Abstract:
The invention relates to a method of detecting Transmissable Spongiform Encephalopathies (TSE) in animals, particularly in animal carcasses, using an anti-PrP SC  antibody in an immunological assay. Also disclosed is a diagnostic kit for detecting TSE comprising the same antibody.

Description:
[0001]    The present invention relates to method of detecting Transmissable Spongiform Encephalopathies and to an immunological assay or test for Transmissable Spongiform Encephalopathies (TSE)  
         BACKGROUND TO THE INVENTION  
         [0002]    Spongiform Encephalopathies are a group of degenerative neurological diseases. There are a number of examples of Spongiform Encephalopathies including BSE (Bovine Spongiform Encephalopathy), Scrapie Creutzfeldt-Jakob Disease (CJD), Gerstmann-Straussler-Scheinker Syndrome, kuru, Transmissable Mink Encephalopathy, Chronic wasting Disease of Mule Deer, Feline Spongiform Encephalopathies and other Spongiform Encephalopathies found in animals such as elk, nyala, greater kudu, gemsbok and tigers. It has also been reported that BSE can be transmitted under laboratory conditions to mice and pigs. This crossing of species barriers by the infective agent has led to increased concern that transfer to humans could occur.  
           [0003]    Bovine Spongiform Encephalopathies (BSE) is a degenerative brain disorder of cattle which is popularly known as “mad cow disease”. It has a slow incubation period, up to four or five years with symptoms of progressive degeneration of the mental state in cows include loss of coordination and staggering gait, lack of interest in their surroundings, disinterest in feed and water, or unpredictable behaviour, including aggressiveness. Affected cattle show symptoms when they are three to ten years old.  
           [0004]    First identified in Great Britain in November 1986, over 100,000 cases have since been recorded there. Post mortems of affected cattle reveal a characteristic pattern of vacuolation in the brain tissue due to destruction of neural cells and the deposition of unusual protein fibres, that give the brain a spongy (spongiform) texture. Similar spongiform diseases have been recognized in humans (for example, Creutzfeldt-Jakob disease or CJD) for over a century and in sheep (scrapie) for over 200 years. The agent thought to be responsible for BSE and its counterparts is an infective protein known as a prion. The prion is an infective particle comprising protein only and no nucleic acid, the presence of nucleic acid being required in the case of a conventional virus. In Scrapie in particular one protein known as prion protein or PrP SC , has been found to co-purify with infectivity and can produce a Scrapie-like condition in brain cell cultures from other animals, such as hamsters under laboratory conditions. PrP SC  is the only known component of the characteristic protein fibres deposited in the brain tissue of Scrapie-infected sheep. This protein, the PrP SC  appears to undergo a structural modification, whereas the term PrP C  is used in respect of the normal cellular counterpart of PrP SC . The natural function of PrP C  is not known, but it appears likely that it has an essential structure or functional role in the organism.  
           [0005]    Recycled animal tissue, which had been routinely fed to British dairy cows as a protein supplement has been identified as the source of the infection. It is believed that BSE was originally spread from sheep&#39;s brains infected with scrapie and that its spread was accidentally accelerated by the ingestion of brain tissue taken from cows that had become infected with BSE. Therefore, the British Government introduced compulsory destruction of suspect animals and their carcasses beginning in 1988. The feeding of animal tissue to cows was banned in Britain in July 1988.  
           [0006]    Since the initial report of the disease, consumers have feared that it might be transferable to humans through milk or beef products, particularly since Kuru, a related disease is known to be spread by ritualistic cannibalism among New Guinea tribesman. In late 1990, consumer concern over the transmission of BSE to humans triggered a collapse in the beef market. A similar scare struck Germany in mid-1994.  
           [0007]    In 1996 ten cases of a newly described type of fatal CJD (Variant CJD) were identified. The victims had distinct brain tissue symptoms, were all under the age of 42, and had no hereditary record of the disease. It has been suggested that the victims may have contracted the disease through contact with BSE-infected cattle before the eradication of suspected animals had taken effect. The identification of Variant CJD led to a dramatic drop in beef consumption in Britain, and the banning of British and in some instances Irish beef imports in various countries worldwide.  
           [0008]    Therefore, for both veterinary and economic reasons, there is an urgent need to provide a method for diagnosis and a diagnostic kit to detect infection with BSE, Scrapie and other related Spongiform Encephalopathies in livestock, animal carcasses and meat generally.  
         OBJECT OF THE INVENTION  
         [0009]    It is therefore an object of the present invention to provide a method of testing cattle, particularly animal carcasses, for the infective agent responsible for BSE. It is also an object that protection method be rapid, with the result being available in a matter of hours, that it should be cheap, reliable and user-friendly.  
           [0010]    Such a detection method would have the advantage that it would prevent the entry of infected meat into the human food chain, thus eliminating the possibility of humans contracting Variant CJD or other related diseases which may be transmitted by eating infected meat. It has the further advantage that it would restore consumer confidence in meat and meat products, which would be advantageous for both the farming community and the meat industry in general.  
           [0011]    At present there is no test available which can identify infected meat carcasses or livestock carcasses, and there is no product available which can allay public fears regarding meat consumption.  
           [0012]    The current invention provides an immunological assay for the putative agent PrP SC  the rogue prion protein believed to be responsible for Spongiform Encephalopathies.  
         SUMMARY OF THE INVENTION  
         [0013]    According to the present invention there is provided a method for detecting the putative agent for TSE in animals comprising taking a body tissue sample from an animal, reacting the sample in a immunological assay with a labelled antibody which is capable of reacting with PrP SC  and determining the amount of labelled antibody bound to the sample.  
           [0014]    Suitably, the antibody used in the assay is raised against a synthetic peptide sequence having the general formula:  
                                X-(R 1 -Lys-His-R 2 )-Ala-Gly-Ala-Ala-Ala-R 3 -Gly-Ala-Val-Val-Gly-G                   ly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R 4 -R 5 )-Y          
 
           [0015]    wherein R 1  is an amino acid residue selected from Met, Leu and Phe;  
           [0016]    R 2  is either Met or Val;  
           [0017]    R 3  is Ala or is absent;  
           [0018]    R 4  and R 5  are independently an amino acid residue selected from Leu, Ile and Met; one or more residues within brackets maybe present or absent with the provisio that if they are present they are attached to the rest of the peptide in sequence; and X and Y may each independently be absent or independently be one or more additional amino acid residues.  
           [0019]    Particularly preferred are prion specific antibodies raised against one or more of the following sequences:  
                       MVKSHIGSWILVLFVVAMWSDVGLCK KRPKPGGGWNTGGSRYPGQ -44                 GSPGGNRYPPQGGGG WGQPHGGGNGQPHGGGWGQPHGGGQGQP-87                 GGGGWGQGGSHSOWNKPSKPPKTNMKHVAGAAAGAVVGG LGGY-131               MLGSAMSSPLIHFGNDYEDRYTRENMYRYPNQVYYRPVDRYSNQNN-177          
 
           [0020]    More particularly preferred are antibodies raised against the underlined sequences shown above.  
           [0021]    The antibody used in the assay is preferably a C5 antibody to PrP SC  which is available from Proteus, England, called herein the differentiation reagent. Other anti-PrP SC  antibodies are also suitable for use in the invention. Suitable antibodies are those directed against the synthetic peptides disclosed in WO 93/11155.  
           [0022]    The immunological assay may be a competitive assay in which a solid support, suitably a microtitre plate, is pre-coated with a carrier protein-peptide conjugate, the animal tissue sample and an anti-peptide antibody are added to the solid support, allowed to react and the support washed, a labelled anti-(anti-peptide antibody) antibody is added, allowed to react, washed, a signal reagent added and the signal read. The label may be horseradish peroxidase.  
           [0023]    The primary antibody used in the assay is preferably a rabbit anti-p r p SC  antibody and the secondary antibody is preferably selected from a goat, sheep, donkey or other anti-rabbit antibody.  
           [0024]    In a particular embodiment an ELISA assay can be used to identify the peptide fragment for PrP SC  but other suitable immunological assays could be used.  
           [0025]    In a preferred embodiment an enhanced chemiluminescence assay is used to aid in detection of the PrP SC  agent. The animals may suitably be cattle, sheep and pigs and the tissue sample may suitably be taken from a carcass of such animals.  
           [0026]    The steps involved are:  
           [0027]    1. Clean up of nervous tissue to allow detection of putative agent for PrP SC  (the rogue prion protein believed responsible for Spongiform Encephalopathies);  
           [0028]    2. Assay priming step involving addition of specific agents (antibodies);  
           [0029]    3. ELISA using wells pre-coated with peptide fragment for PrP SC ;  
           [0030]    4. Enhanced chemiluminescence assay for detection of PrP SC  agent; and  
           [0031]    5. Determination of results.  
           [0032]    In particular the assay involves assay for the PrP SC  protein which is the infective prion protein found in BSE. The assay is capable of distinguishing between PrP SC  and PrP SC  which is the normal BSE prion protein. The PrP SC  protein is a series of peptides in a triple helix whereas in PrP SC  one third of the molecule is in the form of a flat beta-sheet.  
           [0033]    It is particularly important that the assay can discriminate between natural PrP C  and PrP SC , since PrP C  is found in normal subjects while both PrP C  and PrP SC  are found in diseased subjects.  
           [0034]    In a particularly preferred embodiment, a sample of CNS tissue, suitably a cross-section of spinal cord, is removed from an animal carcass, homogenised, filtered and plated onto a microtitre tray. The sample is then reacted in an immunological assay with an antibody as described above.  
           [0035]    The invention also provides a test kit for the detection of TSE in animals comprising an anti-peptide antibody as defined above.  
           [0036]    The method allows the rapid detection of a TSE agent in a carcass, with results being available in a matter of hours, usually about one and a half to two hours. Thus the carcass can be removed from the abattoir before passing into the human food chain.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0037]    The invention will now be described in greater detail with reference to the Examples.  
           [0038]    The requirements for the Enfer TSE immunoassay are as follows:  
           [0039]    a) A sample of CNS tissue  
           [0040]    b) A series of preparative sample treatments  
           [0041]    c) A prion specific antibody  
           [0042]    d) Sensitive method of detection  
           [0043]    a) A sample of CNS tissue is removed from a beef carcass at the point of slaughter using a specialised sampling clavical (available from Medisteel, Dublin, Ireland). This tissue sample must be such that a cross-section of spinal cord can be removed from the sample for confirmatory analysis if necessary. The sample is placed in a universal container and is identified with a traceable identification number i.e. an EU 4 digit carcass number, an Irish Department of Agriculture ear-tag number or a traceable factory kill number. The sample is transported to the laboratory for testing.  
           [0044]    b) On reaching the laboratory the sample is identified using barcodes. The barcode assigned to each sample incorporates information on the factory of origin of the sample, the date the animal is killed and a traceable identification sample number. An amount of each sample, ranging in weight from 0.3 g to 1.1 g, is removed and placed into a stomacher bag for homogenisation. This bag containing a section of the original sample will be assigned an identical barcode to the original sample. This allows full traceability throughout the system. A fixed volume of Homogenising Buffer is added to the stomacher bag and the sample homogenised. The sample is then dispensed in duplicate onto a prepared 96 well microtitre plate. A fixed volume of Differential Buffer is applied to the microtitre plate and incubated. A fixed volume of Priming Buffer is then added to the samples on the plate and incubated. This step completes the sample preparation procedure.  
           [0045]    c) The samples are now ready for immunoassay. This requires a prion specific antibody. This antibody is raised in rabbits to a synthetic prion peptide. A fixed volume of this specific antibody is added to the microtitre plate, incubated and washed. A fixed volume of secondary antibody is added to the plate, incubated and washed. Detection of results is now possible.  
           [0046]    d) Detection of results is by means of Enhanced Chemiluminescence. A fixed volume of a chemiluminescence reagent is added to the microtitre plate and incubated. The light signal is read using a Labsystems Chemiluminometer, the results assigned to the corresponding barcode and a results report printed. 
       
    
    
     EXAMPLE 1  
     Reagents  
     Reagents for Homogenisation of Samples  
       [0047]    Enfer Homogenisation Buffer (HB)  
         [0048]    Reverse Osmosis Water  
         [0049]    Differentiation Reagent  
         [0050]    Positive Control  
         [0051]    Negative Control  
         [0052]    Enfer Immunoassay Priming Buffer (IPB)  
       Reagents for the Immunoassay Procedure  
       [0053]    1.5 M Phosphate Buffered Saline (PBS)  
         [0054]    150 mM Phosphate Buffered Saline (PBS)/Tween 20 (0.05%)  
         [0055]    Sodium Chloride Solution (NaCl)  
         [0056]    Rabbit Anti-PrP peptide in 150 mM PBS/Tween 20 (0.05%) diluted as instructed by the supplier  
         [0057]    Normal Goat Serum  
         [0058]    Donkey Anti-Rabbit IgG-Horse Radish Peroxidase conjugate in 150 mM PBS/Tween 20 (0.05%) diluted as instructed by the supplier Amerlite Reagent™ Johnson &amp; Johnson Clinical diagnostics  
       Equipment  
       [0059]    Bar-code reader and computer database interface  
         [0060]    Bar-code label printer  
         [0061]    Rotating (bottle) mixer  
         [0062]    96 well microtitration plate chemiluminescence reader  
         [0063]    96 well microtitration plate shaking incubator (2)  
         [0064]    96 well microtitration plate washer (2)  
         [0065]    Micro-computer  
         [0066]    Laser Printer  
         [0067]    Micro-computer ‘custom’ software—data processing and reporting  
         [0068]    Deep Freeze Storage −20° C.  
         [0069]    Refrigerated Storage 4° C.  
         [0070]    Reverse Osmosis Water System  
         [0071]    Automated 8 channel pipettes  
         [0072]    Automated microtitre plate strip dispenser  
         [0073]    Vortex Mixer  
         [0074]    Disposable Pasteur pipettes  
         [0075]    Stomacher homogeniser and bags (variable quantity)  
         [0076]    Blades  
         [0077]    Secure sample boxes and transport trailers  
         [0078]    Class II Safety Cabinet system  
         [0079]    Autoclave  
       Samples and Sampling Procedure  
       [0080]    The sample shall be such as to enable the detection of PrP SC  protein in spinal chord.  
         [0081]    The size of the sample will be sufficient to permit the primary analysis and if necessary, a repeat or confirmatory analysis to be carried out.  
         [0082]    Samples will be taken in a manner which will ensure that a fixed amount of tissue will be taken for each sample.  
         [0083]    Samples will be taken in a manner which will permit identification of the sample in the laboratory.  
         [0084]    The method of packaging, preservation and transport to the laboratory will maintain the integrity of the sample such that the results of the analysis is not prejudiced.  
         [0085]    Samples for analysis will be transported to the laboratory, in insulated and sealed containers.  
       Procedure  
     Homogenisation and Preparation of Tissue  
       [0086]    7.5 ml Homogenisation Buffer (HB) is added to each sample.  
         [0087]    The sample is sealed and placed in the homogeniser, homogenise for 3 minutes.  
         [0088]    Remove the sample from the homogeniser and after accumulation of 40 samples, place the samples onto a designated rack.  
         [0089]    Apply 0.02 ml differentiation reagent to each well of the pre-coated microtitre plate except wells A1, A2.  
         [0090]    Transfer, in order, the identification bar-code of each sample to the microcomputer with a bar-code scanner.  
         [0091]    Apply 0.18 ml of sample to each well except A1, A2, A3, A4 (controls).  
         [0092]    Cover the plate with a microtitre plate sealer.  
         [0093]    Incubate the microtitre plate for 1 hour at 37° C., shaking.  
         [0094]    Wash the microtitre plate wells 8× with 0.4 ml NaCl solution.  
         [0095]    Dispense 0.25 ml of the Immunoassay Priming Buffer to each well.  
         [0096]    Incubate the microtitre plate for 15 minutes at 37° C., shaking.  
         [0097]    Wash the microtitre plate wells 4× with 0.4 ml PBS/Tween 20 (0.05%) solution.  
       Chemiluminescent Immunoassay  
       [0098]    Dispense 0.2 ml primary antibody into each well of the microtitration plate.  
         [0099]    Incubate the microtitration plate for 40 minutes, with shaking, at 37° C.  
         [0100]    Wash the microtitration plate wells 4× with 0.4 ml of PBS/Tween 20 (0.05%) solution.  
         [0101]    Dispense 0.2 ml secondary antibody into each well of the microtitration plate.  
         [0102]    Incubate the microtitration plate for 30 minutes, with shaking, at 37° C.  
         [0103]    Wash the microtitration plate wells 4× with 0.4 ml of PBS/Tween 20 (0,05%) solution.  
         [0104]    Dispense 0.15 ml Amerlite™ reagent into each well of the microtitration plate.  
         [0105]    Incubate the microtitration plate for 3 minutes, with shaking, at 37° C.  
         [0106]    Read the luminescence in the reader.  
         [0107]    Data reduction and interpretation.  
       Calculation of Results  
       [0108]    N/A  
       Antibodies, Homogenisation Buffer, Immunoassay, Priming Buffer, Quality Controls  
     Antibodies  
       [0109]    rabbit Anti-PrP peptide, stored frozen and diluted to working strength as instructed by the supplier.  
         [0110]    donkey Anti-Rabbit IgG-Horse Radish Peroxidase (HRP) conjugate, stored at 4° C., to be diluted as instructed by the supplier.  
         [0111]    normal goat serum, stored at 4° C., to be diluted as appropriate.  
       Quality Controls  
       [0112]    Supplied by Veterinary Research Laboratories, Abbotstown, Dublin.  
       Source of all Reagents  
       [0113]    Enfer Scientific Ltd. Co. Tipperary, Ireland.  
                         
 
       EXAMPLE 2  
       [0114]    1. Homogenisation:  
         [0115]    1.1 1 g of CNS tissue is removed from the sample and placed in a stomacher bag.  
         [0116]    1.2 15 ml of Homogenising Buffer is added to the section.  
         [0117]    1.3 This mixture is homogenised for 3 minutes using a stomacher homogeniser.  
         [0118]    1.4 The resultant homogenate is filtered through a 1 micron filter.  
         [0119]    2. Plating:  
         [0120]    2.1 96-well microtite plate is prepared by dispensing 200 ul of Adhering Agent into each well and incubating overnight at 37° C.  
         [0121]    2.2 Wash the plate 4×PBST (5× Phosphate Buffered Saline tablets, supplied by Sigma, U.K., to 1 liter reverse osmosis H 2 O/1% Tween 20) (150 mM) before use.  
         [0122]    2.3 200 ul of blank control (such as water, saline solution or buffer) is dispensed in duplicate onto the plate, positions A1, 2.  
         [0123]    2.4 200 ul of negative control (known negative BSE homogenate) is dispensed—4 replicates—onto the plate, positions B1, 2 C1, 2. The known negative BSE homogenate is supplied by the Veterinary Research Laboratory, Abbotstown, Castleknock, Dublin, Ireland.  
         [0124]    2.5 200 ul of positive control (known positive BSE homogenate, available from the Veterinary Research Laboratory, Abbotstown, Castleknock, Dublin, Ireland) is dispensed—4 replicates—onto the plate, positions D1, 2 E1, 2.  
         [0125]    2.6 200 ul of each filtered homogenate is dispensed in duplicate onto the plate, remaining positions.  
         [0126]    2.7 50 ul of Differential Buffer is dispensed onto the plate bringing the well volume to 250 ul.  
         [0127]    2.8 The plate is covered with a microtitre plate sealer and incubated at 20° C. for 30 minutes.  
         [0128]    2.9 The plate is centrifuged for 30 minutes at 2000 rpm.  
         [0129]    2.10 The plate is then washed 4 times with 150 mM PBST.  
         [0130]    2.11 50 ul of Priming Buffer are added to each well of the microtitre plate and the plate incubated for 1 hour at 37° C.  
         [0131]    2.12 The plate is washed 4 times with 150 mM PBST.  
         [0132]    3. Immunoassay:  
         [0133]    3.1 250 ul of primary prion specific antibody, a rabbit anti-Prion peptide antibody at a dilution of 1:2000, is dispensed onto the plate.  
         [0134]    3.2 The plate is incubated at room temperature for 40 minutes.  
         [0135]    3.3 The plate is washed 4 times with 150 mM PBST.  
         [0136]    3.4 250 ul of secondary antibody, a donkey-anti-rabbit HRP at a dilution of 1:2000 is dispensed onto the plate and the plate incubated at 37° C. for 30 minutes.  
         [0137]    3.5 The plate is washed 4 times with 150 mM PBST.  
         [0138]    4. Detection:  
         [0139]    4.1 250 ul of Enhanced Chemiluminescent reagent (amerlite reagent, supplied by Johnson &amp; Johnson Clinical Diagnostics, U.K.) is added to the plate.  
         [0140]    4.2 The plate is incubated at room temperature for 10 minutes.  
         [0141]    4.3 The light signal is read using a Labsystems Chemiluminometer (supplied by Medical Supply Co., Dublin, Ireland), which is a scanning wavelength reader. The device reads the luminescence from each well of the plate by scanning the entire IR-visible-UV spectra and extrapolating results.  
         [0142]    4.4 The light signals from the plate are transferred to a customised software package (available from G.K.S. Software, Dublin, Ireland).  
         [0143]    4.5 Each light signal is assigned to a corresponding barcode and a report printed.  
         [0144]    4.6 Results are quoted in chemiluminescent light unit L.U.  
       Source of Reagents  
       [0145]    1. Plate Adhering Agent, Homogenising Buffer, Priming Buffer and Differential Buffer are supplied by Enfer Products Ltd.  
         [0146]    2. Prion Specific Antibodies—anti-PrP—are supplied by Enfer Products and are rabbit antibodies raised to the following synthetic prion peptides.  
         [0147]    Prion Sequence  
         [0148]    N-Terminal  
                       MVKSHIGSWILVLFVVAMWSDVGLCK KRPKPGGGWNTGGSRYPGQ -44                 GSPGGNRYPPQGGGG WGQPHGGGWGQPHGGGWGQPHGGGQGQP-87                 GGGGWGQGGSHSQWNKPSKPPKTNMKHVAGAAAGAVVGG LGGY-131               MLGSAMSSPLIHFGNDYEDRYTRENMYRYPNQVYYRPVDRYSNQNN-177          
 
         [0149]    All the sequences used herein are given using standard l.U.P.A.C. three letter code abbreviations for amino acid residues to find as follows:  
         [0150]    A - Alanine, C - Cysteine, D - Aspartic acid, E - Glutamic acid, F - Phenylalanine, G - Glycine, H - Histidine, I - Isoleucine, K - Lysine, L - Leucine, M - Methionine, N - Asparagine, P - Proline, Q - Glutamine, R - Arginine, S - Serine, T - Threonine, V - Valine, W - Tryptophan and Y - Tyrosine.  
         [0151]    Both underlined sequences, (a 34 amino-acid peptide and a 40 amino acid peptide) are used to raise rabbit anti-PrP antibodies. The peptides are conjugated to activated ovalbumin and injected intra-muscularly in Freund&#39;s Complete Adjuvant. Booster injections are sub-cutaneous and Freund&#39;s Incomplete Adjuvant is used. The rabbits are bled at 30 days.  
         [0152]    3. Chemiluminescent reagent is supplied by Johnson and Johnson Clinical Diagnostics, U.K. and results are read using a Labsystems Chemiluminometer.  
       EXAMPLE 3  
     Validation Data  
       [0153]    1. Inter-Assay Variation  
         [0154]    Data generated on a positive control and a negative control, in a total of 10 assays are provided in Table 1. These controls have been deemed positive and negative by two unrelated methods—histology (HIS) and immunohistochemistry (ICC), methods which will ultimately be used to confirm results reported using the Enfer test. The inter-assay variations based on these two samples are 19% for the positive control and 93% for the negative control. These data indicate that the assay has acceptable reproducibility. Another control was also included in this study. This was a peptide control which allows study of the variation between assays due to antibody differences from day to day. These data are included in Table 1. Again the inter-assay variation at 14% is satisfactory.  
         [0155]    2. Intra-Assay Variation  
         [0156]    The intra-assay variation was determined using the same type controls as those used for the inter-assay study (positive, negative and peptide) and placing 23 replicates of each control on a single plate. Data generated on these controls are provided in Table 2 showing 9% variation for the positive control. 11% for the peptide control and 57% for the negative control.  
         [0157]    3. Stability Studies  
         [0158]    A number of stability studies were carried out for the purpose of this validation:  
         [0159]    (a) Homogenate stability  
         [0160]    In this case a sample was homogenised and divided into aliquots. Over a seven day period, the same homogenate was tested on four occasions using aliquots stored at −20° C. 2-8° C., and on two occasions using aliquots stored at room temperature and 37° C. The results, outlined in Table 3, show that the light signal is stable, allowing for inter-assay variation, over this period with storage conditions of −20° C. with some deterioration at 2-8° C., room temperature and 37° C.  
         [0161]    (b) Stability of homogenising buffer (HB)  
         [0162]    This study was set up to determine the expiry date of homogenising buffer for production purposes. A similar protocol to that outlined in (a) above was followed with the results provided in Table 4. Again the results show that the HB is stable for use.  
         [0163]    (c) Stability of primary antibody—working strength  
         [0164]    Undiluted antibody can be stored frozen for an indefinite length of time. However antibody diluted to working strength using PBS/Tween 20 (0.05%) is not as stable as concentrated antibody and a stability study was, therefore carried out. Antibody dilutions of 1:1K were made on 9 occasions over a 72 day period. On the final day of the study, each of the 9 preparations was applied to an antigen coated plate and an ELISA carried out. The results are presented in Table 5 and from these data it can be seen that the working strength antibody is stable for at least a 70 day period.  
         [0165]    (d) Stability of sample in homogenising buffer before homogenisation  
         [0166]    This stability test was carried out to ensure that the assay would not be affected if some factor resulted in a delay in homogenising the sample after the HB had been added. Homogenising buffer was added to a sample in the ratio of 1 g of brain to 15 ml of buffer and the mixture left at room temperature overnight (15 hours). The sample was homogenised after this time and an immunoassay carried out. The results are shown in Table 6 (−2 samples tested one without and one with a delay). The treatment made no difference to the eventual result.  
                                                           TABLE 1                           Inter-assay variation data, generated on a single positive and negative       control sample, in 10 assays.            Assay       ‘Neg’   ‘Pos’   Peptide       Number   Blank   Control   Control   Control                    1   2   4   30834   110524       day 1       2   2   13   33789   105242       day 1       3   2   12   35319   104293       day 1       4   15   23   32533   101227       day 2       5   13   15   35424   114081       day 2       6   17   15   31749   124122       day 3       7   28   50   22732   146110       day 3       8   7   13   29143   114355       day 4       9   18   22   28565   111084       day 4       10    13   12   36213   100920       day 4                  
 
         [0167]    The value stated at each data point is the mean value of replicates of each control in the assays. The overall mean stated below is calculated using every individual replicate value. The values are quoted in chemiluminescence light units—LU  
                                           Blank   ‘Neg’ Control   ‘Pos’ Control   Peptide Control                   Mean = 14   Mean = 16   Mean = 31927   Mean = 111691       SD = 10   SD = 15   SD = 5954   SD = 15331       CV = 91%   CV = 93%   CV = 19%   CV = 14%       n = 46   n = 51   n = 51    n = 42                  
 
         [0168]    [0168]                                                           TABLE 2                           Intra-assay variation data, generated on a single positive and negative       control sample, 23 replicates of each on a single plate.            Replicate       ‘Neg’   ‘Pos’   Peptide       Number   Blank   Control   Control   Control                    1   34   69   19857   96632       2   49   34   17738   95999       3   22   39   18354   112017       4   29   87   19645   121987       5   38   33   18975   119077       6   51   149   19828   118386       7   34   32   16466   103699       8   38   40   18259   106592       9   24   16   19840   119148       10   32   155   22813   101386       11   26   20   23004   88538       12   17   84   22153   99612       13   57   46   21880   99803       14   32   28   21722   92699       15   25   30   21069   117642       16   57   137   18524   82359       17   18   41   19644   88442       18   45   46   22478   97731       19   15   162   21596   92286       20   20   34   22438   115472       21   13   21   21661   118239       22   33   21   20113   97596       23   19   13   20007   93713                            
         [0169]    [0169]                                           Blank   ‘Neg’ Control   ‘Pos’ Control   Peptide Control                   Mean = 32   Mean = 72   Mean = 20351   Mean = 104742       SD = 13   SD = 41   SD = 1753   SD = 11601       CV = 41%   CV = 57%   CV = 9.0%   CV = 11%       n = 23   n = 23   n = 23   n = 23                    
         [0170]    [0170]                                                                                             TABLE 3                           Homogenate Stability Study - mean of 3 replicates at each temperature            Day   −20° C.   2-8° C.   RT   37° C.                    POSITIVE CONTROL            0           1550           2   1073   449   664   205       3   1162   413   —   —       5   2025   887   —   —       7   1661   1198   709   472       30       90       180            NEGATIVE CONTROL            0           39           2   10   15   6   6       3   9   12   —   —       5   17   37   —   —       7   20   31   10   11       30       90       180                            
         [0171]    [0171]                                                                                             TABLE 4                           Homogenising Buffer Stability Study - mean of 3 replicates at each       temperature            Day   −20° C.   2-8° C.   RT   37° C.                    POSITIVE CONTROL            0           24719           2   53526   61248   42873   47088       3   —   31593   24762   —       7   32120   34457   36337   35010       30       60       180            NEGATIVE CONTROL            0           37           2   4   6   3   4       3   —   9   9   —       7   14   16   20   14       30       60       180                            
         [0172]    [0172]                                                           TABLE 5                           Primary Antibody Stability Study                    1° Antibody                       Mean Light   SD-8   %           Day   Signal   Replicates   CV                            1   19849   557   2.8           3   17677   77   0.4           4   21796   340   1.6           9   18283   218   1.2           23   16805   282   1.7           25   21285   377   1.8           30   21050   427   2.0           42   18339   353   1.9           72   17774   511   2.9                        
         [0173]    [0173]                                           TABLE 6                           Study of sample stability in HB for a 15 hour period before       homogenising                    Mean Result       Sample ID   Hours before homogenisation   (LU)                    79   0   73233       POS   15   72578       80   0   18       NEG   15   38                            
         [0174]    [0174] 
     
       
       
         1 
         
           
             5  
           
           
             1  
             45  
             PRT  
             Artificial Sequence  
             
               UNSURE  
               (45)  
               any amino acid  
             
           
            1 

Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe Val Val 
  1               5                  10                  15 

Ala Met Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly 
             20                  25                  30 

Gly Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Xaa 
         35                  40                  45 

 
           
             2  
             43  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequenceprion 
      protein specific synthetic peptide  
             
           
            2 

Gly Ser Pro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp 
  1               5                  10                  15 

Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp 
             20                  25                  30 

Gly Gln Pro His Gly Gly Gly Gln Gly Gln Pro 
         35                  40 

 
           
             3  
             43  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequenceprion 
      protein specific synthetic peptide  
             
           
            3 

Gly Gly Gly Gly Trp Gly Gln Gly Gly Ser His Ser Gln Trp Asn Lys 
  1               5                  10                  15 

Pro Ser Lys Pro Pro Lys Thr Asn Met Lys His Val Ala Gly Ala Ala 
             20                  25                  30 

Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr 
         35                  40 

 
           
             4  
             46  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequenceprion 
      protein specific synthetic peptide  
             
           
            4 

Met Leu Gly Ser Ala Met Ser Ser Pro Leu Ile His Phe Gly Asn Asp 
  1               5                  10                  15 

Tyr Glu Asp Arg Tyr Thr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln 
             20                  25                  30 

Val Tyr Tyr Arg Pro Val Asp Arg Tyr Ser Asn Gln Asn Asn 
         35                  40                  45 

 
           
             5  
             33  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequenceprion 
      protein specific synthetic peptide  
             
           
            5 

Xaa Xaa Lys His Xaa Ala Gly Ala Ala Ala Xaa Gly Ala Val Val Gly 
  1               5                  10                  15 

Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro Xaa Xaa 
             20                  25                  30 

Xaa