Award details

Development of rapid high-throughput pre-clinical diagnostic tests for TSEs

Reference	BB/D004500/1
Principal Investigator / Supervisor	Professor Ian McConnell
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Veterinary Medicine
Funding type	Research
Value (£)	419,876
Status	Completed
Type	Research Grant
Start date	15/08/2005
End date	14/02/2009
Duration	42 months

Abstract

The conversion of a normal membrane glycoprotein, the cellular prion protein (PrPC) to an insoluble aggregated isoform (PrPSc) is thought to be the key process in transmissible spongiform encephalopathy (TSE) pathogenesis. Consequently, the specific detection of PrPSc has formed the basis for the biochemical diagnosis of TSEs which include BSE, scrapie in sheep and CJD in humans. Most often, this specificity has been achieved by the differential proteolysis of PrPC using enzymes such as proteinase K prior to the detection of a protease-resistant core of PrP by Western blotting or other immunochemcal techniques. The use of enzymes, however, is problematic since this step is difficult to control and automate and may be inappropriate in order to develop the sensitive assays necessary to detect the disease in its earliest stages. Although there have been several reports regarding the production of monoclonal antibodies (Mabs) that are specific for the disease-associated isoform of PrP, to date, none of these reagents have made their way into commercially available diagnostic tests. The reasons for this may be manifold, but one of the problems of these reagents might be their relative low affinity for PrPSc, which precludes the possibility of developing a sensitive diagnostic. Recently, however, we have produced a high affinity disease-specific Mab (designated YWH1) that recognises a linear epitope that is only exposed and expressed after the solubilisation of disease-associated aggregated PrP using 8M guanidinium hydrochloride. Remarkably, this epitope does not appear in endogenous PrPC even in the presence of high molarity chaotrope. Accordingly, a very simple one-step extraction procedure has been developed that does not require the prior removal of PrPC. The extraction facilitates the direct quantitative measurement of PrPSc in a rapid two-site DELFIA using the novel Mab YWH1 as capture reagent and a commercially available high affinity Mab to PrPC (SAF32) as the europium-labelled detecting reagent. The aims and objectives of this proposal are to validate and fully automate this robust, rapid and ultra-sensitive diagnostic assay for scrapie and BSE using brain and lymphoid tissue. Subsequently, we will apply this technology to brain, lymphoid tissue and blood taken from animals exposed to natural scrapie at the earliest stages of infection. In addition, we will utilise the same principles which have led to this disease-specific diagnostic and raise new Mabs that are directed against human sequences. We will apply the resulting reagents to develop a simple one-step assay for the diagnosis of CJD using tonsillar lymphoid tissue and blood. Furthermore, it will be very important to understand the reactivity of Mabs in the YWH series and to investigate the involvement of the YWH epitope of PrP in normal function and disease pathogenesis.

Summary

There is still the need for rapid diagnostic tests for diseases such as BSE, scrapie and CJD that are both sensitive and specific. Moreover, with changes to government policy, particularly with the lifting of the Over Thirty Months Scheme, there will be an increasing demand for tests that are robust and which can be fully automated. To date, all the accredited tests for BSE involve the post-mortem detection of a disease associated form of a protein called the prion (PrPSc) which is aggregated and deposited in brain tissue in the terminal stages of the disease. Most often these tests have involved the use of an enzyme called proteinase K (PK) to selectively remove the normal protein prior to the detection of the disease-associated form. Nevertheless, there are several problems associated with the use of PK. Firstly, the conditions employed for the experimental removal of normal protein are somewhat arbitrary. Sufficient PK is required to digest all the normal prion while leaving the majority of the PrPSc to be detected. Some loss of PrPSc may be tolerated when there are sufficiently high levels of aggregated protein. Accordingly, the methods which have been shown to work reliably are those where the target tissue is brain stem collected at post-mortem from animals in the terminal stages of the disease. The application of this approach as an early indication of disease is considerably more difficult. Firstly, in the early stages of disease, there is very little detectable aggregated PrPSc in the central nervous system. Consequently, it is necessary to target alternative tissue such as tonsil to identify disease-associated material. Furthermore, concentrations of aggregated material are relatively low, particularly in the early stages of the disease. Thus, the use of PK in this situation is complicated because of the difficulty of knowing in advance exactly how much enzyme to use. Another problem with the continuing use of PK is the difficulty in developing high throughput automation. Recently, however, we developed a very simple disease-specific test that enables the direct measurement of PrPSc even in the presence of high levels of normal protein. In particular, this rapid diagnostic that does not require the use of PK. The method has been applied to brain tissue and tonsil and has been shown to give results that are in total agreement with the longer and more laborious traditional methods. The aims and objectives of this new proposal are to complete the development, evaluation and automation of this robust and rapid diagnostic assay for scrapie and BSE using brain and lymphoid tissue. Subsequently, we will apply this technology to brain, lymphoid tissue and blood taken from animals exposed to scrapie at the earliest stages of disease. In addition, we will produce similar human-specific reagents in order to develop a simple one-step assay for the diagnosis of CJD. The effective diagnosis of CJD, scrapie and other TSEs has great implications for human and animal health. The early diagnosis of disease in animals (cattle and sheep) can lead to the effective removal of potentially infected material from the human food chain. The implications for the limitation of human exposure to TSE are obvious. In addition, an improvement in animal health and welfare may result by early TSE diagnosis and eradication of BSE and scrapie from the cattle herds and sheep flocks in the UK. The development of effective high-throughput diagnostic tests for scrapie, BSE and CJD has enormous commercial implications. For example, the availability of a test that would identify infection in human blood might be used throughout the National Blood Transfusion Service as a universal rapid screening procedure to minimise the risk of transmission.

Committee	Closed Committee - Agri-food (AF)
Research Topics	Animal Health, Neuroscience and Behaviour, TSEs (transmissible spongiform encephalopathies)
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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