EP1520178A2 - Procede pour enrichir et pour depister des proteines prions pathologiques modifiees (prp sp sc /sp ) - Google Patents

Procede pour enrichir et pour depister des proteines prions pathologiques modifiees (prp sp sc /sp )

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Publication number
EP1520178A2
EP1520178A2 EP03762453A EP03762453A EP1520178A2 EP 1520178 A2 EP1520178 A2 EP 1520178A2 EP 03762453 A EP03762453 A EP 03762453A EP 03762453 A EP03762453 A EP 03762453A EP 1520178 A2 EP1520178 A2 EP 1520178A2
Authority
EP
European Patent Office
Prior art keywords
prp
detection
sheet
solid support
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03762453A
Other languages
German (de)
English (en)
Inventor
Claudia Engemann
Katja Hoeschler
Jörg Lehmann
Jörg GABERT
Ulrike Krummrei
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Priontype & Co KG GmbH
Schleussner Cathrin
Original Assignee
Priontype & Co KG GmbH
Schleussner Cathrin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Priontype & Co KG GmbH, Schleussner Cathrin filed Critical Priontype & Co KG GmbH
Publication of EP1520178A2 publication Critical patent/EP1520178A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/968—Plasmin, i.e. fibrinolysin
    • 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 methods for the detection of pathologically modified prion proteins (PrP Sc ) from living organisms.
  • TSEs Transmissible spongiform encephalopathies
  • PrP Sc pathologically modified prion protein
  • PrP c a conformer of the naturally occurring cellular prion protein
  • the prion replication that occurs in the course of the disease takes place through direct interaction between PrP Sc and PrP c , whereby the conformation of the pathological PrP Sc is forced on the normal PrP c .
  • PrP Sc is characterized by an increased proportion of ⁇ -sheets and a high resistance to proteases (eg proteinase K).
  • PrP Sc bovine spongiform encephalopathy
  • the principle of the current test systems is that tissue parts of the brain stem (Obex region) are homogenized and treated with Proteinase K. The protease treatment completely degrades the normal PrP, while PrP Sc from BSE-infected animals is only shortened by a few amino acids, which results in a reduction in the molecular weight from 33-35 kDa to 27-30 kDa. The remaining PrP is then visualized using monoclonal antibodies in a Western blot or ELISA.
  • the main disadvantage of this test system is its low sensitivity. Since the PrP Sc concentration in BSE-infected cattle for the current test systems is only sufficiently high in the central nervous system (CNS), the previous diagnosis is limited to post-mortem tests and increases the incubation period of the infected organism from at least 18 months to several years ahead.
  • CNS central nervous system
  • the diagnosis is also made p ost mortem here, since it is not possible to remove the necessary brain material from the living organism.
  • the bioassay is able to detect a single infectious unit, but it takes at least several months or even years.
  • the sensitivity of the previous detection methods must be increased significantly and the detection in tissues / body fluids other than the CNS must be made possible.
  • the specific binding of PrP Sc to human plasminogen is used to isolate PrP Sc from CNS material.
  • human plasminogen is immobilized on magnetic particles.
  • this method is only suitable for the detection of PrP Sc in those body fluids and tissues that do not contain plasminogen and is unsuitable, for example, for the detection of PrP S ⁇ in serum. Further disadvantages of this method are the high price and the limited protein binding capacity of the plasminogen-loaded magnetic particles used therein.
  • the invention is therefore based on the object of providing a method which has an increased sensitivity and enables diagnosis of TSE from living organisms.
  • the object is achieved by the subject-matter defined in the patent claims.
  • FIG. 1 shows a graphic representation of the principle of a method according to the invention for the enrichment and detection of PrP Sc using solid-phase-coupled ⁇ -sheet-binding molecules.
  • FIG. 2 schematically shows the structure of the PrP Sc binding assay in microtiter plate (MTP) format (example 1).
  • MTP microtiter plate
  • BLB ⁇ -sheet-breaker
  • FIG. 4 shows an evaluation of the detection of PrP Sc in aqueous humor and CSF samples from BSE-positive cattle.
  • the peptide KLVFF was used in ELISA format as a capture molecule for PrP Sc .
  • Detection was carried out with the anti-PrP Sc monoclonal antibody V5B2 (r-Biopharm, Darmstadt) and a polyclonal peroxidase-conjugated goat anti-mouse IgG antibody.
  • ⁇ -sheet binding molecule describes an organic molecule which, owing to its three-dimensional structure and / or its physical properties, is capable of interacting with ⁇ -sheet structures in proteins, for example in pathological terms modified monomeric / oligomeric prion proteins to occur and bind them due to the interactions.
  • Exemplary ⁇ -sheet binding molecules are listed in SEQ ID NO: 1 to 10.
  • ß-sheet-breaker (BSB) describes short peptides which not only bind to ß-sheet structures of ß-amyloid (protein aggregates in Alzheimer's disease) and amyloid-like structures, but also above them can also block or undo their abnormal folding.
  • pathologically modified prion protein refers to PrP Sc .
  • PrP Sc can exist both in monomeric and or oligomeric form and in fibrillar, amyloid aggregate form.
  • BSE-positive cattle are those animals in whose stem-brain tissue post-mortem proteinase K-resistant PrP Sc can be detected.
  • the present invention relates to a method for the detection of pathologically modified prion proteins (PrP Sc ), comprising the following steps: a) incubating a sample with a solid support, the solid support being coupled to a ⁇ -sheet-binding molecule, b ) Removing the sample components not bound to the ⁇ -sheet-binding molecules, and c) detecting the pathologically modified prion proteins (PrP Sc ) bound to the ⁇ -sheet-binding molecules.
  • the monomeric and / or oligomeric pathologically modified prion proteins contained in body fluids, cell lysates or body tissues are enriched, so that even the smallest previously undetectable concentrations of PrP Sc can be detected.
  • This makes it possible to classify living animals or humans as infected shortly after infection with TSE-triggering prions. Until now, this was not possible because such sensitive tests were not available and, moreover, the evidence could only be carried out post mortem from brain tissue, since the concentration of PrP Sc is high enough there.
  • the method can deliver results in the body tissues, eg brain homogenates, at a much earlier point in time after an infection, which with the current detection methods only show results when the infection has already progressed significantly.
  • the sample to be examined can be a body fluid, for example blood, serum, plasma, CSF, aqueous humor, tear fluid, urine, saliva, lymph, milk, or a cell lysate, for example from leukocytes or from cells of the lymphoid tissue, or a tissue homogenate, for example from central nervous system tissues, from lymphatic tissues (e.g. spleen, tonsils, lymph nodes) or other organs.
  • the sample Before the incubation, the sample can optionally be subjected to sample preparation. This may be necessary for tissue samples in particular. After adding a suitable buffer solution, e.g. 50 mM phosphate buffer, pH 7.5, mechanically comminuted, e.g. by ultrasound or ribolyser treatments, and homogenized to get their components in solution or suspension.
  • a suitable buffer solution e.g. 50 mM phosphate buffer, pH 7.5
  • mechanically comminuted e.g. by ultrasound or ribolyser treatments, and homogenized to get their components in solution or suspension.
  • the sample can also be subjected to a centrifugation and / or filtration step to separate the solid components obtained by mechanical treatment of the tissues or cell suspensions or those present in body fluids.
  • the sample with or without sample preparation described above can additionally or exclusively be subjected to a proteinase treatment for the proteolytic degradation of PrP c before the incubation.
  • a proteinase treatment for the proteolytic degradation of PrP c before the incubation.
  • the PrP Sc to be detected for example the monomeric and or oligomeric form of PrP Sc
  • the sample material is treated with a protease, eg Proteinase K.
  • the protease digestion can be carried out under standard conditions for the respective protease or according to the manufacturer, preferably at 37 ° C. for 1 h.
  • the enzyme concentration used can be in the range from approximately 10 ⁇ g / ml to approximately 1 mg / ml, preferably approximately 50 ⁇ g / ml enzyme is used with a protein content of the sample material of approximately 0.5 to approximately 10 mg / ml.
  • Solid supports can be spherical polymers (eg Sepharose, agarose or latex), plastic surfaces (eg microtiter plates), silica gel-coated glass plates (eg for thin layer chromatography), capillaries or membranes.
  • the spherical polymers can be used as supports in column chromatography or in a batch process (for example magnetic beads). If the polymers are used for column chromatography, they are preferably used in prepacked disposable columns. Besides those here Any solid support that can be used for coupling ⁇ -sheet-binding molecules is also suitable.
  • the sample can be incubated in a closed vessel for about 5 to about 120 minutes at a temperature in the range from about 4 ° C. to about 50 ° C. with the solid support, for example glass plates, microtiter plates.
  • the incubation is preferably carried out at 37 ° C. for 1 h in a shaking incubator with a low rotation frequency (eg 80 rpm).
  • a low rotation frequency eg 80 rpm.
  • the PrP Sc contained in the sample is bound to the ⁇ -sheet binding molecule immobilized on the solid support.
  • a spherical polymer is used as the solid support in a column chromatography, the incubation takes place in the column.
  • the incubation time can vary depending on the column, depending on the connection of the column to an apparatus and the flow rate of the sample.
  • the ß-sheet-binding molecules coupled to the solid support bind PrP So with a much higher affinity than PrP c and, according to the invention, are able to capture the soluble and monomeric or oligomeric form of PrP Sc that occurs in body fluids.
  • the ⁇ -sheet-binding molecules are oligopeptides consisting of 3 to about 30 amino acids, preferably 4, 5 or 6 amino acids. These peptides can be modified at the C- and / or N-terminal, for example in order to achieve better solubility.
  • the ß-sheet-binding molecules can also have the properties of the ß-sheet breaker.
  • BSB properties also have the .beta.-sheet binding molecules according to the invention, however, such binding properties (affinity, reversibility of the bond) to PrP Sc, which enable the capture and enrichment of PrP Sc from solutions.
  • BOD peptides that bind the ß-sheet structures too tightly or irreversibly so that elution is not possible are unsuitable as ß-sheet-binding molecules.
  • BODs that bind PrP Sc with too little affinity or do not bind them permanently eg detachment of the PrP Sc from the BOD after the ⁇ -sheet structure has been broken open.
  • Particularly preferred ⁇ -sheet-binding molecules are listed in Table 1 and as SEQ ID NO: 1 to 10.
  • the ⁇ -sheet-binding molecule can also be a substituted heterocyclic aromatic, advantageously a flavonoid, for example thioflavin T, baicalin or quercitrin.
  • the ⁇ -sheet-binding molecules are preferably immobilized on the solid support via a covalent bond.
  • Functional for coupling to the carrier Groups such as amino, carboxyl or hydroxyl groups are used on the ß-sheet binding molecule. If the ⁇ -sheet-binding molecule is a peptide, the coupling is preferably carried out via the amino group at the N-terminus or the carboxyl group at the C-terminus.
  • the oligopeptide is the pentapeptide with the sequence KLVFF (SEQ ID NO: 2), it is preferably coupled via the carboxyl group at the C-terminus, since when coupled via the amino group the peptide is also fixed to the side chain of the lysine residue would lead to steric hindrance of the PrP Sc bond.
  • a buffered solution with suitable, stringency-increasing additives serves as the washing solution.
  • the pH of the wash solution is in the neutral range, preferably at pH 7.5. 50 mM phosphate buffer is preferably used to buffer the solution. In addition, any buffer with which a pH value in the neutral range can be set is suitable.
  • the stringency enhancing additives can be inorganic salts, e.g. NaCl, as well as detergents, e.g. SDS, Triton X 100 or Tween 20, or chaotropic reagents, e.g. Urea, guanidinium hydrochloride or guanidinium isothiocyanate.
  • a buffered solution with 1 to 4 M NaCl is advantageously used as the washing solution.
  • the PrP Sc bound to the ⁇ -sheet-binding molecules may be eluted from the solid carrier (for example when using spherical polymers in column chromatography). With other supports, such as membranes or plastic surfaces, PrP Sc can be detected directly on the solid support. If desired, however, elution can also be carried out here.
  • the support is rinsed with the smallest possible volume of elution solution.
  • the elution volume is many times smaller than the sample volume.
  • a buffered solution containing additives which break the bond between PrP Sc and the capture molecule is used as the elution solution.
  • the pH of the elution solution is in the range from about pH 6 to about pH 8.5, preferably at pH 7.5. 50 mM phosphate buffer is preferably used to buffer the solution. In addition, any buffer with which a pH in the above described, preferably in the neutral range, can be adjusted.
  • the additives can be, for example, detergents, for example SDS, Triton X 100 or Tween 20, chaotropic reagents, for example urea, guanidinium hydrochloride or guanidinium isothiocyanate, inorganic salts, for example NaCl.
  • the elution solution preferably contains detergents, for example 5% SDS.
  • the PrP Sc enriched in the previous process steps is detected.
  • immunochemical e.g. ELISA, Western blot, immunoprecipitation
  • biophysical e.g. mass spectrometry, fluorescence correlation spectroscopy
  • biochemical e.g. determination of biochemical parameters such as relative molecular weight, N-terminal or C-terminal amino acid sequence, association and Dissociation constants of binding partners
  • biological e.g. cytotoxicity assay
  • the detection is preferably carried out using a method which enables rapid detection of PrP Sc .
  • This can be, for example, an immunological detection method, preferably a sandwich ELISA.
  • the sandwich ELISA is carried out according to a known method.
  • the labeling of the detection antibody can be, for example, an enzyme (for example horseradish peroxidase), a colored compound, a fluorescent dye (for example fluorescein), a gold particle or a nucleic acid (for example a DNA or RNA oligonucleotide).
  • the color intensity is measured photometrically after substrate conversion and is proportional to the amount of PrP Sc contained in the sample. If the antibody label is a colored compound or a fluorescent dye, the color or Fluorescence intensity measured directly. If the antibody label is a nucleic acid, the amount of bound antibody is determined via the absorption of the DNA or RNA label, the signal being amplified by PCR (eg real-time PCR).
  • the present invention further relates to a kit for the detection of PrP Sc in body fluids, cell lysates, tissue homogenates or other fluids.
  • the test kit contains a solid carrier for the enrichment of PrP Sc , an immunological detection system, solubilization, washing and elution buffer concentrates, various controls, an enzyme-labeled anti-PrP antibody and a corresponding substrate and stop solution.
  • the solid supports used are preferably affinity chromatography materials, for example Sepharose, which are used in single-use columns, or plastic surfaces, for example microtiter plates, which are coupled to the ⁇ -sheet-binding molecules according to the invention. If the solid supports are affinity-chromatographic materials with the couplings according to the invention, these can be contained in the test kit as a suspension, in dried form or already packed in disposable columns.
  • the immunological detection system is preferably a sandwich ELISA in which a second solid support, e.g. a microtiter plate is coated with a specific antibody against PrP, preferably with monoclonal anti-PrP antibodies, in particular mouse anti-PrP antibodies.
  • the solid supports are included in the test kit, especially in vacuum packaging.
  • PrP or PrP peptides are preferably used as controls.
  • Horseradish peroxidase is preferably used as the antibody label.
  • the methods and test kits according to the invention enable broad-based investigations with large numbers of samples, as are required in the fields of medicine and agriculture. Automation of the detection process is possible in appropriately equipped laboratories. In contrast to all the methods described so far, the methods according to the invention are also suitable for TSE diagnostics on live animals and humans.
  • Example 1 Isolation of PrP Sc from brain homogenate using various peptides in MTP format
  • An MTP (microtiter plate) (Nunc-Immuno TM Plate Maxisorp TM Surface, F96 (Nunc, Roskilde, Denmark)) was coated with the peptides listed in Table 1 (FIG. 2). The coating was carried out by incubation with 100 ⁇ l peptide solution (10 ⁇ g / ml in 0.1 M carbonate buffer pH 9.6) per cavity for 16 h at 4 ° C. The liquid was then suctioned off and the MTP was washed three times with 300 ⁇ l wash buffer (PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.2); 0.05% Tween 20)) per cavity.
  • PBS 10 mM phosphate buffer, 0.15 M NaCl, pH 7.2
  • Tween 20 300 ⁇ l wash buffer
  • the absorbance measured is proportional to the amount of PrP Sc bound to the peptides and is therefore a measure of the efficiency of the capture molecule.
  • the relative binding efficiencies of the peptides tested are summarized in Tab. 1, the signal of the best ⁇ -sheet binding molecule (peptide 2) being set to 100%.
  • Peptide 2 Lys-Leu-Val-Phe-Phe 00.0 SEQ ID NO: 2
  • Peptide 7 Propionyl-D Arg-D Arg-D Ala-DPhe-DPhe-DVal-amide 76.5 SEQ ID NO: 7
  • PrP Sc The bond between PrP Sc and the capture molecules is very strong, so that for the elution of P PrrPP S cc vvoonn ddeemm ffeesstteenn TTrrääggeerr vveerrgglleeiicchhsswweeiissee ddrraassttiisschchhee. B 'CONDITIONS needed are.
  • the elution conditions were also tested in MTP format. Analogously to Example 1 a MTP with peptide 2 (KLVFF) was coated and loaded with PrP Sc -containing brain homogenate (OD in the Platelia ®> 3.0).
  • PrP Sc was only partially eluted in the presence of chaotropic reagents (eg 6 M urea). In elution buffers with low pH (eg pH 3) or high ionic strength (eg 2 M NaCl), PrP Sc remained almost completely bound to the capture molecule.
  • chaotropic reagents eg 6 M urea
  • PrP Sc remained almost completely bound to the capture molecule.
  • the peptide When coupled via amino groups, the peptide would be fixed both at the N-terminus and on the side chain (Lys), which could lead to a disruption of the three-dimensional structure. For this reason, the ⁇ -sheet-binding molecule KLVFF was specifically bound to the solid support via the carboxyl group at the C-terminus of the peptide.
  • EAH-Sepharose ® 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) served as the carrier material.
  • the EAH-Sepharose was washed with 0.5 M NaCl and the supernatant liquid was completely removed.
  • the ligand the pentapeptide with the sequence KLVFF, was dissolved in HO to a final concentration of 5 mg / ml and the pH was adjusted to 4.5 with HC1.
  • the gel was resuspended in the ligand solution (1 part gel + 2 parts ligand solution) and EDC (N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide) in one Final concentration of 0.1 M was added.
  • EDC N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide
  • the KLVFF-loaded gel was filled into chromatography columns (bed volume 1 ml) and at least three times alternately with 3x 2 ml buffer A (0.1 M Na acetate, 0.5 M NaCl, pH 4) and 3x 2 ml buffer B ( 0.1 M Tris HCl, 0.5 M NaCl, pH 8) and then washed with 10 x 2 ml H 2 O.
  • 3x 2 ml buffer A 0.1 M Na acetate, 0.5 M NaCl, pH 4
  • 3x 2 ml buffer B 0.1 M Tris HCl, 0.5 M NaCl, pH 8
  • PrP Sc from brain homogenate from BSE-positive cattle was bound to the column material and then eluted again.
  • the brain material was processed using the BSE Purification Kit (Bio-Rad Laboratories, Hercules, USA) according to the manufacturer's instructions.
  • the sample material was also taken up in sample digestion buffer R6 (Platelia ® BSE Detection Kit, Bio-Rad Laboratories, Hercules, USA) (OD in Platelia ® 6.0) according to the manufacturer's instructions.
  • a dropping column was made with 1 ml of KLVFF-Sepharose, as indicated in Example 3, filled and equilibrated with PBS at room temperature.
  • FIG. 3 shows the elution profile of this experiment and documents the ability of the KLVFF-Sepharose to reversibly bind PrP Sc and thus the suitability of the matrix for the selective enrichment of PrP Sc from large sample volumes.
  • the PrP Sc contained in the run is due here to an overload of the column capacity, since the brain sample used here had a very high PrP Sc content (OD in the Platelia ® 6.0).
  • Example 5 Isolation of PrP Sc from Body Fluids Using KLVFF in MTP Format (Priontype TM In Vivo BSE Test)
  • An MTP (Nunc-Immuno TM Plate Maxisorp TM Surface, F96 (Nunc, Roskilde, Denmark) was coated with the peptide KLVFF (Fig. 2).
  • the coating was carried out by incubation with 100 ⁇ l peptide solution (10 ⁇ g / ml in 0.1 M carbonate buffer pH 9.6) per cavity for 16 h at 4 ° C.
  • the liquid was then suctioned off and the MTP was washed three times with 300 ⁇ l wash buffer (PBS; 0.05% Tween 20; pH 7.2) per cavity. Free binding sites were blocked by incubation with 0.5% casein in wash buffer at room temperature for 1 h.
  • the coated MTP was incubated with 100 ⁇ l per cavity of the sample containing PrP Sc for 1 h at room temperature covered with foil.
  • eye chamber water samples from 9 BSE-positive and 5-negative cattle as well as CSF samples from 6 BSE-positive and 13 BSE-negative cattle were examined. All samples were previously prepared using the BSE Purification Kit (Bio-Rad Laboratories, Hercules, USA). Unbound sample material was aspirated and the MTP was washed three times with 300 ⁇ l wash buffer per cavity.
  • the absorbance measured is proportional to the amount of PrP c bound to the peptides.
  • the values obtained in the BSE-positive versus BSE-negative animals are significantly increased (t-test).
  • the differences between positive and negative samples are much more pronounced in the aqueous humor samples than in the cerebrospinal fluid.
  • the reason for this is the PrP Sc concentration found in the corresponding body fluids. Based on these results, the aqueous humor is preferred over the CSF as sample material for the detection of PrP Sc in body fluids from living animals.

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Abstract

L'invention concerne un procédé pour dépister des protéines prions pathologiques modifiées (PrP<Sc>) d'organismes vivants.
EP03762453A 2002-07-04 2003-07-04 Procede pour enrichir et pour depister des proteines prions pathologiques modifiees (prp sp sc /sp ) Withdrawn EP1520178A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10230141A DE10230141B4 (de) 2002-07-04 2002-07-04 Verfahren und Kit zur Anreicherung und zum Nachweis von veränderten Prion-Proteinen (PrPSc)
DE10230141 2002-07-04
PCT/DE2003/002249 WO2004005920A2 (fr) 2002-07-04 2003-07-04 Procede pour enrichir et pour depister des proteines prions pathologiques modifiees (prpsc)

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EP1520178A2 true EP1520178A2 (fr) 2005-04-06

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EP03762453A Withdrawn EP1520178A2 (fr) 2002-07-04 2003-07-04 Procede pour enrichir et pour depister des proteines prions pathologiques modifiees (prp sp sc /sp )

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US (1) US20060094071A1 (fr)
EP (1) EP1520178A2 (fr)
JP (1) JP2005531775A (fr)
CN (1) CN1666106A (fr)
AU (1) AU2003250773A1 (fr)
BR (1) BR0312282A (fr)
CA (1) CA2489151A1 (fr)
DE (2) DE10230141B4 (fr)
EA (1) EA200500125A1 (fr)
NO (1) NO20050599L (fr)
PL (1) PL375358A1 (fr)
WO (1) WO2004005920A2 (fr)

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US20060035242A1 (en) * 2004-08-13 2006-02-16 Michelitsch Melissa D Prion-specific peptide reagents
US20090130774A1 (en) * 2005-01-13 2009-05-21 David Peretz Elisa assays using prion-specific peptide reagents
WO2006076497A2 (fr) * 2005-01-13 2006-07-20 Novartis Vaccines And Diagnostics Inc. Isolation de prions pathogeniques
WO2006076683A2 (fr) * 2005-01-13 2006-07-20 Novartis Vaccines And Diagnostics Inc. Isolement et detection de prions pathogenes
EP1931695B1 (fr) * 2005-09-09 2013-04-10 Novartis AG Reactifs peptoides specifiques des prions
WO2007123345A1 (fr) * 2006-04-21 2007-11-01 Peoplebio, Inc. Procédé de détection différentielle d'une forme multimère et d'une forme monomère dans des polypeptides formant des multimères par interactions tridimensionnelles
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CA2489151A1 (fr) 2004-01-15
NO20050599L (no) 2005-04-01
DE10393421D2 (de) 2005-06-09
DE10230141B4 (de) 2004-07-15
DE10230141A1 (de) 2004-02-05
BR0312282A (pt) 2005-04-12
PL375358A1 (en) 2005-11-28
AU2003250773A1 (en) 2004-01-23
CN1666106A (zh) 2005-09-07
WO2004005920A3 (fr) 2004-04-29
WO2004005920A2 (fr) 2004-01-15
US20060094071A1 (en) 2006-05-04
JP2005531775A (ja) 2005-10-20

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