WO2005038464A2

WO2005038464A2 - Method for detecting the presence of an/or strain typing a tse

Info

Publication number: WO2005038464A2
Application number: PCT/GB2004/004396
Authority: WO
Inventors: Kevin Christopher Gough; Roy Jackman; Jonathan Paul Owen; Garry Clark Whitelam
Original assignee: The Secretary Of State For Environment, Food & Rural Affairs
Priority date: 2003-10-16
Filing date: 2004-10-18
Publication date: 2005-04-28
Also published as: GB0324255D0; WO2005038464A3

Abstract

A method for detecting the presence of and/or strain typing a transmissible spongiform encephalopathy (TSE) in an animal comprising the steps of; a) subjecting a sample, suspected of containing PrPSc, obtained from an animal, to a C-terminal protease such as thermolysin; and b) detecting the products of step (a), wherein the presence or absence of certain products is indicative of the presence or strain of PrPSc.The products detected using this method are characteristic of particular strains of TSE. Kits and reagents for use in the method are also described and claimed.

Description

Diagnostic method

The present invention relates to a method of detecting and/or typing strains or forms of transmissible spongiform encephalopathies or prion disease found in infected animals, as well as to diagnostic kits and reagents used in these methods. In a particular embodiment, the invention provides a method for distinguishing between BSE and scrapie in sheep.

The transmissible spongiform encephalopathies (TSEs) comprise a group of progressive neurological disorders characterised by neuroparenchymal vacuolation and accumulation of a disease specific isoform of a host coded cell surface sialoglycoprotein called prion protein (PrP) . Scrapie, bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease belong to this group of disorders. The diseases appear in various forms or strains with the disease form of the prion protein generally being known as PrP^Sc.

Already, numerous strains have been identified following transmission of a range of sources into rodents. BSE has been transmitted to sheep by oral challenge with as little as 0.5g of brain material (Foster et al . , Vet Rec (1993) 133:339-341). The possibility exists that some sheep may be naturally infected with the BSE agent and this is of human and animal health concern.

Current methods of TSE agent strain typing, include those based on the biological properties of an isolate following serial transmission in mice (Bruce et al . , 1994, Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Philosophical Transactions-Royal Society of London . Series B. 343, 405-411) . However, such methods are extremely time consuming and not all strains are readily transmitted to mice. Several other methods have been studied in the hope of providing a more rapid answer to strain identification. These strain typing methods rely on the diagnostic digestion of a sample with proteases and in particular thermostable proteases. In these methods the normal non-disease form of the prion protein, PrP, is fully digested by the proteases, for example protease K, but the diseased form PrP^Sc is resistant to degradation and is only partially degraded to leave a strain specific, protease resistant core PrP^res. This strain specific, protease resistant core can then be tested for strain specific properties such as the molecular weight (Parchi et al . , 1996, Annals of Neurology 39, 767-778) or ratio of glycoforms of the PrP^res fragments (Collinge et al . , 1996, Na ture 383, 685-690; Kuczius et al . , 1998, Journal of Infectious diseases 178, 693-699; Somerville et al . , 1997a, Nature 386, 564-564), or relative protease resistance of PrP^re3 (Kuczius and Groschup, 1999. Molecular Medicine 5, 406-418).

It is known for example that constituent forms of the digested p_rp^res _can be separated by the relative amounts and molecular weights of the di-glycosylated, mono-glycosylated and unglycosylated forms of the protein, using polyacrylamide gel electrophoresis. These are then subsequently detected by Western immunoblotting using antiserum produced against PrP. Such glycoform ratios and molecular weights have been indicated as being characteristic of particular strains of TSE and, for example, similarities in these factors have supported the likelihood of a bovine origin for vCJD human infections, since those derived from sporadic or iatrogenic forms of CJD are quite different from those of vCJD and BSE (Collinge J. et al . Nature (1996) 383:685-690; Hill A.F. et al., Nature (1997) 389, 448- 450) . However, these properties of PrP^res overlap when different strains or isolates are compared and so definitive strain typing based upon these differences is complex. A conformation assay of PrP^ιes has been described and may provide a means of strain typing but the usefulness of this technique has not yet been established (Safar et al . , 1998, Nature Medi cine 4, 1157-1165).

Attempts to distinguish natural sheep scrapie from sheep experimentally infected with the BSE strain by molecular analysis have been previously reported, but have led to conflicting results. In one publication (Hill AF, et al., (1998) Neuroscience Letters 255: 159-162) it was reported that a lower unglycosylated band was found in BSE infected sheep in comparison to natural scrapie cases, but in the other two publications (Hope J, et al., (1999) Gen. Vir. 80: 1-4, Hope J, et al., (2000) Corrigendum 81: 1-4) a lower unglycosylated band was found in seven out of eight natural scrapie cases when compared with BSE in sheep samples. Furthermore, in the latter reports, the authors recorded that BSE in sheep showed similar molecular features with an experimental scrapie strain, CH1641. This strain had been propagated in sheep and was originally isolated from a British Cheviot sheep in 1971 (Foster JD, et al., (1988) Vet. Rec. 123: 5-8).

Alternative diagnostic assays to distinguish BSE from scrapie are being developed. Glycotyping of protease K digested PrP^Sc has been conducted and it is suggested that this method is an invaluable tool for the in vitro differentiation of BSE and scrapie isolates . One drawback of this method is that multiple analyses of individual samples are required to produce reliable results (Kucizus et al . , 1998, Journal of infectious Diseases. 178:693-699). J. Collinge' s lab have also reported a Western blot banding pattern for Protease K digested PrP^Sc which is diagnostic for BSE. The main feature is the relatively low molecular weight of the unglycosylated fragment in BSE strains compared to scrapie strains (Hill et al., 1998. Neuroscience Letters. 255:159-162). The main drawback with these assays is that the difference seen between scrapie strains and BSE is not easily distinguishable and therefore considerable experience of these assays is required to determine strain types. Presently available strain typing methods are therefore complex and difficult to execute reliably and interpret results.

In addition to strain typing and detection of the diseased form of PrP, it is also desirable to be able to detect and/or quantify the non-disease forms of prion protein, known as cellular PrP or PrP^c. It is very difficult to separate PrP^c from a sample containing other proteins, for example a milk sample, where it is present in very low concentrations. Many of the most widely used proteases, for example protease K, have the disadvantage that all PrP^c present in a sample being tested, will be broken down into small undetectable fragments and so residue analysis does not give any meaningful results.

Therefore it is desirable that new and more efficient strain typing and detection methods are developed for both disease and non-disease forms of PrP.

Accordingly, the present invention provides an improved detection and strain typing method.

According to a first aspect of the present invention there is provided a method for detecting the presence of and/or strain typing a transmissible spongiform encephalopathy (TSE) in an animal comprising the steps of; a) subjecting a sample, suspected of containing PrP^Sc, obtained from an animal, to a C-terminal protease ; and b) detecting the products of step (a) , wherein the presence or absence of certain products is indicative of the presence or strain of PrP^sc.

By using this method, fragments characteristic of PrP^Sc, and in particular fragments characteristic of particular strains of

PrP⁵⁰ are detected in step (b) . These results can therefore be related to the presence of a particular disease form in the animal.

As used herein the term "C-terminal protease" shall be taken to mean a protease; which digests PrP primarily within the C- terminal region of PrP, for instance, from the C-terminal end. The C-terminal end is considered to comprise the C-terminal 120 a ino acids of the mature protein. Although such proteases primarily digest within the C-terminal region, or from the C- terminal end, some limited proteolysis may occur within the N- terminal region or from the N-terminal end. The N-terminal end is considered to comprise the N-terminal 90 amino acids of the mature protein. However any such digestion is less than that within or from the C-terminal end, such that, in most cases, most of the N-terminal region of the truncated protein and in particular epitopes within the N-terminal region, would persist.

Preferably the C-terminal protease is thermostable and most preferably is thermolysin or a protease which acts on PrP or PrP^Sc in a similar manner to thermolysin. Thus the use of thermolysin forms a particularly preferred embodiment of the invention.

The animals may be any of those who are known or suspected of suffering from TSE. These include humans, ruminants (such as cattle and sheep) cervids or felines.

The method is readily adaptable to strain typing as well as to detection in these species.

The test can be used simply to detect disease in an animal. However, in a preferred embodiment the method is used also to identify the strain type.

The applicants have found that the digestion of PrP^Sc (the disease form of PrP) using a C-terminal protease which primarily digests PrP within the C-terminal region of PrP or from the C- ter inal end preferentially to within or from the N-terminal end, for example thermolysin, provides a surprisingly efficient strain typing method, over other strain typing methods which are not always able to provide a reliable distinction, in particular in the case of types of TSE found in sheep.

It has been found that by digesting different PrP^Sc strains with a C-terminal protease, different sized fragments are produced in different strains. It may be hypothesized therefore that different strains are susceptible to C-terminal degradation to a varying degree. This does appear to provide a good means of differentiating between strains.

This method is particularly useful for identifying and distinguishing BSE and scrapie strains. It may also provide a useful method of distinguishing between BSE and scrapie in sheep as well as a method to distinguish between different strains of scrapie in sheep. This method may also prove useful for distinguishing other strains of TSE within the same species, for example CJD, CWD, TME or FSE strains in particular in humans, cervids, bovines or ovines . Such tests will be very useful for investigating whether TSE' s can infect different species.

As used herein the term "thermolysin" refers to members of the thermolysin-like protease (TLP) family, a particular example of which is Bacillus thermoproteolyticus neutral proteinase, also known as protease X or protease type X. The term also refers to other species variants of thermolysin, for example from, Mi crococcus caseolticus or Aspergillus oryzae .

The sample used in the methods is preferably a tissue sample, for example a brain tissue, central nervous system tissue, or lymphoid tissue, such as tonsilar tissue, from an animal. Suitably, in step (b) the proteins or peptides, which are a product of step (a) , in the sample are first separated on the basis of molecular weight, for example, on an electrophoretic gel. Thereafter, these proteins or peptides can be detected, for instance by contacting these with an antibody or a binding fragment thereof, which binds to an epitope present in the digested sample, specifically on PrP^res. Visualising the bound antibody or binding fragments detects the presence or absence of

PrP^b

The presence of a particular TSE strain may then be detected by analysing the fragments shown by the bound antibody or binding fragment. This step is suitably effected upon a gel using a technique such as Western blotting, in which bound antibody is visualised, for example by using a detectable label. The antibody or binding fragment thereof used is contacted with the separated sample on the gel, and then visualised to produce a signal for each fragment present which contains the antibody or binding fragments epitope.

The antibody or specific binding fragment thereof, is preferably one which recognizes a peptide sequence found in a part of the N-terminal region of the PrP^Sc. Preferably it recognises a sequence found within the N-terminal 90 amino acids of the mature protein and most preferably the 74 amino acids at the N- terminal end of the mature prion protein. Suitably the antibody is SAF-32 (SPI-Bio France), AG4 (J. M. Manser, Institute for Animal Health, Compton, Newbury, Berkshire. Manser J. et al TSE 2002 Edinburgh poster p3.16) or P4 (a mouse monoclonal antibody raised against amino acids 89-104 of ovine PrP (Hardt et al . , 2000, J. Comp. Pathology, 122, 43-45). Preferably the antibody recognises an epitope in the octapeptide repeat region of the prion peptide, such as SAF-32, or the hexapeptide repeat region of the prion peptide such as AG4. P4 is a particularly preferred antibody for typing ovine BSE PrP^Sc vs ovine scrapie PrP^Sc. In the context of the present specification, the expression "N- terminal region" of a prion protein refers to a region which comprises the N-terminal 90 amino acids of the mature protein and most preferably the 74 amino acids found at the N-terminal end of the mature protein.

These antibodies are suitably raised to said peptide sequence, which is in the form of discrete peptides in isolation, or as part of a protein or truncated protein.

The presence or absence of particular truncated fragments, after digestion with the C-terminal protease, makes it possible to identify and distinguish the strains. These fragments are detectable using such an antibody, in particular one which is specific for the N-terminal region as discussed above. In many strains the PrP^Sc may be partially digested to leave a resistant core (PrP^res) . However the PrP^res formed by digestion with a C- terminal protease and detected with N-terminal antibodies, depending on TSE strain, is not the conventional 'core' PrP^res as produced by for example Proteinase K. The PrP^Sc appears to have undergone only limited or no truncation when detected with a C- terminal protease. This method therefore produces detectable fragments. References to "PrP^re3" used hereinafter therefore refer to the core remaining after digestion with the specific protease being considered. Thus in relation to the method of the invention, a reference to "p_rp^res" will relate to the core remaining after digestion with C-terminal protease.

However, if the N-terminal epitope has been digested in a particular strain, then no low molecular weight fragments will be detected using an N-terminal specific antibody. Similarly in other strains the degradation within or from the C-terminal end may be minimal or absent, and so again no low molecular weight fragments will be present in the digested material. Using this method, particular digestion products are indicative of the presence or type of TSE in a sample. For example, the presence of protein fragments of between 26 and 40Kda may be diagnostic for the presence of a TSE. The presence of additional 14 to 27Kda protein fragments and in particular 20 to 26Kda protein fragments may be indicative of the presence of scrapie PrP^Sc' . Also, the absence of such additional protein fragments may be indicative of BSE PrP^Sc.

Any scrapie PrP^Sc identified may be further typed by 2D- electrophoresis to, for example, identify the particular strain of scrapie PrP^Sc present in the sample. For example, if protein fragments are detected which are indicative of scrapie PrP^Sc, for example digestion products of, for example, between 14 Kda and 27Kda and in particular between 20 to 26 Kda these fragments may be further characterized by 2D-electrophoresis to identify the particular strain of scrapie PrP^Sc present in the sample.

In particular it is preferred that the method described in the first embodiment of the invention comprises the steps of centrifuging a homogenised sample from an animal suspected of having a TSE, subjecting the product to C-terminal protease, separating the thus formed mixture on a gel, probing the separated mixture with an antibody which binds to, preferably the N-terminal region of PrP^Sc and detecting the products, wherein the presence of certain products is indicative of the presence of PrP^Sc or of a particular strain of PrP^Sc. The protease is preferably thermolysin. Preferably only one blot is used for both the separating and the detection steps.

The amount of protease used in the method is suitably at least sufficient to break down all PrP (specifically the PrP^c), present in the sample, under reasonable conditions. For example, the protease should be able to break down all PrP present within a period of from 10 minutes to 20 hours, preferably within about 5 hours, at temperatures ranging for example of from 20 to 100°C and preferably at 70°C. For example, from 2.5 to 10 enzyme units is added to each lOmg of tissue sample. This amount of enzyme is preferably added every hour during the digestion stage, which preferably lasts for between 1 and 12 hours but may be shorter, lasting between 1 and 7 hours. In this instance, references to breaking down the PrP refers to the digestion of the cellular form of the protein (PrP^c) as fully as possible with the proteases described above.

Under these conditions, PrP^Sc present, in the sample, is broken down until only PrP^res remains .

The digestion step used in the method of the invention is suitably carried out at a pH in the range of from 4.5 to 9.5, for example from 5 to 8 and preferably from 6-7.5. These conditions are suitably created by combining the sample with a suitable buffer.

The particular pH used can affect the results of the tests. In the case of scrapie PrP^Sc for instance, if it is digested at a pH at the lower end of the above-mentioned preferred range, for example between pH 6.0 and 7.0, for instance 6.5, it is either the same as or more protease sensitive than BSE. Whereas at the higher end of the preferred pH range, for example from 7.0 to 8.0 and in particular 7.5, BSE PrP^Sc is more protease sensitive than scrapie PrP^3C.

For direct . comparative purposes, tests are suitably carried out at the same pH, and the relative sensitivities of the various strain types at that pH, and how this is recognised by the particular antibody used, should be understood in order to ensure that accurate results are obtained. This can be carried out using routine methods, such as calibration methods, as illustrated hereinafter. Furthermore, the differential digestion of different strains at different pHs can itself be used as a basis of strain typing. In a particular embodiment, the sample is subjected to an identical digestion process at different pHs and the ratio of the intensities of the signals obtained from the resultant samples is characteristic of a particular strain of TSE. At least two different pHs are suitably employed but more may be used.

Thus a second aspect of the invention provides a method for typing a strain of TSE, said method comprising (i) digesting a sample obtained from an animal suspected of containing a PrP^Sc with a protease at (a) a first pH, and (b) a second pH, (ii) detecting the amount of PrP^res from each digestion step, and (iii) comparing the results.

The comparison of the results, for example by applying the equation: signal at first pH/ signal at second pH, gives a figure which is characteristic of the particular strain. It has been found for instance that the ratio of results at pH 6.5: results at pH 7.5 for BSE is generally higher than that for scrapie. For instance, using thermolysin and detecting resulting PrP^res with an N-terminal specific antibody, the ratio of signal at pH6.5/ signal at pH 7.5 for scrapie was generally less than 1.5 whilst the corresponding ratio for BSE samples was above 1.5.

It has also been found that the second aspect of the invention can be carried out with other proteases such as Pronase E followed by detection with other anti-PrP antibodies that bind to the N-terminal or C-terminal regions of PrP.

The method according to the first aspect of the present invention is readily adaptable to detecting cellular PrP as well as to detection and typing of the diseased form of PrP in animal products. According to a third aspect of the present invention there is provided a method for detecting the presence of cellular PrP in an animal product comprising the steps of; a) subjecting a sample of an animal product, suspected of containing PrP^c, to a C-terminal protease; and b) detecting the products of step (a) , wherein the presence of certain products is indicative of the presence of PrP^c.

By animal product, is meant any biological matrix where PrP^c may be present, especially at low concentrations. These include samples such as milk, urine or saliva, as well as animal tissue such as blood or meat etc. It may be useful to detect the presence of PrP^c in these products for surveillance or diagnostic purposes . In particular surveillance of milk for PrP^c may be desirable.

Again, the C-terminal protease is suitably thermolysin, or a protease which acts in a manner similar to thermolysin. Thermolysin is a preferred example of such proteases.

The applicants have found that the digestion of PrP^c (the cellular form of PrP) using a C-terminal protease, for example thermolysin, provides a surprisingly efficient detection method for PrP^c.

It has been found that by digesting a sample suspected of containing PrP^c with a C-terminal protease, some small N-terminal fragments (less than 14kDa) persist which are indicative of the presence of PrP^c. This is unexpected since proteases that have been utilised in the past have fully digested all PrP present leaving no detectable N-terminal fragments. Being able to detect and isolate the N-terminal fragments that are produced by digestion of PrP^c with a C-terminal protease, for example thermolysin, is beneficial for several reasons. Firstly it allows the identification of animal products, which carry PrP^c for further study. The fate of this protein is clearly of considerable interest in view of its potential conversion to a disease associated isoform.

It also allows the N-terminal fragments left behind after digestion to be concentrated, which is particularly useful if the PrP^c is present in very low concentrations in a sample. For instance, concentration may be effected by immunoconcentration, or NAPTA (sodium phosphotungstic acid) preciptation.

This method also allows N-terminal fragments of PrP^c to be isolated from complex matrices of proteins, which has previously been very difficult and time consuming to carry out . Using the method of this invention a sample of, for example milk, can be treated with a C-terminal protease, for example thermolysin, resulting in digestion of all of the other proteins present in the milk and leaving only the PrP° N-terminal fragment which can be detected using an N-terminal antibody and then concentrated for further study. Other previously utilised proteases for example Protease K digest all proteins fully and therefore cannot be utilised to test for and concentrate PrP^c.

This method may be particularly useful for identifying, concentrating or isolating fragments Nl and N2 produced by in vivo α and β cleavage of PrP^c. These fragments are discussed in more detail in Mange et al, 2004, Biology of the Cell, 96:125- 132. They may be useful in diagnostic tests, and potentially in therapy.

In a fourth embodiment, the invention provides a kit for detecting or typing strains or forms of transmissible spongiform encephalopathies (TSE) , the kit comprising a C-terminal protease and an antibody or binding fragment thereof which binds to PrP^Sc. and in particular, PrP^res. The kit preferably contains an antibody, which can bind to the N-terminal region of PrP^Sc, to produce a detectable signal, which may be indicative of the presence or strain of any PrP^Sc present. The protease is preferably thermolysin. The antibody is preferably SAF32, AG4 or P .

In a fifth embodiment, the invention provides a kit for detecting the presence of or isolating cellular PrP, the kit comprising a C-terminal protease and an antibody or binding fragment thereof which binds to an N-terminal fragment remaining after digestion of PrP^c with said protease. In this case, the N- terminal fragment may be of less than 14kDa in size, and may be the Nl or N2 of Mange et al (supra.).

The kits may be adapted for use in the method of the second aspect of the invention. In particular in this case, the kit may further comprise two buffers at different pHs, in particular a buffer at pH 6.5 and another buffer at pH 7.5.

In a sixth embodiment, the invention provides the use of a C- terminal protease, for detecting or typing transmissible spongiform encephalopathy (TSE) in an animal. Preferably the protease is thermolysin.

In a seventh embodiment, the invention provides the use of a C- terminal protease, for detecting the presence of cellular PrP in an animal product. Preferably the protease is thermolysin.

In an eighth embodiment, the invention provides the use of an antibody which recognises the N-terminal region of PrP^Sc (PrP^re3) for detecting or typing transmissible spongiform encephalopathy (TSE) in an animal.

In a ninth embodiment, the invention provides the use of an antibody which recognises the N-terminal region of PrP^c (PrP^re3) for detecting cellular PrP in an animal. In a tenth embodiment, the invention provides the use of a combination of an antibody, which recognises the N-terminal region of PrP^Sc (PrP^res) and a C-terminal protease, for detecting or typing transmissible spongiform encephalopathy (TSE) in an animal. Preferably the protease is thermolysin.

In an eleventh embodiment, the invention provides the use of a combination of an antibody, which recognises the N-terminal region of PrP^c and a C-terminal protease, for detecting the presence of cellular PrP in an animal. Preferably the protease is thermolysin.

The invention will now be particularly described by way of example and with reference to the following Figures :-

Figure 1, shows a Western Blot of the diagnostic digestion of ovine scrapie PrP^Sc and bovine BSE PrP^Sc with thermolysin for 1 hour and 4 hours . Figure 1 relates to Example 1

Figure 2, shows a Western Blot of the diagnostic digestion of ovine scrapie PrP^Sc and bovine BSE PrP^Sc with thermolysin over a time period of 12 hours. Figure 2 relates to Example 2.

Figure 3, shows a Western Blot of the diagnostic digestion of ovine scrapie PrP^Sc, bovine BSE PrP^sc and ovine BSE PrP^Sc with thermolysin for 1 hour and 3 hours . Figure 3 relates to Example 3.

Figure 4a, shows a Western Blot of the diagnostic digestion of ovine scrapie PrP^Sc and ovine BSE PrP^Sc with thermolysin over an 8-hour time period at pH7.5. Figure 4a relates to Example 4a.

Figure 4b, shows a Western Blot of the digestion of ovine scrapie negative samples and ovine scrapie positive samples with different concentrations of thermolysin. Figure 4b relates to Example 4b.

Figures 5a and 5b, are graphs indicating the effect of pH on the digestion of ovine BSE and ovine scrapie PrP¹³⁰ by thermolysin using two different antibodies. Figures 5a and 5b relate to Example 5.

Figure 5c, shows Western Blots indicating the effect of pH on the digestion of ovine BSE and ovine scrapie PrP^S by thermolysin using two different antibodies. The densitometry results from these gels were used to produce the graphs shown in Figures 5a and 5b.

Figure 6, shows a Western Blot of the diagnostic digestion of brain homogenate, containing a mixture of PrP^Sc and cellular PrP, with thermolysin, illustrating a residue from cellular PrP when the gel is overexposed. Figure 6 relates to Example 6.

Example 1

Method for Distinguishing BSE and scrapie in animals and tissues

Brain material taken from BSE and scrapie infected animals was passed down needles of decreasing diameter in the presence of PBS buffer (pH 7.4) containing NP40 (0.5% v/v) and sodium deoxycholate (0.5% v/v) to produce a 10% (w/v) homogenate.

This homogenate (100 μl) was then digested with thermolysin (1 μl of a 50 mg/ ml stock or 1 μl of a 100 mg/ ml stock) at 70 °C for one hour, with the reaction mixed by inversion every 20 minutes. After this initial digestion further thermolysin was added (1 μl of a 50 mg/ ml stock or 1 μl of a 100 mg/ ml stock) and the incubation repeated, four sequential digestions were carried out removing 5 μl samples at the end of each hour. For all samples the protease was inhibited by the addition of

EDTA to a final concentration of ImM. Samples taken after 1 and 4 sequential digestions were analysed by Western blot using PVDF membrane and detected with the anti-prion antibody SAF-32 (SPI- BIO, France; 1: 2000) followed by affinity purified goat anti- mouse-HRP conjugate (Dako; 1:1000) and chemiluminescence blotting substrate (Roche) .

SeeBlue markers have been used to assess the size of the various bands shown. The SeeBlue markers can be seen in lane 1.

Results

Molecular weight analysis

Scrapie PrP^Sc produces protein fragments between 20 and 26 kDa, which are absent with BSE PrP^Sc. For both strains the presence of protease resistant bands between 26 and 40 kDa are diagnostic for prion disease. Again specificity of the assay can be further increased, if required, by analysing the digests using 2D- electrophoresis .

Example 2

Method for Distinguishing BSE and scrapie in animals and tissues

Example two was conducted using the same method as in example 1, using 1 μl of a 100 mg/ ml stock added every hour for 12 hours.

Results

Molecular weight analysis

Scrapie PrP^Sc produces protein fragments between 20 and 26 kDa, which are absent with BSE PrP^Sc. For both strains the presence of protease resistant bands between 26 and 40 kDa are diagnostic for prion disease. Again specificity of the assay can be further increased, if required, by analysing the digests using 2D- electrophoresis . Example 3

Method for Distinguishing BSE and scrapie in ovine tissue and

BSE in bovine tissues

This homogenate (100 μl) was then digested with thermolysin (1 μl of 100 mg/ ml stock) at 70 °C for one hour, with the reaction mixed by inversion every 20 minutes. After this initial digestion further thermolysin was added (1 μl of a 100 mg/ ml stock) and the incubation repeated, three sequential digestions were carried out removing 5 μl samples at the end of each hour. For all samples the protease was inhibited by the addition of EDTA to a final concentration of ImM. Samples taken after 1 and 3 sequential digestions were analysed by Western blot using PVDF membrane and detected with the anti-prion antibody SAF-32 (SPI-BIO, France; 1 : 2000) followed by affinity purified goat anti-mouse-HRP conjugate (Dako; 1:1000) and chemiluminescence blotting substrate (Roche) .

Results Molecular weight analysis

Scrapie PrP^Sc produced protein fragments between 20 and 27 kDa (below the molecular weight of the unglycosylated PrP band in undigested samples), which are absent with BSE PrP^Sc. For both strains the presence of protease resistant bands between 28 and 40 kDa are diagnostic for prion disease. Again specificity of the assay can be further increased, if required, by analysing the digests using 2D-electrophoresis . Example 4a

Method for Distinguishing ovine scrapie from ovine BSE

Brain material taken from BSE and scrapie infected animals was passed down needles of decreasing diameter in the presence of

100 mM PBS buffer (pH 7.5) containing NP40 (0.5% v/v) and sodium deoxycholate (0.5% v/v) to produce a 10% (w/v) homogenate. This homogenate (100 μl) was then digested with thermolysin (1 μl of 100 mg/ ml stock) at 70 °C for one hour, with the reaction mixed by inversion every 20 minutes. After this initial digestion further thermolysin was added (1 μl of a 100 mg/ ml stock) and the incubation repeated, Eight sequential digestions were carried out removing 5 μl samples at the end of each hour. For all samples the protease was inhibited by the addition of EDTA to a final concentration of ImM. Samples taken after 1, 4, 6 and 8 sequential digestions were analysed by Western blot using PVDF membrane and detected with the anti-prion antibody SAF-32 (SPI- BIO, France; 1:2000) or P4 (Harmeyer et al., J. Gen. Virol. 1998; 79:937-945) followed by affinity purified goat anti-mouse-HRP conjugate (Dako; 1:1000) and chemiluminescence blotting substrate (Roche) .

Results

Molecular weight analysis

After 4 or more sequential digestions with thermolysin, ovine scrapie PrP^Sc produces protein fragments between 16 kDa and 27 kDa (below the molecular weight of the unglycosylated PrP band in undigested samples) , which are absent with ovine BSE PrP^Sc. Furthermore, the BSE PrP^Sc is more protease sensitive than ovine PrP^Sc. For both strains the presence of protease resistant bands between 28 and 40 kDa are diagnostic for prion disease. Again specificity of the assay can be further increased, if required, by analysing the digests using 2D-electrophoresis . Example 4b

Method for detecting the presence of PrP^bc

Figure 5 shows the results when this method is used to distinguish ovine scrapie from healthy ovine. Brain material taken from healthy and scrapie infected animals was passed down needles of decreasing diameter in the presence of 10 mM PBS buffer (pH 7.4) containing NP40 (0.5% v/v) and sodium deoxycholate (0.5% v/v) to produce a 10% (w/v) homogenate. This homogenate (100 μl) was then digested with thermolysin (1 μl of 5, 25, 50 or 100 mg/ ml stock) at 70 °C for one hour, with the reaction mixed by inversion every 20 minutes. For all samples the protease was inhibited by the addition of EDTA to a final concentration of ImM.

Samples taken after digestion were analysed by Western blot using PVDF membrane and detected with the anti-prion antibody SAF-32 (SPI-BIO, France; 1 : 2000) followed by affinity purified goat anti-mouse-HRP conjugate (Dako; 1:1000) and chemiluminescence blotting substrate (Roche) .

Results

Molecular weight analysis

It was found that 25 μg/ml of thermolysin was sufficient under these conditions to break down all PrP^c between 14 and 40 kDa in healthy ovine samples as detected with SAF32 antibody. Under the same conditions (and thermolysin concentrations up to at least 100 μg/ml) PrP^ce3 between 14 and 40 kDa is detected in scrapie affected animals. Example

Effect of pH on digestion of ovine scrapie and ovine BSE by thermolysin

Brain material taken from BSE and scrapie infected animals was passed down needles of decreasing diameter in the presence of 100 mM PBS buffer (pH 7.5 or pH 6.5 as indicated) containing NP40 (0.5% v/v) and sodium deoxycholate (0.5% v/v) to produce a 10% (w/v) homogenate. This homogenate (100 μl) was then digested with thermolysin (1 μl of 100 mg/ ml stock) at 70 °C for one hour, with the reaction mixed by inversion every 20 minutes. After this initial digestion further thermolysin was added (1 μl of a 100 mg/ ml stock) and the incubation repeated. Eight sequential digestions were carried out removing 5 μl samples at the end of each hour. For all samples the protease was inhibited by the addition of EDTA to a final concentration of ImM. Samples taken after 4 and 8 sequential digestions were analysed by Western blot using PVDF membrane and detected with the anti-prion antibody SAF-32 (SPI-BIO, France; 1 : 2000) or P4 (Harmeyer et al., J. Gen. Virol. 1998; 79:937-945) followed by affinity purified goat anti-mouse-HRP conjugate (Dako; 1:1000) and chemiluminescence blotting substrate (Roche) .

Results

As can be seen in Figure 5c, after 8 sequential digestions with thermolysin, BSE PrP^Sc is more protease sensitive than ovine PrP^Sc at pH 7.5, however at pH 6.5 BSE PrP^Sc appears to be equally or slightly more protease resistant that scrapie PrP^Sc. Looking at each TSE strain individually, scrapie PrP^Sc is digested approximately equally at pH7.5 and pH6.5 when detection is carried out with P4 and when SAF32 is used the digestion of scrapie is more at pH6.5 than at pH7.5. For BSE PrP^Sc digestion detected with both SAF32 and P4, more digestion takes place at pH7.5 compared to pH 6.5. This demonstrates that TSE strains can be identified as their digestion (as described) is affected differentially by pH. This effect can be clearly expressed by quantifying the signals produced on the western blots by densitometry (Quantiscan software from Biosoft was used) and then expressing these quantities as the signal produced at pH 6.5 / signal produced at pH 7.5 for each of the TSE strains (as shown in Figures 5a and 5b) .

Densitometry readings were taken from lanes 6 and 10 for ovine scrapie and lanes 8 and 12 for ovine BSE to produce the ratios shown in Figures 5a and 5b. Figures 5a and 5b clearly show that for ovine scrapie this equation gives a result less that 1.5 for instance around 1 or below (0.5 for SAF32 detection and 1.03 for P4 detection) whereas for ovine BSE the result is greater than 1.5 (21 for SAF32 detection and 2.8 for P4 detection).

Example 6 Diagnostic Method

10 % (w/v) brain homogenate (produced as described in the strain typing assay) was digested with 100 μg /ml thermolysin for 1 h at 37°C resulting in the production of an N-terminal fragment of PrP^c that can be detected with N-terminal antibodies such as SAF32 or AG4 when the gel is overexposed.

Results

Molecular weight analysis

The results are illustrated in Figure 6.

In this experiment, when the gel is overexposed a fragment of less than 14kDa, estimated at about lOkDa was observed. This is believed to be a residue of cellular PrP.

Claims

1. A method for detecting the presence of and/or strain typing a transmissible spongiform encephalopathy (TSE) in an animal comprising the steps of; a) subjecting a sample, suspected of containing PrP^Sc, obtained from an animal, to a C-terminal protease ; and b) detecting the products of step (a), wherein the presence or absence of certain products is indicative of the presence or strain of PrP^Sc.

2. A method according to claim 1 wherein the C-terminal protease is thermostable.

3. A method according to claim 1 or claim 2 wherein the C- terminal protease is thermolysin or a protease which acts on PrP or PrP^Sc in a similar manner to thermolysin.

4. A method according to claim 3 wherein the C-terminal protease is thermolysin.

5. A method according to any one of the preceding claims which is used to identify the strain type of the TSE.

6. A method according to claim 5 which is used to distinguish BSE and scrapie strains in bovines or ovines.

7. A method according to any one of the preceding claims wherein the sample is a tissue sample from an animal.

8. A method according to claim 7 where the tissue sample is a brain tissue, central nervous system tissue, or lymphoid tissue.

9. A method according to any one of the preceding claims wherein, in step (b) , the products in the sample are separated on the basis of molecular weight.

10. A method according to claim 9 wherein the separation is carried out on an electrophoretic gel.

11. A method according to any one of the preceding claims, wherein in step b) the products are contacted with an antibody or a binding fragment thereof, which binds thereto.

12. A method according to claim 11, wherein PrP^Sc is detected by visualizing the bound antibody or binding fragment.

13. A method according to claim 11 or 12, wherein the antibody or specific binding fragment thereof, recognises an epitope found within the N-terminal 90 amino acids of the mature PrP protein

14. A method according to any one of claims 11 to 13, wherein the antibody is SAF-32, AG4 or P4.

15. A method according to claim 14 wherein the antibody is SAF- 32.

16. A method according to claim 1, wherein the presence of protein fragments of between 26 and 40Kda is indicative of the presence of a PrP^Sc.

17. A method according to any one of the preceding claims, wherein the presence of 14 to 27Kda protein fragments is indicative of the presence of scrapie PrP^Sc and the absence of such protein fragments is indicative of BSE PrP^Sc.

18. A method according to claim 17, wherein any scrapie PrP^Sc identified is isolated and further typed by 2D-electrophoresis to identify the particular strain of scrapie PrP¹"" present in the sample .

19. A method according to any one of the preceding claims, which comprises the steps of centrifuging a homogenised sample from an animal suspected of having a TSE, subjecting the product to C-terminal protease, separating the thus formed mixture on a gel, probing the separated mixture with an antibody which binds to, the N-terminal region of PrP^Sc and detecting the products, wherein the presence or absence of certain products is indicative of the presence of PrP^Sc or of a particular strain of PrP^Sc.

20. A method according to any one of the preceding claims wherein only one blot is used for both the separating and the detection steps.

21. A method according to any one of the preceding claims wherein step (a) is suitably carried out at a pH in the range of from 4.5 to 9.5.

22. A method according to claim 21 wherein step (a) is carried out at a pH in the range of 6-7.5.

23. A method according to any one of the preceding claims wherein in step (a), the sample is subjected to an identical digestion process at at least two different pHs and the ratio of the intensities of the signals obtained from the resultant samples is measured and related to a particular strain of TSE.

24. A method according to claim 23 wherein the at least two different pHs are pH 6.5 and pH 7.5.

25. A method for typing a strain of TSE, said method comprising (i) digesting a sample obtained from an animal suspected of containing a PrP^Sc with a protease at (a) a first pH, and (b) a second pH, (ii) detecting the amount of PrP^es from each digestion step, and (iii) comparing the results.

26. A method according to claim 25 wherein the first pH is 6.5 and the second pH is 7.5.

27. A method according to claim 25 or claim 26 wherein the protease is a C-terminal protease.

28. A method according to claim 27 wherein the C-terminal protease is thermolysin.

29. A method for detecting the presence of cellular PrP in an animal product comprising the steps of; a) subjecting a sample of an animal product, suspected of containing PrP^c, to a C-terminal protease ; and b) detecting the products of step (a) , wherein the presence of certain products is indicative of the presence of PrP^c.

30. A method according to claim 29 wherein the animal product is milk.

31. A method according to claim 29 or claim 30 wherein the C- terminal protease is suitably thermolysin, or a protease which acts in a manner similar to thermolysin.

32. A method according to any one of claims 29 to 31 wherein N- terminal fragments remaining after step (a) are concentrated or isolated.

33. A kit for detecting or typing strains or forms of transmissible spongiform encephalopathies (TSE) , the kit comprising a C-terminal protease and an antibody or binding fragment thereof which binds to PrP^res.

34. A kit for detecting the presence of or isolating cellular PrP, the kit comprising a C-terminal protease and an antibody or binding fragment thereof which binds to an N-terminal fragment remaining after digestion of PrP^c with said protease.

35. A kit according to claim 33 or claim 34 wherein the protease is thermolysin.

36. The use of a C-terminal protease, for detecting or typing transmissible spongiform encephalopathy (TSE) in an animal.

37. The use of a C-terminal protease, for detecting the presence of cellular PrP in an animal product.

38. The use according to claim 36 or claim 37 wherein the protease is thermolysin.

39. The use of an antibody which recognises the N-terminal region of PrP^Sc (PrP^res) for detecting or typing transmissible spongiform encephalopathy (TSE) in an animal.

40. The use of an antibody which recognises the N-terminal region of PrP^c (PrP^res) for detecting cellular PrP in an animal.

41. The use of a combination of an antibody, which recognises the N-terminal region of PrP^Sc and a C-terminal protease, for detecting or typing transmissible spongiform encephalopathy (TSE) in an animal.

42. The use of a combination of an antibody, which recognises the N-terminal region of PrP^c and a C-terminal protease, for detecting the presence of cellular PrP in an animal.

43. The use according to claim 41 or claim 42 wherein the protease is thermolysin.

44. A kit according to any one of claims 33 to 35 which further comprises two buffers at different pHs .

45. A kit according to claim 44 wherein one buffer is at pH 6.5 and the other is at pH 7.5.