title / Assessment and validation of emerging methods and reagents for BSE diagnosis.
/ DEFRA
project code / SE1762
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Assessment and validation of emerging methods and reagents for BSE diagnosis.
DEFRA project code / SE1762
Contractor organisation and location / Veterinary Laboratories Agency,
Woodham Lane,
New Haw, Addlestone, Surrey.
KT15 3NB
Total DEFRA project costs / £ 569,664
Project start date / 01/04/99 / Project end date / 31/03/03
Executive summary (maximum 2 sides A4)
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CSG 15 (1/00) 4
Projecttitle / Assessment and validation of emerging methods and reagents for BSE diagnosis.
/ DEFRA
project code / SE1762
New techniques, methods and (immuno)reagents for the diagnosis of BSE and the detection of the BSE agent (PrPsc) are being produced at VLA, other laboratories worldwide and by commercial companies. It is essential that there is a facility for the assessment of these methodologies and reagents, on a selective basis, so that claims can be examined and comparisons made with Gold Standard protocols in order to provide advice and consultancy to aid the formulation of Defra policy on BSE diagnostics. As the Community Reference Laboratory and a major centre for BSE diagnosis, VLA must also maintain its ability to incorporate the latest technology (when appropriate) and expertise in the diagnostic process to ensure that Defra is provided with up to date advice and data on BSE.
In order to be able to achieve these goals the original proposal set out a four phase approach, primarily for PrPsc - based diagnostics but also for alternative (surrogate) markers of disease.
1. To compare directly the quantification and limits of detection of Western blot, SAF, and immunohistochemistry with infectivity titres in pooled CNS to provide a standard analytical matrix for evaluation of other methods.
2. To continue to assess and evaluate new reagents and techniques for use in existing immunohistochemical, Western blot and ELISA based methodologies on an annual basis.
3. To evaluate new methods for BSE diagnosis, mainly for PrPbse detection but also for surrogate markers.
4. To improve the sensitivity for PrPsc detection by immunohistochemistry and to overcome the restrictions currently apparent on the use of IHC for the determination of PrPsc distribution in non- neural tissues.
Over the 4 years of the project, the following techniques, reagents and claims have been evaluated and, where appropriate, further developed and optimised or incorporated into new, separate, funding proposals.
Year 1, 1999 / 2000.
1. ImmunoCapillary Electrophoresis (ICE) for live animal scrapie testing (on blood samples)
Study output: Evaluation completed; Subsequent proposal funded (SE1757)
2. DELFIA development and application to the analysis of CSF
Study output: Evaluation and development completed; Studies extended into TSE5003
3. S-100 Protein determination by DELFIA
Study output: Evaluation completed; Method adopted for trial
4. A rapid Dot-Blot method for PrPsc
Study output: Evaluation completed; system insufficiently robust
5. Improvements and developments to the Immunohistochemical diagnosis of TSEs
Study output: Developments completed; Adopted into routine TSE diagnosis.
6. Evaluation of the claims for a 14-3-3 Protein diagnostic for BSE
Study output: Evaluation completed; Inconclusive data, study to be continued.
Year 2, 2000 / 2001.
1. Further 14-3-3 Protein evaluation and development
Study output: Evaluation completed; Insufficient test specificity for diagnostic use.
2. Development of IDEXX / Caprion collaboration. Investigation of claims of PrPsc – specific antibodies
Study output: Evaluation completed; Subsequent proposal funded (SE1776)
3. Initiation of links with CEA, Paris (Jaques Grassi) and subsequent evaluations of BIO-RAD systems
Study output: Evaluation completed; Real diagnostic potential. Further studies planned
4. Evaluation of heparan mimetics as diagnostic reagents for PrPsc in collaboration with the University of Paris.
Study output: Evaluation completed; Subsequent proposal funded (SE1778)
5. Continuing IHC development and optimisation – use of C-terminus PrP-peptide antibodies raised at VLA (R486 and R145) for TSE diagnosis. Improvements to antigen retrieval and epitope demasking on neural and LRS tissue sections.
Study output: Developments completed and incorporated into routine diagnostic process for IHC.
6. Other contacts.
Year 3, 2001/2002.
1. Attempted reproduction of the findings of PrP in urine from BSE-affected cattle
Study output: Evaluation completed; failure to reproduce original findings
2. Biochemical strain typing studies (Molecular weight of the unglycosylated PrP band in Western blots)
Study output: Developments completed as groundwork for subsequent “BSE in sheep” test
3. Improvements to the analytical sensitivity of PrP Western blotting:
- Use of NaPTA for sample preparation.
Study output: Evaluation and developments completed; method adopted for diagnostic Western Blotting
b. Cyclic amplification of PrPsc
Study output: Evaluation completed; Failure to reproduce original report.
4. Initial contacts and evaluation regarding SEPRION – a new synthetic ligand for PrPsc-specific extraction.
Study output: Evaluation completed; Promising results leading to submitted proposal (turned down)
5. Screening of new antibodies from IDEXX / Caprion for PrPsc specificity.
Study output: Evaluation completed; New antibodies available.
6. IHC development including improvements to turnround times for the diagnostic process, observations on possible differentiating procedures for BSE and scrapie in sheep and antibody comparisons (EU).
Study output: Evaluations and development completed; Processes incorporated into diagnostic system.
7. Other contacts
Year 4, 2002/2003. Unlike previous years, the programme of evaluations and developments for 2002 / 2003 was presented to defra on 13th May 2002 and formally agreed in September of that year. It included:
1. Characterisation of brain-pool materials for the comparison of diagnostic tests. First comparison exercise
Study output: Biochemical evaluation completed, infectivity titres on-going.
2. Evaluation and validation of BIO-RAD Western blot system for confirming Platelia positives in bovine BSE diagnostis.
Study output: Study completed, claims upheld by data.
3. Comparison of analytical sensitivity between NaPTA precipitation and alcohol sedimentation for PrPsc recovery from brain tissue.
Study output: Comparison completed;
4. Further evaluation of SEPRION. Double-blinded trial in comparison to 2 commercial PrPsc detection kits and an in-house Western blot.
Study output: Evaluation completed; performance successful
5. Provision of information and advice to defra regarding emerging TSE diagnostic methods and reagents.
CSG 15 (1/00) 4
Projecttitle / Assessment and validation of emerging methods and reagents for BSE diagnosis.
/ DEFRA
project code / SE1762
Scientific report (maximum 20 sides A4)
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Projecttitle / Assessment and validation of emerging methods and reagents for BSE diagnosis.
/ DEFRA
project code / SE1762
For years 1 –3 of the project, a summary of achievements are given below. More detailed
reports on these studies have been included in the Annual Interim Reports for 1999 / 2000, 2000 / 2001 and 2001 / 2002. For year 4 (2002 / 2003) a full report is given here and in the appended Annexes 1 - 4.
Year 1:
A. Evaluation of the claims for the ImmunoCapillary Electrophoresis procedure of Schmerr et al for the detection of PrPsc in the blood of sheep infected with scrapie. These claims were investigated at the inventer’s laboratory and by the analysis of VLA-supplied samples and this formed the basis of a collaborative funding application to DEFRA (SE1757) for the further development of the technique.
B. Evaluation of the DELFIA technique from IAH for PrPsc determination was completed and funding
continued under a Surveillance Project TSE5003. The DELFIA system was also applied to CSF
analysis.
The DELFIA technology developed at the VLA and Carmarthen R.C, for use with brain
homogenate, proved to be a useful TSE diagnostic test but failed to exhibit the sensitivity
expected when considering the detection system employed. This problem was initially thought to
be the result of the unlikely antibody pairing used, that is, 3F4 and FH11, antibodies perceived to
recognise epitopes found either side of the presumed PK proteinase K cleavage site in the prion
protein. Much work was performed, within this project, on the optimisation of the DELFIA
technology, using new antibodies and sample preparation procedures. Despite the unlikely
antibody pairing currently used in this system, no other combination was found to even match the
performance of the FH11 and 3F4 used.
This DELFIA assay is targeted at BSE detection in adult cattle in the terminal stages of disease
by PrPbse determination in brainstem. The DELFIA technology was adapted within this project to analyse body fluids that are readily available from the live animal, that is CSF and serum. Initial studies carried out, on both bovine CSF and serum, usilised the same 3F4 and FH11 antibody pairing as that used in the bovine brain assay. The results showed that bovine CSF was capable of generating a strong signal whereas serum failed to elicit any detectable response above background and so efforts, within SE1762, were focused on the further development of the CSF assay. A small scale study on 6 negative and 5 positive CSF samples resulted in 5 of the 6 negatives giving a significantly higher response without proteinase K (PK) digestion and when treated with 10ug/ml of the enzyme, but all the PrP, in both positive and negative samples, was digested at 100ug/ml PK. Having shown reproducible differences in the behaviour of positive and negative samples, further DELFIA studies were performed in order to optimise the assay and enhance the differences already seen. After exhaustive assessment of CSF dilutions, guanidine concentrations and PK levels, a method was selected. Larger sample numbers were assayed, to determine whether, under the conditions of the analytical procedure applied, there really was a greater concentration of antigen present in the CSF of BSE-negative cattle, which is immunoreactive to the antibodies used. This distribution was confirmed but is as yet unexplained and has not been pursued further.
The DELFIA system was also developed for the analysis of S-100 Protein in CSF and compared to the system used at the Institute of Neurology and subsequently at the National CJD Surveillance Unit, Edinburgh which utilised a more simple peroxidase chromogen. The methods were applied to CSF samples from experimentally challenged cattle in project no. SE1749 and, surprisingly there was no indication of increased sensitivity using DELFIA when compared to the original peroxidase – based detection system. The latter proved to be more robust and reproducible and was used to publish the first paper on S-100 Protein in cattle CSF (Green AJE, Jackman, R Marshall TAV & Thompson EJ 1999. Increased S-100b in the CSF of some cattle with BSE. Veterinary Record. 145 107 – 109)
C. Initial investigation was carried out of a Dot-Blot technology developed in collaboration with
Leicester University as a spin-off from a DEFRA project SE1740. Initial evaluation of the
technology allowed a decision to be taken on whether to proceed with a blind trial using this
methodology.
This included visits to the originating laboratory, scrutiny of the techniques involved and
concluded with the blind trial being undertaken under the project code TS5004.
Fig.1: Example of the dot blots developed at Leicester University
However, further progress has been slow primarily due to inconsistent supplies of the necessary prion antibody. Recently, this has been overcome and the results reported at the recent Antibodies in Agri-Foods in Uppsala, Sweden (Owen JP, Gough KC, Jackman R & Whitelam GC. 2003). A Dot-Blot for the Rapid Detection of PrPsc in Ovine LRS). These were also used as the basis for continued funding in a proposal to the Joint Funders Call for TSE Diagnostics (2003).
D. Developments with the immunolocalisation of PrPsc (IHC) were concentrated on the recently developed VLA antibodies to selected PrP-peptide immunogens. Those targeted to the C-terminus sequence especially, showed considerable promise in the sensitivity of PrPsc detection and in the low level of background staining – of particular importance in the immunostaining of non-neural tissue sections.
E. The claims of a BSE test based on 14-3-3 protein developed at the University of Mainz (Prof. Schroeder) were evaluated through provision of blinded samples and were encouraging but incomplete.
Year 2:
A. The 14-3-3 evaluation was continued by further sample exchange and visits to the laboratories in Mainz and by bringing in a further collaboration with the Institute of Neurology, London / CJD Surveillance Unit, Edinburgh. Although the analyte proved to be a useful diagnostic for differentiating sCJD and vCJD, the required 100% sensitivity and specificity has not been achieved in challenged cattle prior to the development of clinical disease. Modifications to the technique were carried out and a trial of CSF samples from the BSE-challenged animals in SE1737 and SE1749 was planned.
B. A major aim of SE1762 was to create working contacts with other TSE research groups and, where necessary, to undertake small pilot studies that could lead to future joint funding. A close collaboration was initiated during the 2000 – 2001 financial year with Caprion Pharmaceuticals Inc., Montreal and IDEXX Laboratories Inc., Westbrook, Maine, U.S.. Representatives of the company worked at the VLA using the Group’s category III facilities, due the unavailability of such facilities and suitable tissue samples in the U.S. In exchange for the facilities provided and assistance given, IDEXX have disclosed much of the data generated during their initial antibody evaluations. The collaboration was subsequently put on a more formal footing and after meetings at IDEXX headquarters, confidentiality agreements were signed and proposals for future joint funding finalised.
The project entitled “Antibodies specific for the abnormal isoform of the prion protein: their
application to TSE diagnostic tests” (SE1776) was funded by DEFRA, with work commencing
August 2002. Prior to the onset of this project, no diagnostic antibody or other ligand has been