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WO2006002563A2 - Technique de dosage pour trouble neurodegeneratif chez un animal - Google Patents

Technique de dosage pour trouble neurodegeneratif chez un animal Download PDF

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Publication number
WO2006002563A2
WO2006002563A2 PCT/CH2005/000360 CH2005000360W WO2006002563A2 WO 2006002563 A2 WO2006002563 A2 WO 2006002563A2 CH 2005000360 W CH2005000360 W CH 2005000360W WO 2006002563 A2 WO2006002563 A2 WO 2006002563A2
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WO
WIPO (PCT)
Prior art keywords
rna
cysteine proteinase
proteinase inhibitor
cystatin
protein
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PCT/CH2005/000360
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English (en)
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WO2006002563A3 (fr
Inventor
Adriano Aguzzi
Harald Seeger
Gino Miele
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Universität Zürich
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Publication of WO2006002563A2 publication Critical patent/WO2006002563A2/fr
Publication of WO2006002563A3 publication Critical patent/WO2006002563A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G01N2333/8121—Serpins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases

Definitions

  • the present invention relates to a method of diagnosis for identifying a neurodegenerative disorder, in particular for identifying a transmissible spongiform encephalopathy, Parkinson's disease, Huntington's disease, multiple sclerosis or Alzheimer ' s disease in an animal, wherein a sample of said animal is provided to determine the protein and/or RNA level(s) of specific protein(s) expressed differentially in tissues and/or body fluids of animals suffering from neudegenerative disorders at early stages of the disease.
  • the present invention relates to a kit of parts for identifying a neurodegenerative disorder in an animal, comprising specific detection means for determining the protein and/or RNA level(s) of one or more of the above-mentioned compounds.
  • Neurodegenerative diseases in mammals include, for example, Alzheimer's Disease, Parkinson's Disease, multiple sclerosis, Huntington's disease, and also transmissible spongiform encephalopathies (TSEs) such as, for example, Scrapie in sheep, Bovine Spongiform Encephalopathy (BSE) in cattle, Kuru, Creutzfeldt-Jakob disease (CJD), new- variant (nv) CJD, Gerstmann-Straussler Sheinker Syndrome (GSS) and Fatal Familial Insomnia in humans.
  • TSE diseases also manifest themselves in other species such as elk and deer (Chronic Wasting Disease), mink, cats and exotic zoo species such as Nyala, Arabian Oryx, Cheetah and greater Kudu.
  • the problem associated with the diagnosis of TSE's lies in the fact that all currently existing methods of TSE diagnosis are based on detection of an abnormal prion protein isoform (PrP Sc ) of a host-encoded glycoprotein, PrP c . Conversion of normal, proteinase K-sensitive cellular prion proteins (PrP 0 ) into the abnormal, partially proteinase K-resistant isoform PrP Sc , is a hallmark common to most of the TSEs.
  • prions are devoid of informational nucleic acids and consist primarily, if not exclusively, of an "infectious" protein (PrP Sc ) capable of converting the normal host protein (PrP c ) into a likeness of themselves. Whilst following peripheral prion entry, replication of the infectious agent occurs to high titres initially in lymphoid tissues (such as spleen, lymph nodes, etc.) prior to entry of the agent into the peripheral or central nervous system (CNS). The only organ system in which histopathological damage and its clinical manifestation can be demonstrated as a consequence of infection with prions is the nervous system (Brandner, S., S. Isenmann, et al. (1996).
  • PrP Sc protein The problem associated with TSE-diagnosis is the unique biology of the prion protein.
  • PrP Sc protein The only disease-specific macromolecuie identified to date (PrP Sc protein) has exactly the same primary amino acid sequence as the normal cellular protein PrP c . Distinguishing disease- specific PrP Sc from endogenous PrP c is time-consuming, laborious and is not always possible in all forms of TSE and in easily accessible body tissues and fluids. In other words, currently the PrP Sc protein is the only currently known surrogate marker for presence of TSE disease.
  • PrP c and PrP Sc are identical, with PrP c being a membrane-bound protein that is more or less ubiquitously expressed and found in many cell types in healthy individuals. Whilst PrP c and PrP Sc do differ in a number of physical and chemical attributes, primarily with respect to tertiary structure, it has proven extremely difficult to develop immunological reagents which reliably differentiate between these two isoforms.
  • PrP Sc in tissues other than CNS tissue (for example in blood or CSF) is extremely difficult, if not impossible.
  • PrP Sc is not always detectable in all forms of TSE even in CNS tissue of humans and animals clearly affected by TSE disease. Therefore, an effective pre-mortem diagnostic means of assessing TSE infection is still lacking.
  • CD11 b+ cells cultured for 12-18 hours have the drawback that they reflect in vivo processes poorly because the process of enrichment and the time period in culture places these cells in an activated state, regardless of the cause being direct interaction of microglia with prions, a secondary response to proinflammatory molecules, or a direct effect of accumulating pathologic PrP. Also, this cell model is lacking in that the stage of pathogenesis is undefined, but most likely being the terminal stage of pathogenesis.
  • lysozyme M represents the most dramatic change identified in their analysis.
  • this finding was not confirmed in more relevant in vivo screens that led to the conclusion that lysozyme is not differentially expressed in either CNS tissue, spleens, or lymph nodes of TSE-infected mice or in chronically infected cultured cells as assessed by microarray analysis (data not shown).
  • This provides further evidence that the system utilised by Baker & Manuelidis is extremely artefactual and does not necessarily reflect the situation in vivo.
  • Cystatin F which is known to inhibit viral replication in some systems, is induced in both CJD and IFN- ⁇ -treated microglia in the above described system. With respect to Cystatin F and in spite of the undefined cell culture status, Baker & Manuelidis conclude that Cystatin F levels represent a non-specific response to amyloid deposition.
  • the sample material necessary for diagnosing such disorders should be easily accessible and assayable, preferably body fluids (such as blood, urine, sera, CSF), in addition to more conventional tissues such as CNS.
  • the diagnostic method should allow assessment of neurodegenerative disorders in live individuals at an early stage to still allow for therapeutic intervention or preventative measures.
  • the diagnostic method should also be suited for routine screening of blood and blood products for use in transfusion medicine.
  • the present invention relates to a method of diagnosis for identifying a neurodegenerative disorder in an animal, comprising providing a sample of said animal to determine the protein and/or RNA levels of one or more of the following compounds:
  • cysteine proteinase inhibitor Cystatin F a) cysteine proteinase inhibitor Cystatin F, b) target of cysteine proteinase inhibitor Cystatin F c) serine or cysteine proteinase inhibitor SerpinA3, d) target of serine or cysteine proteinase inhibitor SerpinA3.
  • cystatin F refers to any member of the Cystatin family of cysteine proteinase inhibitors.
  • the cystatin superfamily encompasses proteins that contain multiple cystatin-like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never acquired this inhibitory activity. There are three inhibitory families in the superfamily, including the type 1 cystatins (stefins), type 2 cystatins and the kininogens.
  • the type 2 cystatin proteins (which includes Cystatin F) are a class of secreted cysteine proteinase inhibitors found in a variety of human fluids and secretions.
  • Type 2 cystatin proteins are preferred as marker compounds of the present invention (Vray, B. Et al. (2002). Cell MoI. Life Sci. 59:1503-1572, J. Saklatvala et al. Biochem. Soc. Symp. 2003 70).
  • the cystatins are a superfamily of similar proteins present in mammals, birds, fish, insects, plants and protozoa. In general, they are potent peptidase inhibitors.
  • the type 1 cystatins (A and B) are mainly intracellular, the type 2 cystatins (C, D, E/M, F, G, S, SN and SA) are extracellular, and the type 3 cystatins (L- and H-kininogens) are intravascular proteins.
  • Cystatins are thiol protease inhibitors.
  • target of proteinase inhibitor Cystatin F refers to the molecular and physiological target of Cystatin F.
  • SerpinA3 refers to any Serpin A3 member of the family of Serine proteinase inhibitors.
  • Serpins are proteins that are primarily known as irreversible serine protease inhibitors active against S1 (INTERPRO database accession number: IPR001254), S8 (INTERPRO database accession number: IPR000209) and C14 (INTERPRO database accession number: IPR002398) peptidases. There are both extra- and intracellular serpins, which are found in all groups of organisms.
  • Serpins and their homologues are a group of high molecular weight (40 to 50 kDa) structurally related proteins involved in a number of fundamental biological processes such as blood coagulation, complement activation, fibrinolysis, angiogenesis, inflammation, tumour suppression and hormone transport. All known serpins have been classified into 16 clades and 10 orphan sequences. The vertebrate serpins can be conveniently classified into six sub-groups (Irving et al., Genome Res. 2000 Dec; 10(12):1833-5). In human plasma they represent approximately 2% of the total protein, of which 70% is antitrypsin.
  • target protease of serine or cysteine proteinase inhibitor SerpinA3 refers to the molecular and physiological target of Serpin A3.
  • the method of the present invention makes use of the set of genes and/or proteins of the marker compounds mentioned above that are profoundly differentially expressed, for example, in neural tissue of animals with neurodegenerative disorders, in particular TSE- infected animals, preferably at early and also the later stages of the disease.
  • the profoundly increased levels of these marker compounds in neural tissue and secretion of these into body fluids allows for an entirely novel approach for non-PrP Sc -based diagnostic screening for neurodegenerative diseases including, but not limited to, TSEs.
  • the diagnosis of neurodegenerative diseases at early stages of the disease is a preferred embodiment.
  • stage of the disease means in this context a stage of pathogenesis at which pathological hallmarks of disease are not pronounced and clinical symptoms are not yet evident.
  • neurodegenerative disease refers to a disorder caused by the deterioration of certain nerve cells (neurons). Changes in these cells cause them to function abnormally, eventually bringing about their death. Neurodegeneration in the central nervous system accompanies these diseases, which include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's disease, multiple sclerosis, and TSEs such as Creutzfeldt-Jakob Disease, variant Creutzfeldt-Jakob Disease, variant Creutzfeldt-Jakob Disease, Kuru, Fatal Familial Insomnia, Gerstmann-Straussler Sheinker Syndrome in humans, and scrapie, BSE, CWD.
  • RNA refers to any ribonucleotide products derived from animal DNA in the course of transcription.
  • RNA-level refers to the amount of RNA by weight or copy number or abundance of a specific RNA in a sample of an animal suspected to have acquired a neurodegenerative disorder in comparison to the amount of RNA by weight or copy number or abundance of the same specific RNA in a sample of the same source of the same animal species suspected of being healthy in this respect.
  • RNA of the marker compounds may be any RNA resulting under suitable conditions in a biologically functional marker compound upon transcription.
  • sequence(s) of the RNA(s) of the RNA level(s) determined is (are) set forth in any one of SEQ ID NO: 1-5, which are presented further below.
  • sequence(s) of the RNA(s) of the RNA level(s) determined has (have) at least 50, preferably at least 70, more preferably at least 90, and most preferably at least 95 percent sequence identity with those sequences set forth in any one of SEQ ID NO: 1-5, and wherein the RNA(s) result(s) in expression products which are functionally equivalent to those resulting from the RNA set forth in any one of SEQ ID NO: 1-5.
  • protein refers to any peptide products derived from animal RNA in the course of translation.
  • protein-level refers to the amount of protein by weight or abundance of a specific protein in a sample of an animal suspected to have acquired a neurodegenerative disorder in comparison to the amount of protein by weight or abundance of the same specific RNA in a sample of the same source of the same animal species suspected of being healthy in this respect.
  • the protein of the marker compounds may be any protein resulting under suitable conditions in a biologically functional marker compound upon translation or secretion.
  • sequence(s) of the protein(s) of the protein level(s) assayed is (are) set forth in any one of SEQ ID NO: 6-10.
  • sequence(s) of the protein(s) of the protein level(s) determined is (are) set forth in any one of SEQ ID NO: 6 - 10 and/or
  • sequence(s) of the protein(s) of the protein level(s) determined has (have) at least 50, preferably at least 70, more preferably at least 90, and most preferably at least 95 percent sequence identity with those sequences set forth in any one of SEQ ID NO: 6-10 and wherein the proteins are preferably functionally equivalent to those resulting from the RNA set forth in any one of SEQ ID NO: 1-5.
  • sequence identity which is well known to the person skilled in the art designates the degree of relatedness among two or more polynucleotide or polypeptide molecules, which is determined by the agreement between the sequences.
  • the percentage "identity” is calculated from the percentage of identical regions in two or more sequences, taking account of gaps or other sequence features.
  • the identity of related polynucleotides or polypeptides can be determined by means of known procedures. As a rule, special computer programs with algorithms taking account of the special requirements are used. Preferred procedures for the determination of identity firstly generate the greatest agreement between the sequences studied.
  • Computer programs for the determination of the identity between two polynucleotide or two polypeptide sequences include, but are not limited to, various implementations of the Basic Local Alignment Search Tool (BLAST or BLAST2; Tatusova TA, Madden TL FEMS Microbiol Lett.
  • BLAST Basic Local Alignment Search Tool
  • Tatusova TA Madden TL FEMS Microbiol Lett.
  • Blastn for example Blastn, Blastp, Blastx, tBlastn, tBlastx, Phi-Blast, Psi-Blast, in particular bl2seq (which utilises the Blastn or Blastp implementations of the BLAST2 algorithm specifically for alignment of two sequences) preferably using, but not limited to, standard default settings for Blastn (strand option, both strands; Open gap, 5; extension gap, 2; gap x_dropoff, 50; expect, 10; word size, 11 , no filter) and for Blastp (Matrix, either BLOSUM62 or PAM30 or PAM70 or PAM250 or BLOSUM90 or BLOSUM50; Open gap, 10; extension gap, 1 ; gap x_dropoff, 50; expect, 10; word size, 3; no filter).
  • Blastn strand option, both strands
  • the invention has already demonstrated its effectiveness in a number of neurodegenerative disorders, evidence thereof being provided further below in the experimental section.
  • the neurodegenerative disorder to be assayed is selected from the group consisting of Alzheimer's Disease, Parkinson's Disease, Huntington's disease, Multiple sclerosis and Transmissible spongiform encephalopathies (TSE).
  • TSE Transmissible spongiform encephalopathies
  • TSEs Transmissible Spongiform Encephalopathies
  • BSE Bovine Spongiform Encephalopathy
  • Kuru Creutzfeldt-Jakob Disease
  • nv new variant
  • s sporadic
  • GSS Gerstmann-Straussler Scheinker Syndrome
  • FSE diseases are characterised by the eposition and progressive accumulation of PrP Sc , an abnormal isoform of a host- encoded glycoprotein, PrP 0 .
  • TSEs Transmissible Spongiform Encephalopathies
  • said transmissible spongiform encephalopathy is selected from the group consisting of Scrapie, Bovine Spongiform Encephalopathy (BSE), Chronic Wasting Disease (CWD), Creutzfeldt-Jakob Disease (CJD) new-variant (nv) CJD 1 sporadic (s) CJD 1 Gerstmann-Straussler Scheinker Syndrome and Fatal Familial Insomnia.
  • Animals affected by TSE include mammals, such as ovines, bovines, humans, felines, elk, deer, mink, and exotic zoo species such as Nyala, Arabian Oryx, Cheetah and Greater Kudu, and potentially avian species, such as poultry, for example, chickens, turkey, guinea fowl.
  • the method of the present invention is used in mammals, more preferably in sheep, bovines, ovines, deer, elk or, more preferably, humans, most preferably to identify neurodegenerative disorders, more preferably to identify TSE ' s.
  • the sample to be determined according to the present invention will typically be a biological sample, for example a body-fluid based sample (e.g. blood, sera, CSF, urine, lymph, saliva) or a fluidized tissue sample.
  • the sample may be whole or fractionated blood (or partly fractionated blood), plasma, sera, a tissue, such as brain, spinal cord.
  • the sample may be subject to the addition of further components in order to optimise sample analysis.
  • substances such as heparin, EDTA and/or sodium citrate may be added to prevent coagulation by means of clot formation.
  • clot formation may be beneficial for the preparation of the blood sample in order to obtain serum.
  • Other sample sources include, but are not limited to, cerebrospinal fluid (CSF), urine, tears, milk, semen, mucous secretions, tissues or organ biopsies, e.g. brain, spinal cord.
  • the sample that is provided from said animal for assay purposes consists of or comprises body fluid(s) and/or fluidised tissue samples.
  • said body fluid(s) consist(s) of or comprise(s) whole or fractionated blood, cerebrospinal fluid, urine, lymph or fluid from central nervous system tissue.
  • Protein and RNA-levels of the marker compounds of the present invention can be determined by any conventional methods available in the art.
  • RNA transcripts of the marker compounds in particular those identified in SEQ ID No:1-5, either individually or in various combinations, in particular RNA transcripts of the inhibitors Cystatin F and/or SerpinA3 (cysteine and/or serine variant) and/or the transcripts which code for the targets of these compounds are measured, preferably quantitatively, and compared to those levels established for a sample derived from healthy donor animals.
  • Establishing a control or normal population value is possible by using standard techniques of sample collection and analysis. Standard statistical techniques can be used in order to define meaningful average values for a population which may require taking into account factors such as age, sex, genotype and may be based on an uninfected, unaffected individual or on a population of uninfected, unaffected individuals.
  • RNA-transcripts of one or more markers of the present invention in the sample of an animal suspected of being TSE-inflicted are quantified in comparison relative to a control or normal population value of that animal species.
  • transcripts of marker compounds of the present invention can be assayed for by using techniques such as, but not limited to, Northern blotting (Thomas, P.S. (1980). Proc. Natl. Acad. Sci. USA. 77; 5201-5205). Briefly, denatured RNA (electrophoresed for Northern blotting, non-electrophoresed for dot/slot-blots for example) from sample tissues and/or cells is transferred onto nitrocellulose or nylon filter membranes for subsequent use in a hybridisation assay.
  • RNA is electrophoresed in a denaturing agarose gel before being transferred onto a membrane either by capillary action or under the action of an electrical field.
  • a radioactively labelled DNA or RNA probe specific for the transcript in question is hybridized to the filter- bound RNA to enable detection.
  • RNA levels can be measured using "Taqman” or "Light-Cycler” quantitative real-time RT-PCR procedures, which are derived from the use of a fluorescent energy transfer (FRET) probe, or by incorporation of fluorescent dyes with affinity for nucleic acids.
  • FRET fluorescent energy transfer
  • Fluorescent signal intensity is directly proportional to levels of synthesised product and therefore within the phase of exponential synthesis, this allows relative or absolute quantification of the amount of target contained in the starting sample.
  • a specific example of how this is achieved is presented in Figs. 4, 7 & 8 (A-C).
  • a further alternative is the use of the "Invader" technology (Third Wave Technologies); which is based on the discovery of a unique class of structure-specific endonuclease enzymes (cleavases). Invader and signal probes are designed to hybridize to overlapping sites on the target DNA/RNA such that the invader probe displaces a portion of the signal probe. This forms a structure that a cleavase enzyme will recognize and cut, thus creating detectable products.
  • the protein level(s) of one or more of the marker compounds preferably those coded for by the RNA set forth in any one of SEQ ID No: 1-5, either individually or in various combinations, in particular the inhibitors Cystatin F and/or SerpinA3 or the targets thereof can be measured, preferably quantitatively, and compared to those levels established for a sample derived from healthy donor animals.
  • this method would involve the use of antibodies specific for protein marker compounds of the present invention, more preferably antibodies specific for those marker proteins coded for by the transcripts set forth in any one of SEQ ID NO: 1-5.
  • antibodies are specific for those proteins as set forth in SEQ ID NO: 6 to 10.
  • These antibodies may be polyclonal or monoclonal antibodies raised by conventional techniques known in the art.
  • Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, chicken, goat, monkey, etc.) when the marker proteins of the present invention are injected into an animal. If necessary, an adjuvant may be administered together with the proteins of the present invention. The antibodies can then be purified by virtue of their binding to the immunized proteins or as described further below.
  • a suitable animal host e.g. a mouse, rat, guinea pig, rabbit, sheep, chicken, goat, monkey, etc.
  • an adjuvant may be administered together with the proteins of the present invention.
  • the antibodies can then be purified by virtue of their binding to the immunized proteins or as described further below.
  • Monoclonal antibodies can be produced from hybridomas or by recombinant synthesis techniques.
  • Hybridomas can be formed by fusing myeloma cells and spleen cells which produce the desired antibody in order to form an immortal cell line (Kohler & Milstein (1975). Nature 256; 52-55).
  • the present invention encompasses derivatives of these which are capable of binding to the marker proteins of the present invention.
  • These antibody derivatives useful in the method of the present invention include antibody fragments and synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Kuby, J. (1997). Immunology. 3 rd Edition, Von Hoffman press Inc. ISBN 0-7167-2868-0.
  • Antibody fragments include, for example, Fab, F(ab) 2 and Fv fragments. Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining V h and Vi regions which contribute to the stability of the molecule.
  • CDR peptides include CDR peptides. These are synthetic peptides comprising antigen binding determinants. Peptide mimetics may also be used. These molecules are usually conformational ⁇ restricted organic rings which mimic the structure of a CDR loop and which include antigen-interactive side chains. Synthetic constructs also include chimeric molecules. An example of a humanized antibody is an antibody having human framework regions but rodent hypervariable regions. Synthetic constructs also include molecules comprising a covalently linked moiety which provides the molecule with some desirable property in addition to antigen binding. For example, the moiety may be a label (e.g. a detectable label, such as a fluorescent or radioactive label) or a pharmaceutically active agent.
  • a label e.g. a detectable label, such as a fluorescent or radioactive label
  • a suitable antibody specific for one or more marker proteins preferably those proteins coded for by the transcripts presented in SEQ ID NO: 1-5, more preferably those proteins as set forth in any one of SEQ ID NO: 6 - 10.
  • suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on Protein A or Protein G columns, or combinations of these techniques.
  • Recombinant antibodies to the marker proteins of the present invention preferably those coded for by the transcripts in any one of SEQ ID NO: 1- 5, more preferably those proteins as set forth in any one of SEQ ID NO: 6 - 10, can be prepared according to standard methods, and assayed for specificity for their targets by procedures generally available, including Western blot, ELISA 1 ABC, dot-blot assays etc.
  • One example of a method for assaying for the aforementioned marker proteins in a sample is Western blotting.
  • An extract of proteins from the sample can be fractionated by electrophoresis through denaturing (or non-denaturing) SDS-polyacrylamide gels.
  • the fractionated proteins can then be transferred and immobilised on a solid membrane of nitrocellulose, nylon or PVDF by electroblotting.
  • the membrane is then incubated with sera or antibody reactive against the protein of interest.
  • the resulting antigen-antibody complex can then be detected by any suitable procedure.
  • a secondary antibody raised to be reactive against immunoglobulins from the species in which the primary antibody was raised, can be linked to a reporter moiety (for example horseradish peroxidase, alkaline phosphatase etc) can be added.
  • the reaction product generated by enzyme action can then be used to indicate the position of the target protein on the membrane. Measurement of the levels of enzyme reaction is indicative of the levels of target protein present in the sample.
  • the sensitivity of the detection system can be improved by using, for example, the biotin-streptavidin system or chemiluminescence detection.
  • marker proteins of the present invention preferably those coded for by the transcripts presented in any one of SEQ ID NO: 1-5, more preferably those proteins as set forth in any one of SEQ ID NO: 6 - 10, can be assayed for by other standard techniques, which include, but are not restricted to, radio-immunoassay (RIA), Enzyme-linked immunosorbent assay (ELISA) and BIACORE technology.
  • RIA radio-immunoassay
  • ELISA Enzyme-linked immunosorbent assay
  • BIACORE BIACORE technology.
  • ELISA for example, an antibody can be linked to a reporter enzyme and then be immobilised on a microtitre plate. A lysate of sample to be measured is then added to allow antibody-antigen complex formation. After washing and provision of substrate, levels of product formation are proportional to the levels of antigen present, i.e.
  • DELFIA DELFIA
  • the Delfia Research System measures the fluorescence of metals from the lanthanide series, including europium, samarium and terbium.
  • Antibodies are labelled and immobilised on microtitre plates. A lysate from the sample to be measured is added to the plate to allow antibody-antigen complex formation. After washing, the level of signal is determined and is proportional to the level of antigen, i.e. target protein, present in the sample.
  • a further method could include the use of immunohistochemistry techniques in which sample tissue is sectioned and immobilised on a slide prior to immunological detection of target antigen.
  • the Cystatin F inhibitor level can be measured by more than one method or marker.
  • the protein or RNA level, preferably the RNA level, of cysteine proteinase inhibitor Cystatin F and/or the target of cysteine proteinase inhibitor Cystatin F is determined.
  • the protein level of cysteine proteinase inhibitor Cystatin F or the target of cysteine proteinase inhibitor Cystatin F is determined by determining the Cystatin F inhibitor activity or the activity of its target of cysteine proteinase.
  • the serine or cysteine proteinase inhibitor SerpinA3 level can be measured by more than one method or marker.
  • the protein or RNA level, preferably the RNA level, of serine or cysteine proteinase inhibitor SerpinA3 and/or the target of serine or cysteine proteinase inhibitor SerpinA3 is determined.
  • the protein level of serine or cysteine proteinase inhibitor SerpinA3 or the target of serine or cysteine proteinase inhibitor SerpinA3 is determined by determining the SerpinA3 inhibitor activity or the activity of its target serine or cysteine proteinase.
  • the protein level of cysteine proteinase inhibitor Cystatin F is determined by determining the cysteine CystatinF inhibitor activity, preferably by the use of a fluorogenic peptide substrate, more preferably by the use of N-carbobenzyloxy-Leu- Arg-7-amido-4-methylcoumarin (Z-L-R-AMC).
  • Levels of activity of the protease inhibitors Cystatin F and/or SerpinA3, and/or activities of their in vivo ligands, either individually or in various combinations of these, can be measured and compared to those levels established for a sample derived from healthy donor animals.
  • Establishing a control or normal population value is possible by using standard techniques of sample collection and analysis. Standard statistical techniques can be used in order to define meaningful average values for a population which may require taking into account factors such as age, sex, genotype and may be based on an uninfected, unaffected individual or on a population of uninfected, unaffected individuals.
  • Cystatin F and SerpinA3 are members of the cysteine and serine or cysteine family of protease inhibitors. It is particularly important to ensure that standard protease inhibitors with a wide-range of action (e.g. Roche Complete Mini cocktail) are not added to samples during preparative steps.
  • a fluorogenic peptide substrate such as N-carbobenzyloxy-Leu- Arg-7-amido-4-methylcoumarin (Z-L-R-AMC) may be employed for activity measurement.
  • Cleavage of Z-L-R-AMC can be measured using excitation and emission wavelengths of 380 nm and 460 nm respectively.
  • Measurement of levels of protease inhibitor activity in a given biological sample can be determined by the degree of efficiency of said sample in cleavage of Z-L-R-AMC.
  • the measurement of activity levels of, for example, Cystatin F cysteine protease inhibitor can be determined by the degree of efficiency of said sample in inhibiting the cleavage of Z-L-R-AMC by, for example, recombinant Cathepsin L, or papain, or lugumain proteases.
  • the method of diagnosis for identifying a neurodegenerative disorder may comprise one or more of the following steps: • A sample collection step, optionally already including general sample preparation and/or pruification steps (e.g. addition of blood clot formation inhibitors, preservatives, fluidisation of tissue sample, etc).
  • a sample collection step optionally already including general sample preparation and/or pruification steps (e.g. addition of blood clot formation inhibitors, preservatives, fluidisation of tissue sample, etc).
  • RNA detection assays QPCR, Northern, Invader etc
  • protein detection assays Western, RIA, ELISA, Biacore, ELISA etc
  • protease inhibition assays and/or (d) target protease assays.
  • Control reference samples from a normal unaffected individual, or group of individuals may include pooled samples from a population of unaffected individuals sufficient to establish a normal range or normal value in statistical analysis.
  • the present invention relates to a kit for identifying a neurodegenerative disorder in an animal, comprising specific detection means for determining the protein and/or RNA levels of one or more of the following compounds:
  • cysteine proteinase inhibitor Cystatin F a) cysteine proteinase inhibitor Cystatin F, b) target of cysteine proteinase inhibitor Cystatin F 1
  • kit further comprises written instructions for performing one of more steps necessary for a method of the present invention.
  • the kit may comprise solvents, buffer substances, components for preparing samples, purifying samples, etc.
  • the detection mean(s) of the kit is (are) selected from the group consisting of antibodies specific for one or more of said marker compounds of the present invention, RNA-probe(s) specific for one or more RNA(s) coding for said marker compounds, and means for determining the biological activity of one or more of said marker compounds.
  • said RNA-probe(s) is (are) specific for one or more RNA(s) as set forth in any one of SEQ ID NO: 1-5.
  • said antibody(ies) is (are) specific for cysteine proteinase inhibitor Cystatin F, and/or serine or cysteine proteinase inhibitor SerpinA3.
  • said antibody(ies) is (are) antibodies specific for one or more proteins as set forth in any one of SEQ ID NO: 6-10.
  • kits according to the invention comprises means for quantitatively determining SerpinA3 inhibitor activity, Cystatin F inhibitor activity, and/or the activity of the target(s) of the SerpinA3 inhibitor and/or CystatinF inhibitor.
  • the present invention relates to the use of an RNA probe specific for an RNA coding for one or more of the following compounds:
  • cysteine proteinase inhibitor Cystatin F a) cysteine proteinase inhibitor Cystatin F, b) target of cysteine proteinase inhibitor Cystatin F,
  • the present invention relates to the use of an antibody specific for one or more of the following compounds:
  • cysteine proteinase inhibitor Cystatin F a) cysteine proteinase inhibitor Cystatin F, b) target of cysteine proteinase inhibitor Cystatin F 1
  • said antibody is polyclonal, monoclonal, recombinant, chimeric and/or humanized or a fragment of such an antibody capable of specifically binding at least one of said compound(s).
  • Fig. 1 shows the time course of PrP Sc accumulation in brain tissue from mice in the C57BI/6 RML murine model of prion pathogenesis.
  • Fig.2 shows those transcripts identified via Affymetrix high-density oligonucleotide hybridisation studies of RNA isolated from whole brains of prion-infected age and sex-matched C57BI/6 mice at 145 dpi stage of pathogenesis as being most dramatically differentially expressed relative to mock-inoculated controls.
  • Affymetrix probeset, Genbank accession identifications and Locuslink identifications are shown. Abbreviations used to further present these are shown.
  • Fig. 3 shows forward and reverse oligonucleotide primers used for quantitative real-time PCR analysis of selected candidate differentially expressed transcripts in Figs. 2 & 4 and Figs. 7 & 8 (A-C) .
  • Fig. 4 shows fold-change in expression level of selected candidate transcripts from Fig 2 in RNA isolated from whole brains of prion-infected mice compared to mock-inoculated controls at 145 dpi and 190 dpi stages of pathogenesis as assessed by quantitative real-time PCR.
  • Fig. 5 shows the results of the assessment of the differential expression of other members of Cystatin family of secreted protease inhibitors in spleen and mesenteric lymph nodes (MLNs) of prion infected mice at 145 dpi stage of pathogenesis and in in vitro cultured cells chronically infected with RML5 strain of prions (by Affymetrix high- density oligonucleotide micorarray analysis).
  • Fig. 6 shows levels of Cystatin F and SerpinA 3N transcripts in CNS tissue at various time points during pathogenesis in the C57BI/6 RML5 murine model of prion pathogenesis as assessed by Northern hybridisation experiments.
  • A Transcript levels in RNA isolated from whole brain at various days post-inoculation (dpi).
  • B Transcript levels in RNA isolated from CNS spinal cord, cerebellum, olfactory bulb and cortex at various days post-inoculation.
  • Fig. 7 shows fold-change in expression level of Cystatin F in RNA isolated from whole brains of prion-infected mice compared to mock-inoculated controls at 100 dpi, 145 dpi and 190 dpi stages of pathogenesis as assessed by quantitative real-time PCR.
  • Fig. 8 shows levels of expression of Cystatin F, SerpinA3 and GFAP transcripts in total RNA isolated from occipital cortex tissue obtained post-mortem from sporadic CJD (sCJD) patients, Alzheimer's Disease (AD) patients and patients in which mortality was a result of a non-neurodegenerative condition (non-neurodgenerative controls).
  • Cystatin F transcript levels (A) Cystatin F transcript levels, (B) GFAP transcript levels, (C) SerpinA3 transcript levels.
  • Fig. 9 shows SerpinA3 levels in urine of human patients.
  • Urine provided by the Swiss National Reference Centre for Prion Diseases (NRPE) and the UK National CJD Surveillance Unit.
  • An ELISA protocol established using commercially available anti- human SerpinA3 (alpha-1 antichymotrypsin) polyclonal antibodies (The Binding Site and Dako).
  • Murine MOE430A and MOE430B high-denisity oligonucleotide microarray GeneChips were purchased from Affymetrix lnc (USA), 3380 Central Expressway, Santa Clara, CA 95051 , USA.
  • Microarray hybridisations were performed at the Functional Genomics Centre Zurich, UNI ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
  • Oligonucleotide primers for quantitative real-time PCR were purchased from Microsynth AG, Sch ⁇ tzenstrasse 15, 9436 Balgach, Switzerland.
  • Full-length cDNA clones for generation of radiolabeled probes for filter hybridisation studies were purchased from BioCat GmbH, Im Neuenheimer FeId 581, D-69120 Heidelberg, Germany. Adevron (3233 15th St.
  • Example 1 Global analysis of gene expression
  • the well-characterised Rocky Mountain Laboratory (RML) strain of prion agent was inoculated intraperitoneally into C57BI/6 mice as a model of prion infection.
  • Age and sex matched mice were intraperitoneally inoculated with either 100 ⁇ l of titred brain homogenate containing high dose (6 LogLD 50 ) RML5 prions or with 100 ⁇ l of brain homogenate from healthy mice (mock-inoculated).
  • Tissues (whole brain, spinal cord, cerebellum, olfactory bulb, cortex, spleen, inguinal and mesenteric lymph nodes, whole blood and sera) were collected from several individual control and prion-infected animals at various defined stages throughout prion pathogenesis (days post inoculation, dpi), representing time points prior to and during appearance of a characteristic pathology. Tissues were also collected at 190 dpi at which stage clinical symptoms are evident. It is well established in this murine model of prion pathogenesis that the incubation period of disease, from the point of inoculation to clinical endpoint, is approximately 200 dpi. Tissues were flash-frozen in liquid nitrogen and stored at -80 0 C until required for further processing.
  • 145 dpi represents a stage of prion pathogenesis in this model in which the early stages of CNS PrP Sc accumulation is evident (Fig. 1 ) but at which neither extensive pathology (vacuolation, neuronal cell loss, astrogliosis) or clinical symptoms are evident.
  • RNA 20 ⁇ g of total RNA was used to synthesise double-stranded cDNA according to procedures outlined in the Functional Genomics Centre Zurich Affymetrix GeneChip protocol manual.
  • Double-stranded cDNA was used to synthesise biotin-labelled cRNA probes which were used to hybridise Affymetrix murine MOE430A and MOE430B high-density oligonucleotide GeneChips according to procedures outlined in the Functional Genomics Centre Zurich Affymetrix GeneChip protocol manual. Washing and scanning of the GeneChips was performed according to procedures outlined in the Functional Genomics Centre Zurich Affymetrix GeneChip protocol manual.
  • High-density oligonucleotide microarray is a powerful molecular tool which allows visualisation of expression levels of many thousands of genes in any particular cell type or tissue simultaneously.
  • MOE430A and MOE430B murine GeneChips it is possible to simultaneously interrogate expression levels of approximately 34 000 transcripts.
  • Affymetrix GeneChip technology and MOE430A and MOE430B murine GeneChips the transcript levels of genes expressed in whole brains of mice at 145dpi stage of prion pathogenesis were compared with transcript levels of genes expressed in whole brains from control animals. For each group, three independent hybridisations for each of the MOE430A and MOE430B GeneChips were performed, corresponding to three independent biological replicate hybridisations for each group.
  • Fig. 2 shows this set of transcripts identified as being differentially expressed in whole brains of prion-inoculated mice relative to controls at 145dpi stage of pathogenesis.
  • Focus was predominantly placed on those transcripts in which increased levels were apparent in whole brains of prion-infected mice at 145dpi stage relative to mock-inoculated controls since these are reasoned to be among the best candidates to form the basis of surrogate marker-based diagnosis of disease. Additionally, focus was also placed predominantly on those transcripts which code for proteins know to be secreted proteins, since these are reasoned to be among the best candidates for surrogate marker-based diagnosis of disease in which sample material would consist of body fluids such as, but not limited to, blood, sera, urine and cerebrospinal fluid.
  • Example 2 Expression levels of selected candidate transcripts in whole brains of prion infected mice at 145 dpi and 190 dpi stages of pathogenesis.
  • RNA samples were collected at various stages during disease pathogenesis (dpi; days post inoculation).
  • Total RNA was isolated from nine brains of mock-inoculated mice and nine brains of prion- inoculated mice at 145 dpi and 190 dpi stages of pathogenesis.
  • total RNA was pooled into groups of three to form three biological replicates.
  • First-strand cDNA was synthesised and used as template for quantitative real ⁇ time PCR.
  • primer pairs were designed such that an amplicon of approximately 250bp would be synthesised in the PCR reaction (Fig. 3).
  • Cystatin F represents the most dramatic change in RNA expression in this set as a result of prion infection, with levels being approximately 70-fold higher in brains of prion inoculated mice at 145 dpi stage of pathogenesis relative to mock inoculated controls.
  • GFAP expression levels classically viewed as the most obvious gene expression change in CNS tissue during prion pathogenesis, are increased to a far lesser degree than those of Cystatin F or SerpinA 3N.
  • Cystatin F (synonym, Cystatin 7, Leuokocystatin, Cmap) is a member of the Cystatin family of cysteine proteinase inhibitors (Genbank accession NM_009977, NM_003650; Locuslink identification 13011 , 8530). Cystatin F protein contains a signal peptide at the N-terminus of the protein and is known to be secreted into the extracellular milieu.
  • the cystatin superfamily encompasses proteins that contain multiple cystatin-like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never acquired this inhibitory activity.
  • the type 1 cystatins stefins
  • type 2 cystatins a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions.
  • the Cystatin F gene encodes a glycosylated cysteine protease inhibitor with a putative role in immune regulation through inhibition of a unique target in the hematopoietic system.
  • the protein encoded by the SerpinA 3N gene is a protease inhibitor and member of the serine protease inhibitor class and is found in a variety of human fluids including, but not limited to, sera and cerebrospinal fluid. These candidates exemplifies the rationale of this analysis in that they are differentially expressed early in disease pathogenesis, levels are increased dramatically during prion disease and the transcripts code for secreted protease inhibitors which are detectable in body fluids.
  • Example 3 Expression levels of members of Cystatin family of protease inhibitors in other tissues of prion infected animals and in an in vitro neuroblastoma cell-culture model of prion replication
  • High-density microarray analysis was also performed using total RNA isolated from spleen and mesenteric lymph nodes (MLNs) of mice at 145dpi of pathogenesis (dpi, days post inoculation) either inoculated intraperitoneally with 6LogLD50 RML5 strain of prions or with brain homogenate from healthy mice. Additionally, array analysis has also been performed using total RNA isolated from a cloned murine neuroblastoma cell line (N2aPK1) chronically infected with RML5 strain of prions or uninfected. In all these models there is accumulation of PrP Sc and high levels of prion infectivity.
  • Cystatin F transcript levels are low in RNA isolated from whole brains of mock-inoculated mice. Increased levels of Cystatin F RNA transcripts in RNA isolated from whole brains of prion inoculated mice are evident by Northern hybridisation analysis by 110dpi and increase dramatically throughout the remainder of pathogenesis (Fig. 6A). Increased levels of SerpinA 3N transcripts are evident by 130dpi and are increased throughout the remainder of disease pathogenesis (Fig. 2A).
  • Cystatin F Expression levels of Cystatin F were investigated in whole brain RNA samples from timepoints 100 dpi, 145 dpi and 190 dpi by the more sensitive Quantitative real-time PCR technique to determine whether increased levels of Cystatin F was evident in whole brain RNA at 100 dpi.
  • total RNA from several individual whole brains were pooled into groups of three to form three biological replicates.
  • First-strand cDNA was synthesised and used as template for quantitative real-time PCR. Primer pairs were designed such that an amplicon of approximately 250 bp would be synthesised in the PCR reaction.
  • Example 5 Expression levels of Cvstatin F and SerpinA3 in human Alzheimer's disease and sporadic CJD patients
  • Total RNA was isolated from occipital cortex tissue from eight sporadic CJD (sCJD) patients (3 males, 5 females; Mean age 72.88; sd 2.83), eight Alzheimer's disease patients (5 males, 3 females; Mean age 71.5; sd 2.06) and eight non-neurodegenerative disease patients (4 males, 4 females; Mean age 71.25; sd 11.65). Numbers represent patient identifications.
  • nFirst-strand cDNA was synthesised and used as template for quantitative real-time PCR.
  • Primer pairs were designed such that an amplicon of approximately 250 bp would be synthesised in the PCR reaction.
  • Signal intensity at each PCR cycle was measured (in triplicate) by degree of incorporation and fluoresence of the fluorophore SybrGreen, which is directly proportional to PCR product level. Normalisation was performed relative to ⁇ -actin levels and transcript levels were expressed as arbitrary values for each individual patient sample (Fig. 8). Mean expression levels for each experimental group are also presented. Error bars represent the standard error of the mean of the expression values for each experimental group.
  • SerpinA3 RNA transcript levels are higher in some Alzheimer's disease occipital cortex samples compared to controls although there is no statistically significant difference in average Alzheimer's disease SerpinA3 RNA levels compared to average control Cystatin F RNA levels in occipital cortex samples (Fig. 8C).
  • Example 6 Raising antisera to Cvstatin F and SerpinA3 proteins
  • Representative nucleotide sequences of murine, human and bovine Cystatin F cDNAs are presented herein. Locus link identifications for murine and human Cystatin F are 13011 respectively.
  • the nucleotide sequence presented for bovine has not been officially recognised or assigned as bovine Cystatin F. This has been identified by us as the bovine homologue of murine and human Cystatin F by computer-assisted homology searches.
  • Antisera reactive against human SerpinA3 are currently commercially available, for example via R&D Systems (R&D Systems Europe Ltd., 19 Barton Lane, Abingdon Science Park, Abingdon, Oxon OX14 3NB, United Kingdom).
  • Urine was provided by the Swiss National Reference Centre for Prion Diseases (NRPE) and the UK National CJD Surveillance Unit. An ELISA protocol was established using commercially available anti-human SerpinA3 (alpha-1 antichymotrypsin) polyclonal antibodies (The Binding Site and Dako). Samples provided by the UK CJDSU were coded, studied blind and decoded following the analysis. Urine samples were from 10 healthy subjects (control), 10 patients with Alzheimer's Disease (AD) 1 4 NRPE sporadic CJD (sCJD) patients, 6 UK sCJD patients, 6 UK variant CJD 8 (vCJD) patients and 6 patients with non-CJD pathologies (Fig. 9). Values obtained for SerpinA3 were normalised by Cystatin C levels. The levels of SerpinA3 from the different patient groups were compared among each other as well as with the healthy subjects control.
  • AD Alzheimer's Disease
  • sCJD sporadic CJD
  • the present invention identifies a set of transcripts which are differentially expressed as a result of prion pathogenesis.
  • the marker compounds identified show increased levels during prion pathogenesis and are secreted proteins present in a variety of body fluids.
  • Expression analysis of transcripts coding for the Cystatin F cysteine protease inhibitor member of the Cystatin family of protease inhibitors revealed that increased levels of Cystatin F transcripts are evident in whole brain of prion infected mice at 100 dpi and rise profoundly throughout the remainder of pathogenesis. Differential expression of Cystatin F in spinal cord of prion infected animals is evident between 90 dpi & 100 dpi and by 140 dpi increased expression is also profound in cortex and cerebellum.
  • Cystatin F levels represent the most dramatic gene expression change yet identified in prion pathogenesis.
  • Expression analysis of transcripts coding for the SerpinA 3N serine or cysteine protease inhibitor member of the Serpin family of protease inhibitors revealed that increased levels of SerpinA 3N transcripts are evident in whole brain of prion infected mice between 120 dpi and 130 dpi and rise profoundly throughout the remainder of pathogenesis.
  • Differential expression of SerpinA 3N in spinal cord of prion infected animals is profound by 140 dpi stage, being increased in cerebellum, spinal cord and cortex.
  • PrP Sc accumulation was not detected at 100 dpi in whole brains. This, together with the determination that neither Cystatin F or SerpinA 3N transcript differential expression is evident in spleen or MLN tissues of prion infected mice or in prion-infected neuroblastoma cells (where in all cases there is accumulation of PrP Sc ) strongly supports that the increased levels of these transcripts in prion disease are not simply a result of the presence of PrP Sc .
  • Sequence ID 1 Murine Cystatin F cDNA (Genbank accession number NM_009977; Locuslink accession number 13011)
  • the underlined sequence represents the known open reading frame.
  • Sequence ID 2 Human Cystatin F cDNA (Genbank accession number NM_003650; Locuslink accession number 8530)
  • the underlined sequence represents the known open reading frame.
  • Sequence ID 3 Bovine Cvstatin F cDNA (Identity determined from Genbank accession number CK775683)
  • the underlined sequence represents the predicted open reading frame.
  • Sequence ID 4 Murine SerpinA 3N cDNA cDNA (Genbank accession number NM_009252; Locuslink accession number 20716)
  • the underlined sequence represents the known open reading frame.
  • Sequence ID 5 Human Serpin A3 cDNA (Genbank accession number NM_001085; Locuslink accession number 12)
  • the underlined sequence represents the known open reading frame.
  • Sequence ID 6 Murine Cystatin F (Leukocystatin) pro-protein (Locuslink number 13011 ; Genbank accession number NP_034107)
  • This sequence represents the sequence of the pro-protein prior to cleavage of the N- terminal signal sequence.
  • Sequence ID 7 Human Cystatin F (Leukocystatin) pro-protein (Locuslink number 8530; Genbank accession number NP 003641 )
  • This sequence represents the sequence of the pro-protein prior to cleavage of the N- terminal signal sequence.
  • This sequence represents the sequence of the pro-protein prior to cleavage of the N- terminal signal sequence.
  • Sequence ID 9 Murine SerpinA 3N pro-protein (Genbank accession number NP_033278; Locuslink number 20716)
  • This sequence represents the sequence of the pro-protein prior to cleavage of the N- terminal signal sequence.
  • SequenceID 10 Human SerpinA3pro-protein(Genbankaccession numberNP_001076; Locuslinknumber12)
  • Thissequence representsthesequenceofthepro-proteinpriortocleavageoftheN- terminalsignalsequence.

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Abstract

La présente invention concerne une technique de diagnostic permettant d'identifier un trouble neurodégénératif, en particulier d'identifier une encéphalopathie spongiforme transmissible, une maladie de Parkinson, une maladie de Huntington, une sclérose en plaques ou une maladie d'Alzheimer chez un animal, un échantillon prélevé sur cet animal permettant de déterminer la protéine et/ou le(s) niveau(x) d'ARN de protéine(s) spécifique(s) exprimée(s) de manière différenciée dans des tissus et/ou des fluides anatomiques d'animaux souffrant de troubles neurodégénératifs à des stades précoces de la maladie. Par ailleurs, cette invention concerne un kit d'éléments permettant d'identifier un trouble neurodégénérative chez un animal, comprenant un organe de détection spécifique qui permet de déterminer la protéine et/ou le(s) niveau(x) d'ARN d'un ou de plusieurs des composés susmentionnés.
PCT/CH2005/000360 2004-07-06 2005-06-30 Technique de dosage pour trouble neurodegeneratif chez un animal WO2006002563A2 (fr)

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CN107881178A (zh) * 2017-11-20 2018-04-06 辽宁师范大学 八目鳗口腔腺CystatinF、制备方法及应用
CN111965353A (zh) * 2020-08-18 2020-11-20 四川农业大学 绵羊痒螨组织蛋白酶l的应用以及一种elisa试剂盒
US11214835B1 (en) 2017-06-06 2022-01-04 University Of South Florida Methods and compositions for diagnosis and management of neurodegerative diseases

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11214835B1 (en) 2017-06-06 2022-01-04 University Of South Florida Methods and compositions for diagnosis and management of neurodegerative diseases
CN107881178A (zh) * 2017-11-20 2018-04-06 辽宁师范大学 八目鳗口腔腺CystatinF、制备方法及应用
CN111965353A (zh) * 2020-08-18 2020-11-20 四川农业大学 绵羊痒螨组织蛋白酶l的应用以及一种elisa试剂盒
CN111965353B (zh) * 2020-08-18 2022-08-19 四川农业大学 绵羊痒螨半胱氨酸蛋白酶抑制剂的应用以及一种elisa试剂盒

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