since the first description of variant CJD (vCJD) (Will et al., 1996), there have been 161 cases of vCJD in the UK (http://www.cjd.ed.ac.uk/figures.htm) and an increasing number of other countries are now reporting cases. Among the human prion diseases, vCJD appears to have the broadest tissue distribution of PrPSc at the post-mortem stage, including the lymphoreticular system (tonsil, spleen, appendix and lymph nodes), ganglia of the autonomic and peripheral nervous system (trigeminal, dorsal root, celiac and stellate ganglia), gastrointestinal tract, adrenal gland and thymus (Wadsworth et al., 2001; Haïk et al., 2003; Head et al., 2004a). In vCJD, peripheral accumulation of PrPSc appears to precede the onset of clinical symptoms (Hilton et al., 1998, 2002) and this fact, coupled with the known infectivity of these tissues at the post-mortem stage (Bruce et al., 2001), has prompted fears that individuals in a pre- or subclinical state might pose a risk of secondary infection to others by iatrogenic means (Hilton et al., 2004).
The risk associated with blood was considered to be theoretical in nature until the demonstration that BSE and scrapie could be transmitted between sheep by blood transfusion, even when the donor sheep were in the preclinical phase of infection (Hunter et al., 2002). Fears that similar events could occur in humans have been realized by the description of vCJD in two recipients of blood from donors who went on to develop vCJD (Llewelyn et al., 2004; HPA, 2006). A case of transfusion-associated transmission has also been described in which the recipient died of another cause, but showed evidence of vCJD infection in the peripheral tissues (Peden et al., 2004).
These events have added considerable urgency to the need for a blood test for CJD. Any tests capable of detecting PrPSc in blood would require excellent biochemical sensitivity (Brown & Cervenakova, 2004; Minor, 2004) and any test with such biochemical sensitivity would also need to have an extremely high degree of test specificity in order to be implemented by the transfusion services (Johnson et al., 2001; Blajchman et al., 2004).
Claims for a high degree of biochemical sensitivity have been made for a number of PrP assays, including conformation-dependent immunoassay (Safar et al., 2002; Bellon et al., 2003), immuno-PCR (Barletta et al., 2005), immunocapillary electrophoresis (ICE) (Schmerr et al., 1999; Yang et al., 2005a, b; Jackman et al., 2006) and PrPSc-selective ligand-based assays (Lane et al., 2004; Grosset et al., 2005). We have evaluated one of these, ICE, by using clinical blood specimens from CJD patients [both sporadic CJD (sCJD) and vCJD] and diagnostically relevant neurological controls. We have also sought to determine whether post-mortem vCJD brain or spleen tissue might provide a source of positive-control material to validate and calibrate the ICE assay.
The ICE method has been described previously (Schmerr & Jenny, 1998; Schmerr et al., 1999; Yang et al., 2005a). Buffy-coat fractions of around 10 ml starting volume of whole blood are lysed by cycles of freezing and thawing and digested sequentially with DNase and proteinase K. Extraction is then performed by using a patented procedure and the material is purified by using hydrophilic-interaction chromatography as described previously (Schmerr & Alpert, 2000; Yang et al., 2005a). The competitive immunoassay involves titration of a fluorescently labelled prion protein sequence synthetic peptide (FP) and a polyclonal antibody (Ab) raised to that sequence, such that free-zone capillary electrophoresis (CE) followed by laser-induced fluorescent (LIF) detection shows equivalent peak heights for the antibody-bound and free peptide. Test samples added to the developing immunocomplex are scored as positive if the bound/free peptide values are <70 % of the control values (Schmerr & Jenny, 1998; Schmerr et al., 1999).
In this study, the ICE method was applied to archival buffy-coat fractions of clinical blood specimens taken with consent for research from 15 patients suspected of having CJD. Of these 15 patients, five were cases of definite or probable vCJD, four were definite cases of sCJD and the remaining six (in whom a diagnosis of sCJD or vCJD was initially considered) were given an alternative final diagnosis. Buffy-coat fraction had been prepared previously from EDTA-treated whole blood by using Histopaque-1077 (Sigma) and stored at –70 °C. DNase and proteinase K were used at 50 and 100 µg ml–1, respectively, and the assay employed the ovine FP 223RESQAYYQRGASVIL237. Immunocomplex formation and CE-LIF analysis conditions were as described previously (Schmerr et al., 1999). The peak heightbound/peak heightfree for each sample was calculated and expressed as a percentage of the peak heightbound/peak heightfree values obtained in the absence of extracted blood samples. The results are shown graphically (Fig. 1). By using the predetermined 70 % value to define a positive result (Schmerr et al., 1999), none of the samples from the neurological-control group tested positive, although one was borderline positive. By using the same criteria, two of the sCJD samples were positive and a third was borderline positive. Lastly, three out of the five vCJD samples were strongly positive. The assay therefore appears to detect the presence of protease-resistant PrP in the blood of a proportion of the patients with CJD, which is absent from blood samples from patients with other neurological conditions. However, the assay uses relatively large volumes (
10 ml) of an extremely limited resource (CJD patient blood specimens) and there is, as yet, no confirmatory assay for the presence of abnormal prion protein in CJD patient blood. Hence, we sought to investigate further the potential of ICE by employing FP sequences from different regions of the human prion protein and attempted to calibrate the assay by using autopsy vCJD brain and spleen reference materials that have been prepared by the National Institute of Biological Standards and Control (NIBSC) expressly for such purposes (Minor et al., 2004).
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1 pmol ml–1) with the appropriate dilution of Ab for 16 h at 7 °C. The CE injection volume was 3.6 nl, corresponding to <4 amol FP analysed. The competition of relevant unlabelled peptide (UP) for the binding of antibody in all assays was found to be concentration-dependent (Fig. 2a). Complete competition was obtained with the addition of the relevant UP at a concentration of <20 nM in each case and the 70 % cut-off was obtained at 0.41 nM for FP1, 0.25 nM for FP2 and 0.27 nM for FP5. As expected, the Ab/FP reactions were specific. Competition with irrelevant UP did not exceed 20 %, even when added to a concentration of up to 1 µM (Fig. 2b). Surprisingly, when recombinant human PrP (rHuPrP) was added in the 11–546 nM range, no competition was observed in the FP1 and FP5 assays. There was concentration-dependent competition when rHuPrP was added to the FP2 assay and, although complete competition was not observed in the 11–546 nM range, the 70 % cut-off was reached at 109 nM rHuPrP. A typical example of such an assay is shown in Fig. 2(c).
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PrPSc was prepared from vCJD, sCJD and neurological control (Lewy body dementia; LBD) brain homogenates by repeated rounds of proteinase K digestion and centrifugal concentration (Head et al., 2004b). Western blotting with 3F4 and 6H4 showed a tenfold enrichment for PrPSc. The PrPSc was also detectable on Western blots by the FP5 Ab. SDS-PAGE and Coomassie blue staining showed that the protein had been partially purified. These preparations were diluted (4x10–2–4x10–8 in 0.5 ml PBS) and the extraction and ICE analysis were performed. Neither the standard assay nor any methodological variation attempted produced evidence for PrPSc detection in these samples by ICE. Finally, direct extraction and analysis were undertaken of the NIBSC vCJD and AD spleen samples (100 µl 10 % extract). This attempt was confounded by the apparent presence in vCJD and AD spleen of proteins or other moieties that interacted with the FP, producing artefactual distortion of the fluorescent peaks.
In an effort to determine at what point during the extraction procedure the PrPSc was lost, samples were taken from key steps (Fig. 3) in the extraction process of the semi-purified vCJD and LBD brain homogenates and analysed by Western blotting (with 3F4, 6H4 and FP5 Ab) and dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA; 6H4 and 3F4 combination). These experiments indicated consistently that detectable PrPSc failed to reach the hydrophilic interaction-chromatography step, but were rendered insoluble during the prior organic solvent-extraction steps.
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; Llewelyn et al., 2004). However, this proposition is only necessarily true if PrPSc itself is indeed the infectious agent. Moreover, the physical forms of PrPSc in blood and brain need not be identical. The finding that the rodent scrapie brain and murine vCJD plasma contain a fraction of protease-sensitive PrPSc or protease-sensitive infectivity complicates matters further (Safar et al., 2002; Yakovleva et al., 2004). Any test that uses protease resistance to distinguish between PrPC and PrPSc may miss putative protease-sensitive forms of PrPSc. Conversely, sensitive methods that rely on concentration techniques to detect low levels of PrPSc may erroneously detect residual PrPC that escapes proteolytic digestion. Techniques such as Western blotting identify analytes by immunological reactivity and confirm this by showing their electrophoretic mobility. Techniques such as ELISA and the ICE assay have no such additional inbuilt confirmatory aspects. The challenge for a first-generation blood prion assay is considerable as there is, by definition, no confirmatory test.
The simplest approach to test evaluation is therefore to screen sufficiently large numbers of samples from confirmed cases and a negative-control group to establish the sensitivity and specificity of the test in as close to the desired setting as possible. This may be done in sheep flocks ‘at risk’ of developing scrapie or animals infected experimentally with the agent of interest (Jackman et al., 2006). Such studies are not possible when considering human prion diseases and the alternatives are to use blood specimens from vCJD patients or to spike normal blood specimens with infectious material from other sources.
We have performed both types of experiment. None of the experimental protocols that we tried were able to detect vCJD brain- or spleen-derived PrPSc in the ICE assay; however, this appears to be a problem associated with sample processing rather than the competitive immunoassay or the analytical CE-LIF component. This is unfortunate, as the NIBSC reference materials are designed specifically to establish the analytical sensitivity of assays under development. In addition, matrix-associated problems were observed consistently with the spleen, involving interference with the competitive immunoassay, a problem that remains unresolved. The tests performed on clinical CJD blood specimens and relevant controls show some promise. By using the predetermined criteria for positivity, over half of the CJD blood specimens tested positive and none of the neurological controls were positive. It should, however, be noted that, when taken as a group, the results from the sCJD (mean, 77±23) and vCJD (mean, 76±49) patients differ little from the control group (mean, 78±5.7), a result similar to that found in the only other published study of ICE applied to CJD blood specimens (Cervenakova et al., 2003).
Given the relatively large volume (10 ml) of blood required for this assay, the extremely limited archives of blood available from patients with vCJD and the increasing demands on these resources from other assays under development, it seems unlikely that a larger ICE study on vCJD blood specimens will be performed in the near future. Since this work on vCJD blood was performed in 1999, there have been improvements made to both the ICE sample preparation and the analytical assay (Jackman & Schmerr, 2003; Yang et al., 2005a, b). For these reasons, the validation of ICE technology may prove more readily achievable in the veterinary rather than the medical context.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 February 2006;
accepted 25 May 2006.
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, Unlabelled peptide 2; 
, unlabelled peptide 5. (c) Representative competition plots with recombinant human PrP protein (rHuPrP) in the three FP/Ab pair assays: FP1, FP2 and FP5. rHuPrP dilution points: 11, 55, 109, 273 and 546 nM. Vertical bars denote SD (n=2–8). 
, FP2 assay; 
, FP5 assay.