A METHOD FOR THE DETERMINATION OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES IN MAMMALS FIELD OF THE INVENTION This invention relates to a method for the determination of transmissible spongiform encephalopathies in mammals. More particularly, the invention concerns a novel method for the determination of the percentage of disease related prion protein in a sample by differential extraction using two solutions of a chaotropic agent. Moreover, the invention concerns the use of this percentage as a diagnostic marker for the transmissible spongiform encephalopathies (TSE).

BACKGROUND OF THE INVENTION The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

The TSE family of diseases Scrapie, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Sheinker (GSS) syndrome and related diseases of mink (transmissible mink encephalopathy), mule deer and elk (chronic wasting disease) are classified as the transmissible degenerative (or spongiform) encephalopathies (TSE's). New species have been affected in recent years including cattle (bovine spongiform encephalopathy), cats (feline spongiform encephalopathy) and a variety of captive zoo felines and antelope and a new form of CJD in man has recently emerged. latrogenic transmission of CJD in man occurs and these diseases can be transmitted from affected to healthy animals by inoculation or by feeding diseased tissues. The following text describing these diseases is taken from a review by Hope, 1998 (see reference 31).

Scrapie Scrapie of sheep has been known in Europe for centuries and has spread to most parts of the world, excluding Australasia and Argentina, with the migrations of man and his livestock. It is characterised by altered behaviour, hypersensitivity to sound or touch, loss of condition, pruritus and associated fleece loss and skin abrasions and in-coordination of the hind limbs. Diagnosis is confirmed post- mortem by examination of brain tissue for a triad of histopathological signs- vacuolation, loss of neurones and gliosis'.

Scrapie has been reported in most breeds of sheep and, within a flock, it appears to occur in related animals. The natural clinical disease has a median peak incidence in flock animals of 3.5 years, with a range of 2.5 to 4.5 years covering the vast majority of cases2. For most of this period, the infected animal is clinically normal and indistinguishable from its un-infected flockmates. The within-flock incidence of clinical disease is usually 1-2 cases per 100 sheep per year but there have been several instances of 40-50% of animals of a flock succumbing to the disease within a year. A number of genetic markers have recently been identified as risk factors and the introduction of gene typing has greatly facilitated interpretation of field studies on the incidence of natural and experimental disease3.

Creutzfeldt-Jakob disease Creutzfeldt-Jakob disease is a progressive dementia with clinical signs suggesting dysfunction of the cerebellum, basal ganglia and lower motor neurones. It is associated with gradual mental deterioration leading to dementia and confusion, and a progressive impairment of motor function. Most patients die within six months of onset of clinical signs and there are no verified cases of recovery. Pathologically the lesions of the brain included variable vacuolation of

the neuropil, astrocytosis and, in about 10% of CJD cases, amyloid plaques.

Gerstman-Straussler syndrome is a familial variant of CJD with an extended, clinical time course.

The incidence of CJD-related disease in man is remarkably constant at 0.5-1 cases per million of population per year throughout the world and so is not linked to the incidence of any of the animal diseases. This low incidence casts doubt on the role of infection in its propagation within the population (but see below).

About one in seven of cases are familial and linked to mutations in the open reading frame of the PrP (prion protein) gene. There has been a large amount of clinical and pathological studies on human cases of neurological disease which seem to be associated with these rare mutations of the PrP gene (for a review, see4). In some families, there is complete penetrance of the phenotype and so the mutation is regarded as the cause of the disease. Apart from iatrogenic cases induced by transplantation of infected tissues or inoculation of contaminated pharmaceuticals of human origin, there is no epidemiological evidence for horizontal transmission of the disease. A stochastic event involving conversion of the PrP protein to its disease-associated isoform or the chance mutation of a benign, ubiquitous viral-like agent are two mechanisms which have been suggested to explain the incidence of sporadic cases (see section on the nature of the agent). There is no cure for the clinical condition although genetic counselling, where applicable, may effectively prevent transmission of disease from one generation to the next.

There is considerable clinical and pathological heterogeniety in the human prion diseases and although genetic typing and nucleotide sequencing of the PrP ORF (ORF = Open reading Frame) has provided some unifying concepts, mutation in the PrP protein does not appear to be the whole story. Other genetic factors including linkage to the E4 allele of ApoE gene have been implicated as risk factors for the occurrence of CJD.

Emerging human TSE's Fatal familial insomnia (FFI) has emerged as the newest member of the human transmissible encephalopathies. It is characterised by a dysfunction of the autonomic nervous system usually presenting with insomnia and problems of appetite, temperature and blood pressure regulation. At post-mortem, the pathology of the brain is mostly neuronal loss and degeneration of the thalamus with little or no vacuolation of the neuropil. Its classification as a prion disease was originally based on its association with an asparagine (N) to aspartic acid (D) mutation at codon 178 of the PrP gene, a mutation which is also linked to a classical form of CJD. Which of the two phenotypes prevails appears to depend on the amino-acid encoded by codon 129 of the same PrP allele: in FFI, codon 129 encodes methionine while in CJD, codon 129 encodes valine. Homozygosity at codon 129 also appears to be risk factor in the development of sporadic CJD, but each polymorphism at this codon is fairly common and not thought to be pathogenic per se. The classification of FFI has been confirmed by transmission of disease to laboratory mice5.

In March 1996, the UK government announced their concern about a new variant of CJD (nvCJD) and details of these cases have subsequently appeared6. To date, more than 40 cases of nvCJD have been identified in the UK with a new neuropathological and clinical profile-extensive PrP deposition, cerebellar amyloid plaques, spongiform change most evident in the basal ganglia and thalamus, prolonged duration (up to 2 years), atypical EEG, early ataxia, behavioural and psychiatric disturbances. Similar cases have not been identified in archival patient files or elsewhere in Europe (apart from a single French case7) and there would appear to be a risk factor for this variant unique to the UK. Its co-incidence with a novel bovine TSE (BSE) which is largely restricted to the UK has led to speculation, as yet unproven, that this new form of CJD represents a cross-species transmission of infection from cattle.

Bovine spongiform encephalopathy Bovine spongiform encephalopathy (BSE) has devastated the UK cattle industry for the past decade8. From isolated cases first reported in 1986 and some retrospectively identified in May 1985, a major epidemic was underway by 1988 which has to date claimed over 180 000 cattle within the British Isles. Some other countires have also confirmed cases: Switzerland (450+), Ireland (700+), Portugal (300+), France and Germany with one or two cases in Italy, Denmark, Canada, the Netherlands, Oman and the Falkland Islands.

The disease produces a progressive degeneration of the central nervous system and was named because of the sponge-like appearance of BSE-brain tissue when seen under the light microscope9. Warning signs of the illness include changes in the behaviour and temperament of the cattle. The affected animal becomes increasingly apprehensive and has problems of movement and posture, especially of its hindlimbs. The cow (or bull) has increased sensitivity to touch and sound, loss of weight and, as the disease takes hold of its nervous system, a creeping paralysis sets in. This clinical phase of BSE lasts from a fortnight to over six months. Although the majority of animals affected have been dairy cows, this neurological disease can occur in either sex with a modal age of onset of 4-4.5 years (range 1.8-18 years). Most cases of BSE have occurred in cattle between the ages of 3 and 5 years and for most of its development time the disease gives no tell-tale sign of its presence10.

The neurological lesions in BSE-affected cow brains are virtually identical to those found in scrapie-affected sheep and include the spongiform change which gives BSE its name. From its clinical and neuropathological signs, BSE was immediately suspected to belong to the scrapie family of transmissible spongiform encephalopathies. This has been confirmed by biochemical studiesll

and by experimental transmission of BSE to mice'2, sheep and goats'amongst other species.

The prion protein (PrP) The conversion of a normal membrane glycoprotein, the cellular prion protein or PrPC, to an aggregated, insoluble isoform, PrPsc, is a key process in the pathogenesis of BSE, scrapie and other transmissible spongiform encephalopathies (TSE's). The specific detection of PrPsc forms the basis for biochemical diagnosis of these diseases. Folding differences in the abnormal isoform of the prion protein (PrPSc) can be investigated by probing the conformation of the protein in diseased tissues by proteolysis under conditions where the normal protein is either destroyed and drastically reduced in amount prior to detection by SDS-PAGE/immunoblotting or ELISA techniques.

The prion protein is expressed in many different cells but is found in greatest abundance associated with the neurones of the central nervous system.

Consequently, the abnormal form of PrP accumulates predominantly in the brain although it can also be detected in extra-neural tissues such as the tonsil and spleen early in the development of disease.

In cell culture, the PrP protein is cycled to and from the cell surface via the endosome-lysosome system; during this process the protein appears to undergo proteolytic cleavage between residues 109 and 112. To what extent this cleavage occurs in vivo is unknown although C-terminal fragments of PrP similar to those expected to result from the lysis of this peptide bond have been seen in deposits of mouse and human PrPsc. The exact site of cleavage may be related to the phenotype of disease or strain of infectious agent; this is an area of current investigation.

Previous work on detection of PrP Ever since the prion protein (PrP) was discovered, measurements of prions and PrP have been made relating the number of protein molecules per infectious particle. This is usually quoted as 100 000 PrP molecules per infectious particle if measured by intracerebral inoculation in an homologous rodent bioassay; for example, using the 263K strain of agent in hamsters or the ME7 strain in mice, etc. The detection of PrPSc as fibrils 14, 15 or by immunochemical methods""' can be used as a diagnostic test for TSE's either at post-mortems or in the live animal.

A preliminary validation of this approach using fibril or PrP ICC (ICC = immunocytochemistry) to monitor the oral pathogenesis of BSE in cattle has recently been published23. Western or dot blotting can currently detect 10-100 pg (108-109 molecules) of PrPSc and these methods are about the same sensitivity as an heterologous bioassay of bovine or human tissue for BSE infectivity in mice.

For example, typically, 1 gram of BSE-affected brain contains 1 microgram of PrPSc ll and 1035 LDso units/g of infectivity24. This is equivalent to a specific infectivity of 1 infectious particle/109 molecules. Higher specific infectivities may be found in transmissions between the same species 25, 26 but from these calculations it is clear mouse bioassays offer no greater sensitivity than PrP immunochemical assays for detection of BSE infectivity. Improving the sensitivity of the assay system may shed light on the scientific conundrum of why lateral or maternal transmission of BSE occurs26 in the (apparent) absence of infectivity (and PrPsc) in mils27, blood, placenta and other peripheral tissues of the BSE-infected cow28.

The immuno-diagnosis of a TSE invariably involves the identification of deposits of prion protein (PrP) using specific antibodies (e. g. Senetek 3F4) in tissue preparations (Immunohistochemistry) or Western Blot analysis. In the latter case, prior proteolysis of the sample with an appropriate concentration of enzyme (e. g. proteinase K) will result in the digestion of normal non-aggregated PrP (PrP) while leaving disease-associated aggregated and deposited PrP (designated

PrPSC) largely intact. The residual core of the PrP molecule (designated Prpres) may be detected after polyacrylamide gel electrophoresis and subsequent antibody detection (e. g. Prionics 6H4).

More recently, there has been the perceived need for more rapid procedures which allow for a greater throughput. Consequently, several immunoassays have been developed and reported. Many of these employ proteinase K to digest away 'normal'PrP leaving the residual core of the molecule (PrPreS) to be detected in the immunoassay. The problem with this approach (together with Western Blot analysis) is the somewhat arbitrary nature of the proteolysis.

By its very nature, aggregated (i. e. deposited) PrP is more resistant to proteolysis but this resistance will be directly related to the concentration, specificity and potency of the proteolytic enzyme employed in the experiment. For example, proteinase K (PK) is a widely used laboratory reagent since the enzyme has a broad specificity and retains a high activity even in the presence of the chaotropes and detergents used in the experimental extraction of PrP prior to detection.

The concentration of PrPreS, however, is not an absolute. The extent of proteolysis of the deposited prion protein will be related to the concentration and activity of the enzyme that has been added experimentally to the extracted protein. The more enzyme added the more proteolysis and vice versa.

Consequently, the presence of Prpres is totally dependent on the experimental conditions employed. In other words, Prpres is an experimental artefact.

In practice, this has led to the development of routine Western blots which are essentially'yes-no'tests. In other words, careful optimisation is undertaken to discover the exact conditions of proteolysis to ensure that the detectability of prpres is a reflection of the presence of disease-associated aggregated and deposited PrP. Because of this'yes-no'approach, conditions that have been

carefully established for one type of tissue (e. g. brain stem) from an animal in the terminal stages of the disease will be totally unsuitable for the detection of Prpres from other tissues (e. g. spleen) and for the detection of pre-clinical disease.

By definition, pre-clinical disease must be categorised by little deposited and/or aggregated PrP. In this situation, less PK will be required to digest the material since the enzyme is no respector of protein. Consequently, the measurement of Prpres following PK digestion will always be technically difficult. Furthermore, it may turn out to be an inappropriate parameter to measure in order to diagnose a TSE from a wide variety of tissues taken from animals at different stages of the disease.

There is thus a great need for the development of an easier and more accurate method for the diagnosis of TSE in a mammal.

SUMMARY OF THE INVENTION Thus, the present invention concerns a method for determining the solvent resistence of prion protein (PrP) in a tissue or body fluid sample of a mammal, comprising the steps of 1) subjecting said body fluid or tissue sample to a solution of a chaotropic agent having a low molarity of said chaotropic agent, 2) separating the supernatant (supernatant 1) from the solid residue, and subjecting said supernatant 1 to an immunoassay for the determination of its content of PrP, wherein this amount of PrP is considered as soluble PrP, 3) subjecting said solid residue to a solution of a chaotropic agent having a high molarity of said chaotropic agent, and 4) separating the supernatant (supernatant 2) from the solid residue, and subjecting said supernatant 2 to an immunoassay for the determination of its content of PrP, wherein this amount of PrP is considered as sparingly soluble PrP (solvent resistent PrP).

According to another aspect, this invention concerns a method for diagnosing a transmissible spongiform encephalopathy (TSE) in a mammal, said method comprising the determination of prion protein (PrP) in a body fluid or tissue sample from said mammal, by a method for determining the solvent resistence of prion protein (PrP) according to this invention, wherein the percentage of sparingly soluble PrP (solvent resistent PrP) calculated as (amount of PrP in supernatant 2) x 100 (amount of PrP in supernatant 2 + amount of PrP in supernatant 1) is used as indication of TSE in said mammal.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the percentage of solvent resistent PrP in 160 samples of bovine brain tissue, using the differential extraction method described in the Example (NZ = New Zealand; animals never exposed to BSE; CVL = Central Veterinary Laboratories Archive material), Figure 2 shows the percentage of solvent resistent PrP, using the differential extraction method described in the Example, in paired samples of bovine brain tissue taken from the caudal and rostal regions of the brain stem from 9 clinically positive animals, Figure 3 shows the percentage of solvent resistent PrP, using the differential extraction method described in the Example, in paired samples of bovine brain tissue taken from the caudal and rostal regions of the brain stem from 9 clinically negative animals, Figure 4 shows the"within assay variation",

Figure 5 shows the"between assay variation" Figure 6 shows the solubility of normal PrP and disease related PrP in an aqueous solution of guanidine hydrochloride versus molarity, and Figure 7 shows the solubility of normal PrP and disease related PrP in an aqueous solution of urea versus molarity.

DETAILED DESCRIPTION OF THE INVENTION According to this invention, a method has been developed that quantifies disease- associated deposited and aggregated PrP as a percentage of the total PrP on the premise that aggregated and deposited PrP (PrPSC), characteristic of a TSE, is much less soluble in low molarity chaotropic agent (e. g. 1M guanidine hydrochloride) than non-aggregated PrP. In addition, a measure of total PrP can be obtained by using high molarity chaotrope (e. g. 6M guanidine hydrochloride) to extract the residual aggregated prion from the tissue homogenate.

Consequently, by the use of differential extraction using low and then high molarity chaotrope, a measure of solvent resistance can be established which is both independent of protein concentration and directly related to the concentration of disease-associated aggregated and deposited PrP.

Definitions and preferred embodiments: The term"chaotropic agent"means generally a reagent that denatures another substance. In the case of an aggregated polymer (e. g. an aggregated protein), the chaotrope causes both disaggregation and unfolding (i. e. loss of both quaternary and tertiary structure).

As examples of preferred chaotropic agents to be used in the present invention can be mentioned highly soluble guanidine salts such as guadinine hydrocloride; urea; and certain detergents such as sodium dodecyl sulphate (SDS).

The solvent for the chaotropic agent is preferably water, but not limited thereto.

Also organic solvents may be useful. The solution of a chaotropic agent can also contain a chaotropic agent dissolved in another chaotropic agent, or two or more chaotropic agents dissoved in another solvent.

The terms"low molarity"and"high molarity"solutions of chaotropic agents will need some, but not undue, experimentation to define, because the ranges of"low" and"high"molarity will depend i. a. on the chaotropic agent and the solvent used.

Reference is made to Figures 6 and 7. A low molarity solution is defined as a solution which does not substantially affect (i. e. dissolve) the disease related prion protein. As seen from Figure 6, the upper limit of"low molarity"for an aqueous solution of guanidine hydrochloride is below 2 M. The preferable upper value for"low molarity"is about 1 M. For an aqueous solution of urea (Figure 7), the preferable upper range for"low molarity"is about 2 M. The lower limit for the"low molarity"solution of the chaotrope is the concentration where the normal, non-disease related PrP is essentially soluble. For many chaotropes, this point is rather close to zero.

From Figures 6 and 7 it can also be seen that the lower limit for the range of "high molarity"solutions of the chaotropic agent is achieved at a point where the maximum solubility of the disease related prion protein is essentially achieved.

For an aqueous solution of guanidine hydrochloride (Figure 6), this point is reached at about 3 M and for an aqueous solution of urea (Figure 7), the point is reached at about 6 M. The upper limit of the"high molarity"chaotrope solution is determined by the solubility of said chaotrope and is the saturation point.

Using the procedure disclosed by Figures 6 and 7 it is possible to establish the suitable ranges for"low molarity"and"high molarity"solutions of the chaotrope for any chaotropic agent and any solvent.

According to a preferred embodiment, the low molarity chaotrope solution is an aqueous solution of less than 2 M guanidine hydrochloride, and the high molarity chaotrope solution is a is an aqueous solution of at least 3 M guanidine hydrochloride. Using these solutions, a percentage of sparingly soluble PrP that is about 10 % can be used as indication of TSE in said mammal. A clinical disease may be associated with levels of 60 % or more.

The method be carried out on a body fluid, e. g. a blood sample, a or tissue sample. The sample can thus be, for example, brain tissue, spinal cord, lymphoid tissue, spleen, tonsil, whole blood or a blood fraction. For post-mortem analyses, brain tissue is a particularly preferred sample. For investigation of living individuals, less invasive tissue samples or body fluid samples must be used. As examples of body fluid samples can be mentioned whole blood or fractions thereof.

The results of the immunometric method according to this invention can be used for many different purposes. They can be used for diagnosing a transmissible spongiform encephalopathy (TSE) in the mammal, wherein the mammal can be a living individual or a deceased individual.

As an especially important field of use can be mentioned routine control of animal bodies, especially bovine bodies in slaughterhouses. A sample can be derived from each slaughtered body and analysed. Bodies showing TSE infection will be destroyed. This would be an effective method for preventing infected meat and other animal-based products from being commercialised. On the other hand, unnecessary destruction of healthy bodies can be avoided. Therefore, this control method is of great economical value.

Another important field of use is the screening of blood samples, particularly blood samples obtained from human blood donors, to investigate whether a sample is infected by a transmissible spongiform encephalopathy (TSE). This

method comprises determination of prion protein (PrP) in a sample of whole blood or blood fraction from said blood donor. Blood showing presence of an abnormality in PrP, which indicates TSE infection, will be destroyed.

The invention is described in more detail by the following, non-limiting experiments.

EXAMPLE 1. Homogenisation of sample Approximately, 1 gram of brain tissue (rostral or caudal to the obex region of bovine brain) is accurately weighed into a sterile 20 milliliter Falcon tube. Four volumes of 0.1 M Tris buffered saline (0. 01M TBS; pH 7.5) is added and the mixture homogenised with Camlab homogeniser using a disposable probe. Care is taken to avoid the presence of lumps (non-homogenised tissue) in the resulting 20% homogenate.

2. Differential extraction of sample The 20% homogenate is vortexed and 50 microliter is pipetted into a 1.5 milliliter Eppendorf tube. Fifty (50) microliter of freshly prepared 2M GdnHCl (guadinine hydrochloride) is added and the tube stoppered and vortexed.

Subsequently, 900 microliter of Wallac assay buffer added, tube stoppered and vortexed and centrifuged at 13,000 g for 10 minutes. The Wallac assay buffer (Catalogue No. 1244-106 is a ready for use Tris-HCl buffered (pH 7.8) salt solution with bovine serum albumin, bovine globulin, Tween 40, an inert red dye, and < 0.8 % sodium azide as preservative.

The supernatant (designated supernatant 1) is carefully removed and transferred to a separate test tube taking great care not to dislodge the pellet. A filter paper

wick is placed into the Eppendorf to remove the remaining supernatant. After a few moments, the filter paper wick is removed and discarded. One hundred (100) microliter of freshly prepared 6M GdnHCl in water is added to pellet. The tube is stoppered and the pellet carefully vortexed into solution. A further 900 microliter of Wallac assay buffer is added, the tube stoppered, vortexed and centrifuged at 13,000 g for 5 minutes. This supernatant is designated supernatant 2.

3. Immunoassay The lyophilised human platelet enriched plasma standard is reconstituted by the addition 1 milliliter distilled water. Six standards (including a blank) are prepared by serially diluting this stock material 1: 5 (v/v) with Wallac assay buffer. Two hundred (200) microliter of standard or sample (supernatants 1 and 2) added in duplicate to the microtitre plate coated with an excess amount of anti-PrP capture antibody (clone: FH11). The plate is incubated on shaker at 4°C for 60 minutes (Note: alternatively, plate may be sealed and placed at 4°C overnight).

Subsequently, the plate is washed three times and 200 microliter of europium- labelled 3F4 (diluted at 1: 1000 v/v in Wallac assay buffer) is added to each well.

The plate is incubated on shaker in fridge for 60 minutes and then washed six times. Two hundred (200) microliter of Wallac enhancement solution is added using the dispenser, the plate is shaken for 5 minutes at room temperature and the fluorescence measured. Wallac enhancement solution, Catalogue No. 1244-105, is ready for use with Triton X-100, acetic acid and chelators.

4. Calculation % PrP retained in pellet (i. e. % solvent resistance) is calculated according to the formula: (amount of PrP in supernatant 2) x 100 (amount of PrP in supernatant 2 + amount of PrP in supernatant 1)

It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

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