Rapid screening and confirmatory methods for biochemical diagnosis of human prion disease

https://doi.org/10.1016/j.jviromet.2011.05.016Get rights and content

Abstract

Transmissible spongiform encephalopathies (TSEs) are characterised by accumulation of an abnormal isoform of prion protein (PrPsc), mainly in the brain but also in various peripheral tissues. Home-made assays consisting of non-standardised protocols are used currently for laboratory diagnosis of human TSE. The purpose of the present study was to test the ability of two commercial assays, TeSeE™ CJD ELISA and TeSeE™ Western blot, to detect PrPsc in cerebral and lymphoid tissues of TSE patients. Both tests detected a PrPsc-significant signal in the brains of 54 affected patients and not in 51 controls, yielding 100% specificity and 100% sensitivity. Furthermore, three post-mortem spleens and two pre-mortem tonsils from three patients with variant Creutzfeldt–Jakob disease (vCJD) were detected correctly. The expected PrPsc molecular patterns were found in TSE patient brain tissue and in the tonsils and spleens of the three vCJD patients. In conclusion, these rapid and robust in vitro tools were suitable for routine human TSE diagnosis and characterisation. CJD could also be diagnosed during the patient's lifetime by detection of PrPsc in the tonsil. A diagnostic strategy associating TeSeE™ CJD ELISA screening to biochemical confirmation by TeSeE™ Western blot is proposed.

Highlights

► We test commercial ELISA and Western blot originally designed for animal diagnosis. ► Both assays are suitable for human diagnosis in brain and lymphoid tissue. ► We obtain the expected molecular pattern of pathological prion protein. ► Commercial kits are robust assays and good alternative to time consuming methods. ► We propose a diagnostic strategy associating screening to confirmatory methods.

Introduction

Transmissible spongiform encephalopathies (TSEs) include bovine spongiform encephalopathy (BSE), scrapie in sheep and goats, chronic wasting disease (CWD) in deer and elk and Creutzfeldt–Jakob disease (CJD), fatal familial insomnia (FFI), and Gerstmann–Straüssler–Scheinker syndrome (GSS) in humans. In all these diseases, the conversion of the protease-sensitive cellular form of the prion protein (PrPc) into a pathological isoform (PrPsc) appears to be a key process in pathogenesis. The protease resistance of PrPsc leads to the formation of a residual core of 27–30 kDa (PrPres) after proteinase K (PK) treatment (Prusiner, 1998).

This property is used mainly in assay protocols for residual PrPres detection by antibodies targeting the resistant core and/or the carboxylic moiety of PrP. For routine TSE diagnosis in animals, assays include the Western blot technique with or without concentration step and immunoassays such as LIA, IEA, ELISA or DELPHIA (Grassi et al., 2008). Development strategies for commercial tests are very different between TSE diagnosis in animals and humans. Whereas the BSE epidemic led to the development of rapid and robust commercial diagnostic assays for BSE and then scrapie and chronic wasting disease in veterinary programs, there are still no commercial kits for detecting PrPres in CJD in humans. In most cases, laboratories use home-made methods based on Western blot which is the “gold standard” for biochemical diagnosis of CJD.

Various assay protocols for Western blot detection of PrPres in CJD have been reported. The first described Western blot detection after PrPres purification and concentration by an ultracentrifugation step (Brown et al., 1986). Direct PrPres detection from 10% sample homogenates treated with PK then denatured in Laemmli buffer was recommended for its ease of use (Collinge et al., 1996, Parchi et al., 1996). Later, concentration steps using centrifugation, NaPTa precipitation, ultracentrifugation or streptomycin were introduced to enhance sensitivity, a key point in tissue other than brain (Favereaux et al., 2004, Lee et al., 2000, Quadrio et al., 2009, Wadsworth et al., 2001). All these methods, however, present certain disadvantages, lacking reproducibility, standardisation and robustness, and are time-consuming or make demands on operator experience to recover the ultracentrifugation pellet.

In the animal field, 12 different tests were validated successively by the European Commission (Regulation (EC) N° 999/2001 of 22 May 2001, amended by Regulations N° 1053/2003 of 19 June 2003, N° 260/2005 of 16 February 2005 and N° 162/2009 of 26 February 2009 for the prevention, control and eradication of certain TSEs). In addition to its rapid tests for animal TSE screening (TeSeE™ SAP and TeSeE sheep and goat ELISA), Bio-Rad has also developed a commercial test for confirmation of suspected animal TSE (TeSeE™ Western blot). The maximum operating times from tissue homogenisation to plate reading in the ELISA protocol and to film development after immunoblotting is about 3 and 7 h, respectively. These relatively short times could enable further rapid and robust tools to be implemented in the diagnostic field. The present study assessed the performance and feasibility of these kits for detecting PrPres in cerebral and lymphoid tissue in human TSEs.

Section snippets

Post-mortem samples and pre-mortem tonsils

For the detection of PrPres in brain, cerebral fragments (see Table 1) were treated in the Department of Neurochemistry BSL-3 laboratory dedicated to prion diseases at the Hospices Civils de Lyon. Necropsy brain tissues were collected from Lyon, Besançon and Marseille and treated according to the French legislation on ethical and technical issues.

For the retrospective study, 28 diagnosed patients (neuropathological lesions, immunohistochemistry and immunodetection of PrPres on the frontal

Diagnostic sensitivity

Brain tissue: In the absence at the time of study of a predetermined cut-off value adapted for human brain tissue, a CJD and a TSE-negative population were compared.

From the cohort of 28 patients, the suitability of the TeSeE™ CJD ELISA test was confirmed, and an initial cut-off value was set (Fig. 1). For the negative samples (n = 14), tested neat, the mean and standard deviation (SD) were calculated at respectively 0.039 OD and 0.018 OD. OD values for positive samples were all between 1.796 and

Discussion

TeSeE™ Western blot and TeSeE™ CJD ELISA permitted clear discrimination of the 54 TSE patients and the 51 without TSE, with no equivocal results. These data, obtained both on a well characterised panel of brain tissue examined retrospectively and on a prospective blind patient cohort, showed that the full process used for animal TSE was transferred successfully to human application. The monoclonal antibodies used in these assays were selected for their sensitive detection of the PK-treated and

Conclusion

These commercial kits, supplied initially for animal TSE diseases, were validated successfully for detection of PrPres in human brain for diagnostic purposes. In lymphoid tissue, PrPres was detected in the few samples available, and completed the results reported by Clewley et al. (2009) in tonsil study.

The feasibility study data demonstrated that the TeSeE™ CJD ELISA and TeSeE™ Western blot kits are suitable for detection of PrPres in human brain and lymphoid tissue. They seem to be a good

Acknowledgments

We wish to thank F. Didier, R. Plantier and C. Radenac for their technical assistance and G. Besson, J. Boulliat, E. Diot, F. Philippeau, L. Ribouillard and Ph.Vion for help in the collection of tissues. We also thank the families who permitted the use of tissue for research.

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