1 NIBSC, Blanche Lane, South Mimms, Potters Bar EN6 3QG, UK
2 National Transfusion Microbiology Reference Lab, National Blood Service, Colindale Avenue, London NW9 5BG, UK
3 University of Toronto, CRND, Tanz Building, 6 Queen's Park Crescent, West Toronto, Ontario, Canada, M5S 3H2
4 VA Medical Centre, Mailstop 151, Room 3C-128, 10N Green Street, Baltimore, MD 21201, USA
5 FDA CBER, Division of Emerging and Transfusion-transmitted Diseases, OBRR, CBER, FDA HFM310, 1401 Rockville Pike, Rockville, MD 20852-1448, USA
6 Sanquin Research, Plesmanlaan 125, PO BOX 9190, 1066 AD Amsterdam, The Netherlands
7 Institute of Neurology, Medical University of Vienna, Austrian Reference Centre for Human Prion Diseases, AKH 4J, A-1097 Vienna, Austria
8 Aventis Behring GmbH, Postfach 1230, 35002 Marburg, Germany
9 Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0EG, UK
10 National CreutzfeldtJakob Disease Surveillance Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
Correspondence
P. Minor
pminor{at}nibsc.ac.uk
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ABSTRACT |
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Published online ahead of print on 19 March 2004 as DOI 10.1099/vir.0.79959-0.
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INTRODUCTION |
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CJD is classified into sporadic, iatrogenic, familial and variant forms, depending on causation (Prusiner, 1997). In addition, a number of different forms are recognized based on clinical and histopathological presentation, and different molecular forms of PrPSc have been recognized, depending on the size and properties of the residual fragments after treatment with proteinase K, including the cleavage site and the glycosylation profile (Parchi et al., 1996
; Wadsworth et al., 1999
). The differences observed imply a difference in the conformation of the protein linked to the strain of CJD involved. Finally, the human PrP gene has a polymorphism at codon 129, which may encode either methionine or valine; the genotype modulates the expression of the disease (Palmer et al., 1991
). Met/Met homozygotes are over-represented in cases of sporadic CJD and all cases of variant (v) CJD to date have been of this genotype. All of these factors interact to some extent and it is possible that standard preparations suitable for one particular type will not be suitable for others.
This report describes materials for use in the calibration of results of assays of PrPSc in preparations of CJD brains of the Met/Met genotype. This type was chosen as vCJD is of major concern and has thus far always been of this type. However, at least two molecular types of PrPSc in cases of sporadic CJD are recognized in this genotype. Specimens of sporadic and vCJD were therefore sought. Under the auspices of the World Health Organization (WHO), the materials were homogenized and distributed in identical aliquots in ampoules. The preparations have been characterized in the preliminary study reported here, dealing in the first instance with in vitro assays of PrPSc content. The objective was to measure PrPSc content in some commonly used assays that might differ in sensitivity and therefore benefit from the use of a calibrating reference material. No attempt was made to include all assay methods and formats, the study being intended to demonstrate the value of the reference materials and to characterize them in part, rather than to assess the assays used to examine them. The design of the study was discussed and agreed by the WHO Working Group on International Reference Materials for the Diagnosis and Study of TSEs, whose membership is given in the Acknowledgements.
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METHODS |
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Design of study.
Participants were requested to use routine in-house methods to titrate the amounts of PrPSc in the preparations four times from separate vials, using 10-fold serial dilutions for the first run to establish approximate end-points and 3-fold serial dilutions for the remainder. The highest dilution containing detectable PrPSc was reported and raw data were returned with technical details of the method employed.
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RESULTS |
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In the second format (b), samples were diluted in 0·5 log10 steps in human plasma containing 2 % lauryl sarcosine, after which 1 ml was precipitated with phosphotungstic acid, treated with proteinase K and split into two aliquots as above. The aliquots were treated as for method (a) and divided between three wells in a microtitre plate. The wells were coated with a monoclonal antibody specific for human PrP. Each well thus received the equivalent of one-third of 500 µl of each dilution, or 167 µl. The data are given in Tables 2 and 3 under Laboratory 7b. The end-point titres were 9·7x104, 2·3x104 and 1·7x105 for sp1CJD, sp2CJD and vCJD, respectively (Table 2
), equivalent to a minimum detectable volume of 10 % brain homogenate of 0·0017, 0·0073 and 0·00098 µl, respectively, approximately 50-fold lower than any other method. However, the potencies measured by method 7b expressed as the ratio of the end-point dilutions relative to that of the vCJD preparation were similar to those determined for the immunoblot methods (Table 4
), suggesting that the use of the vCJD preparation as a calibrant would make assay results comparable.
The other in vitro method employed by the eighth laboratory was qualitative in nature in the format used and was based on the extraction of PrPc by 2 M guanidinium hydrochloride followed by the extraction of PrPSc by 6 M guanidinium hydrochloride (Barnard et al., 2000). Extracted PrP was assayed by DELFIA (dissociation enhanced lanthanide fluorescence immunoassay) and the results expressed as the ratio of PrPSc to total PrP. A result in excess of that of normal brain indicated the presence of PrPSc. The results from one run are shown in Table 5
. Provided sufficient sample was examined, the ratio was independent of dilution. It can be seen that the normal brain gave a figure of 15·5 % as a 1 : 10 or 1 : 100 dilution of the 10 % homogenate, the sp1CJD a mean figure of 50·5 % as a 1 : 10 or 1 : 100 dilution, sp2CJD a figure of 22·9 % at a dilution of 1 : 10 or 1 : 100 and vCJD a mean figure of 65·7 % up to a dilution of 1 : 1000. Presumably the data could be reinterpreted to provide a truly quantitative estimate, but the assay was not designed for use in this way. However, it is of interest that the vCJD gave the highest percentage of PrPSc, closely followed by sp1CJD, and sp2CJD was the least potent, in keeping with the results presented in Tables 2 and 3
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DISCUSSION |
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The initial characterization of the materials reported here concerned in vitro methods, mostly immunoblotting. Differences in sample preparation and the volumes loaded onto the gels resulted in a range of detectable end-point dilutions among laboratories of the order of 100-fold. Within-laboratory reproducibility was of the order of threefold except for Laboratory 3 where the assay was less well controlled at the time of the study. When all results were included and expressed in terms of minimum volume of homogenate containing detectable PrPSc, the results between laboratories were surprisingly uniform with a range of 4·4- to 9·0-fold depending on the homogenate concerned. This approximates to the dilution steps employed and to the variation within a laboratory. Thus, as might be expected, expressing the results as a potency relative to the vCJD sample did not greatly improve reproducibility, but gave a range of 5·7- and 6·3-fold for the sp1CJD and sp2CJD samples, respectively. It was striking that the details of the assays, specifically dilution before or after digestion in brain or buffer, had no detectable influence on the results obtained.
Data from the other quantitative in vitro method, CDI, showed that it was of high sensitivity, but comparable to the most sensitive of the immunoblot methods in terms of minimum detectable volume of brain homogenate when used in the initial format. Differences in sensitivity expressed in terms of end-point dilutions arose chiefly from the sample volume assayed, with the most sensitive able to concentrate dilute preparations in a quantitative manner. However, the modified CDI format, involving dilution in plasma and the use of a monoclonal antibody capture method, gave a signal from 50- to 100-fold less material. The potencies of the sp1CJD and sp2CJD preparations relative to the vCJD preparation were within the same range as for the other assays, however, and the vCJD preparation could be used as a calibrator of potencies.
The semi-quantitative assay method gave results consistent with those of the other methods, with the vCJD and sp1CJD specimens giving a higher PrPSc content than the sp2CJD material.
The observation that both sp1CJD and sp2CJD preparations contained PrPSc of both type 1 and type 2 could not be attributed either to accidental mixing of samples from more than one individual or to gel artefacts and is interpreted to mean that the patients' brains actually contained both types. This may be either because both brains were somehow atypical or because the presence of both types of PrPSc in a single patient is more common than has been previously thought. Even when small tissue samples (<100 mg) have been assayed, two laboratories have independently reported the presence of type 1 and type 2 PrPSc in individual spCJD brains (Puoti et al., 1999; Kovacs et al., 2002
). The specimens used in this study involved 100 g of each brain, so that material was derived from a larger brain area than is usually examined. The vCJD brain preparation was uniform. While the reproducible behaviour of the sp1CJD and sp2CJD brains in the study suggests that the mixture of types does not present a problem for the types of assays performed here, the suitability of the materials from the sporadic CJD cases as reference preparations for some other purposes might be questioned and that was the reason why two laboratories stopped work after preliminary characterization. This issue will only be settled by further studies using other methods, including infectivity assays that are currently under way or planned. Ultimately the materials described here, with others in development, will be examined by a range of procedures, enabling a correlation between in vitro and in vivo potencies and providing well-characterized materials by which new procedures, including new infectivity assays in lines of transgenic mice, can be compared quickly. The candidate Biological Reference materials are intended for diagnostic assessment, not as seed materials or for use in spiking studies, and are now available for this purpose. It is intended to expand the available materials to include suspensions of brain tissue from CJD patients with other PrP genotypes and samples of other tissues including lymphoid tissues and blood.
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ACKNOWLEDGEMENTS |
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Dr D. Asher, FDA CBER, Office of Blood Research and Review, Laboratory of Special Pathogens, FDA HFM470, 1401 Rockville Pike, Rockville, MD 209852-1448, USA.
Dr Henry Baron, Senior Director, Prion Research, Aventis Behring, Aventis Behring SA, 15 rue de la Vanne, Tri T1, 92545 Montrouge Cedex, France.
Professor H. Budka, Institute of Neurology, Medical University of Vienna, Austrian Reference Centre for Human Prion Diseases, AKH 4J, A-1097 Vienna, Austria.
Dr F. A. C. van Engelenburg, Plesmanlaan 125, PO Box 9190, 1006 AD Amsterdam, The Netherlands.
Professor John Collinge, MRC Prion Unit, Department of Neurogenetics, Imperial College School of Medicine at St Mary's, Norfolk Place, London W2 1PG, UK.
Dr D. Dormont, Service de Neurologie, Direction de Sciences du Vivant, Departement de Recherche Medicale, Centre d'Etudes Nucleaires (CEA), Fontenay-aux-Roses, France.
Dr F. van Engelenburg, Sanquin Research, Plesmanlaan 125, PO Box 9190, 1006 AD Amsterdam, The Netherlands.
Dr P. Gambetti, Division of Neuropathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, USA.
Dr A. Giulivi, Associate Director, Bureau of Infectious Diseases, Blood-borne Pathogens Division, Laboratory Centre for Disease Control, Health Protection Branch, LCDC Building, AL 0601E2, Ottawa, ON, Canada, K1A 0L2.
Dr M. Groschup, Federal Research Centre for Virus Diseases of Animals, Institute of Immunology, Paul Ehrlich Strasse 28, 72076 Tübingen, Germany.
Dr J. Hope, VI Technologies Inc., 134 Coolidge Avenue, Watertown, MA 02472, USA.
Dr J. Ironside, Consultant Neuropathologist, National CreutzfeldtJakob Disease Surveillance Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.
Dr J. C. Manson, Instititute for Animal Health, AFRC, Ogston Building, West Mains Road, Edinburgh EH8 3JF, UK.
Dr J. Hope, Institute of Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK.
Dr D. Matthews, TSE Programme Manager, Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, UK.
Dr A. Padilla, World Health Organisation, Geneva 1211, Switzerland.
Professor M. Pocchiari, Registry of Creutzfeldt Jakob Disease, Laboratory of Virology, Istituto Superiore di Sanita, Viale Regina Elena 299, I-00161 Rome, Italy.
Dr R. G. Rohwer, Molecular Neurovirology Laboratory, Medical Research Service, VA Maryland Health Care System, 10N Greene Street, Baltimore, MD 21201, USA.
Dr J. Safar, Institute of Neurodegenerative Diseases, University of California, San Francisco, HSE 774, Box 0518, San Francisco, CA 94143, USA.
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Received 7 January 2004;
accepted 2 March 2004.