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WO2005052585A1 - Procede de detection de proteines du prion (prpsc) a modification pathologique - Google Patents

Procede de detection de proteines du prion (prpsc) a modification pathologique Download PDF

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Publication number
WO2005052585A1
WO2005052585A1 PCT/DE2004/002563 DE2004002563W WO2005052585A1 WO 2005052585 A1 WO2005052585 A1 WO 2005052585A1 DE 2004002563 W DE2004002563 W DE 2004002563W WO 2005052585 A1 WO2005052585 A1 WO 2005052585A1
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WO
WIPO (PCT)
Prior art keywords
prp
buffer
sample
detergent
concentration
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.)
Ceased
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PCT/DE2004/002563
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German (de)
English (en)
Inventor
Claudia Engemann
Ulrike Krummrei
Jörg Lehmann
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Priontype & Co KG GmbH
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Priontype & Co KG GmbH
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Publication of WO2005052585A1 publication Critical patent/WO2005052585A1/fr
Anticipated expiration legal-status Critical
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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
    • G01N33/68—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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 for the preparation of body fluids as a prerequisite for the detection of pathologically modified prion proteins (PrP c ) in 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 , which forces the normal PrP c on the conformation of the pathological PrP Sc .
  • PrP Sc is characterized by an increased proportion of ⁇ -sheets and a high resistance to proteases (eg proteinase K).
  • This resistance of PrP Sc is currently used in z> zv / tro diagnostics for the detection of bovine spongiform encephalopathy (BSE).
  • BSE 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 breaks down the normal PrP c , 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 detected using monoclonal antibodies using the Western blot or ELIS A method.
  • the decisive disadvantage of these test systems is their 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 tesis and increases the incubation period of the infected organism from at least 18 months to several years ahead. According to the current state of knowledge, the disease is also transferable to humans and, after an incubation period of one to several years, causes the new variant of Creutzfeldt-Jakob disease (nvCJD). The relatively long Incubation time runs the risk that infected people pass on the pathogens, for example, through blood transfusions or organ transplants. This results in a high need for the early diagnosis of TSE.
  • CNS central nervous system
  • aqueous humor is particularly suitable for in-v / vo diagnostics of the TSE, since only a minor veterinary intervention, the puncture of the aqueous humor, is necessary for sample collection.
  • WO 02/066987 does not list any examples for carrying out the method described.
  • the proposed sample preparation proved for detection of PrP sc in mind as unfit and did not lead 'to success.
  • the invention is therefore based on the object of providing a method for the detection of PrP Sc in body fluids which is sensitive enough to detect PrP Sc without the potentially infected organisms having to be killed.
  • Figures la and lb show the need for proteinase K treatment as part of the sample preparation procedure for body fluids.
  • Six CSF and aqueous humor samples from ' BSE-positive and BSE-negative cattle were subjected to the sample preparation procedure with and without Proteinase K treatment.
  • the resulting PrP Sc content was determined in a PrP Sc -specific ELISA.
  • KW means fermentation water.
  • FIG. 2 shows the results of the sample preparation method with and without a heat step during the resolubilization of the sample.
  • 4 CSF and 2 eye water samples from BSE-positive and BSE-negative cattle were tested Subjected to sample preparation procedures.
  • the resolubilized PrP Sc was detected in a PrP Sc -specific ELISA.
  • KW means eye chamber water.
  • BSE-positive cattle animals are referred to as "BSE-positive cattle" in whose brain tissue after mortem proteinase K-resistant PrP S can be detected.
  • Body fluids from BSE-positive cattle were taken from the live animals, stored and used as sample material after post mortem evidence.
  • the present invention relates to a method for the detection of pathologically modified prion proteins (PrP Sc ) in body fluids, comprising the steps
  • the anionic detergent is preferably present in a buffer solution.
  • the anionic detergent can be N-lauroyl sarcosine, sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, sodium cholate or sodium deoxycholate.
  • the anionic detergent is N-lauroyl sarcosine or SDS.
  • the pathologically modified prion protein (PrP Sc ) contained in body fluids is processed. Furthermore, the naturally occurring PrP c is removed from the sample. Due to the sample preparation according to the invention, PrP Sc can be detected in body fluids, for example with an immunological detection using anti-PrP antibodies.
  • the method enables detection with a high sensitivity, so that even the smallest previously undetectable concentrations of PrP Sc in body fluids can be detected. This makes it possible to examine living animals or humans at a very early point in time after infection with the TSE-triggering prions, preferably within the first 6 months, and to classify them as infected if PrP Sc is detected. This has not been possible until now such sensitive tests were not available and, moreover, the evidence could only be carried out post mortem from brain tissue.
  • the body fluids to be examined can come from all organisms that can become infected with prions.
  • the body fluids preferably originate from mammals, in particular from cattle, sheep, goats, mules, red and fallow deer, mice, hamsters or humans.
  • the body fluids to be examined can also be plasma, blood or serum samples which are to be used as preserves for administration to other people or patients.
  • the sample preparation procedure is for all body fluids, e.g. Blood, serum, plasma, tear fluid, aqueous humor, liquor, urine, saliva, lymph, peritoneal fluid or milk are suitable, the process with aqueous humor or liquor being preferred.
  • body fluids e.g. Blood, serum, plasma, tear fluid, aqueous humor, liquor, urine, saliva, lymph, peritoneal fluid or milk are suitable, the process with aqueous humor or liquor being preferred.
  • the isolated sample is treated with a detergent-containing solution.
  • a detergent-containing solution This ensures that PrP Sc is dissociated.
  • An anionic detergent selected from N-lauroyl sarcosine, sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, sodium cholate or sodium deoxycholate, preferably N-lauroyl sarcosine or SDS, in a concentration of at least 0.1%, preferably in the range of 0.1%, is used as the detergent. to 30%, particularly preferably in the range from 10% to 30%, particularly preferably used with a concentration of at least 20%.
  • the detergent-containing solution can contain a buffering substance which preferably buffers in the alkaline range (pH 8.0 to 11.0), such as, for example, TRICIN-NaOH buffer, BICIN-NaOH buffer, glycine / NaOH buffer, Tris- HCl buffer, phosphate buffer or carbonate buffer.
  • a Tris HCl buffer at pH 7.4 or a Na acetate buffer at pH 5.0 can also be used.
  • the anionic detergent is preferably present in a 50 mM glycine / NaOH buffer at pH 9.5.
  • the sample is subjected to a protease treatment after the sample preparation described above.
  • a protease eg Proteinase K.
  • the protease digestion can be carried out under standard conditions for the respective protease or as specified of the manufacturer, preferably at 37 ° C for 10 min.
  • the enzyme concentration used can be in the range from approximately 1 ⁇ g / ml to approximately 200 ⁇ g / ml, preferably in the range from approximately 10 ⁇ g / ml to approximately 100 ⁇ g / ml, particularly preferably approximately 50 ⁇ g / ml enzyme is used.
  • PrP c can then be enriched by first performing a precipitation step with organic solvents and then centrifuging the solution.
  • a mixture of organic solvents such as methanol, ethanol, propanol, butanol, acetone, isopropanol, isoamyl alcohol, is used to precipitate PrP Sc , preferably isoamyl alcohol plus acetone in a ratio of 70:30.
  • the precipitation can also be carried out with the individual solvents listed, preferably with butanol.
  • the sample solution is mixed with the solvent or the solvent mixture in a ratio of 2: 1.
  • the suspension of sample solution and detergent-containing solution is clarified by vigorous mixing. This is necessary in order to be able to sediment PrP Sc by centrifugation.
  • the centrifugation takes place for 2 to 20 min at 2000 to 22000 xg and 0 to 37 ° C, preferably for 5 min at 16000 xg and 4 ° C.
  • Non-ionic or zwitterionic detergents are not suitable for this.
  • the concentration of the anionic detergent should be at least 20% so that the precipitation step can be carried out.
  • the concentration of the anionic detergent is preferably at least 20% in step a), but can also be increased to at least 20% after this step.
  • the buffer used contains a detergent, a chaotropic reagent and a chelating agent.
  • the following buffer is preferably used: 50 mM Tris, pH 7.4, 8 M urea, 2% CHAPS, 10 mM EDTA.
  • the selection of the substances contained in the buffer is selected such that aggregation of PrP Sc is prevented in order to get the maximum amount of PrP Sc back into solution.
  • the fricubation of the sample is carried out at 100 ° C for at least 2 min and a maximum of 20 min.
  • the PrP Sc prepared in the previous method steps is then detected in any method.
  • 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 present invention further relates to a kit for the preparation / isolation of PrP Sc from body fluids.
  • the preparation kit contains the required solutions and enzymes.
  • Example 1 Composition of the detergent-containing buffer solution
  • the detergent-containing buffer solution (hereinafter referred to as denaturing buffer) has the function of dissociating PrP Sc from masking proteins of the respective body fluid.
  • the denaturation buffer should keep as many foreign proteins in solution as possible in the subsequent PrP Sc precipitation step in order to achieve an effective enrichment of the PrP Sc .
  • the efficiency of the denaturation buffer is shown after the addition of a protein precipitation reagent. If the sample solution is then clear, apart from the PrP Sc, almost all sample components have remained in solution. Consequently, only PrP Sc can be sedimented by subsequent centrifugation and thus enriched. If the sample solution remains or becomes cloudy after adding the precipitation reagent, this means that a large part of the foreign components contained in the sample could not be kept in solution and thus sedimented together with the PrP c during centrifugation.
  • the suitability of the various solutions as a denaturing buffer was therefore assessed using turbidity measurements.
  • the same volume of denaturing buffer was added to each 100 ⁇ l sample (liquor, aqueous humor and plasma from cattle) and mixed thoroughly.
  • 100 ⁇ l of precipitation reagent either isoamyl alcohol plus acetone, 7: 3, or butanol
  • the turbidity of 100 ⁇ l of the denatured samples was determined photometrically at 620 nm in microtiter plates (Tecan Sunrise, Tecan, Switzerland). The results are summarized in Table 1.
  • Lauroyl sarcosine or SDS in high concentrations are most suitable. This effect is independent of the pH of the solution. Buffering the solution is therefore not necessary.
  • one is alkaline buffered solution with 20% N-lauroyl sarcosine (50 mM glycine pH 9.5, 20% N-lauroyl sarcosine) or 20% SDS (50 mM glycine pH 9.5, 20% SDS) is most suitable.
  • Table 1 also shows that detergents other than anionic are sometimes also suitable, but not in all body fluids.
  • Denaturation buffers which result in a turbidity of OD 62 o ⁇ 0.07, are considered suitable in the corresponding sample, with OD 62 o 0.07 to 0.15 as conditionally suitable and with OD 620 > 0.15 as unsuitable.
  • SDS sodium dodecyl sulfate
  • NDSP non detergent sulfobetaine
  • Example 2 Composition of the precipitation reagent
  • TCA trichloroacetic acid
  • PrP Sc is present in body fluids masked by other proteins. Therefore, PrP Sc cannot be detected in untreated body fluids such as CSF, plasma and aqueous humor from BSE-positive cattle.
  • a protease treatment eg proteinase K
  • 6 BSE-positive and BSE-negative CSF and eye aqueous humor samples were mixed with denaturing buffer (50 mM glycine pH 9.5, 20% N-lauroylsarcosine) and for 10 min at 37 ° C with proteinase K (50 ⁇ g / ml) incubated.
  • denaturing buffer 50 mM glycine pH 9.5, 20% N-lauroylsarcosine
  • proteinase K 50 ⁇ g / ml
  • the reaction was stopped by protein precipitation with an isoamyl alcohol-acetone mixture (70:30).
  • the precipitated protein was sedimented by centrifugation for 5 min at 16000 xg and 4 ° C. The supernatant was discarded.
  • the pellet obtained was resolubilized in 50 mM Tris pH 7.4, 8 M urea, 2% CHAPS, 10 mM EDTA.
  • PrP Sc contained in the sample was detected as follows:
  • An MTP (Nunc-Irnmuno TM Plate Maxisorp TM Surface, F96 (Nunc, Roskilde, Denmark) was coated with the peptide KLVFF.
  • 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.
  • PBS wash buffer
  • the coated MTP was incubated with 100 ⁇ l per cavity of the sample containing PrP So for 1 h at room temperature covered with foil. Unbound sample material was aspirated and the MTP was washed three times with 300 ⁇ l wash buffer per cavity. Incubation with the detection antibody (2 ⁇ g / ml F89, Sigma-Alderich, Taufkirchen, Germany ⁇ ) was carried out for 1 h at room temperature.
  • the plate was then washed again three times with 300 ⁇ l washing buffer and for 1 h at room temperature with a goat anti-mouse IgG-peroxidase conjugate (1: 20000 in wash buffer, Jackson, USA), excess conjugate was removed by washing five times with 300 ⁇ l wash buffer per cavity, and after addition of the substrate solution (TMB) the color development was stopped after 15 min by adding 1 MH 2 SO stopped and the color intensity registered by absorbance measurement at 450 nm (reference 620 nm) The measured absorbance is proportional to the amount of PrP Sc bound to the peptides. From Fig. La and lb it can be seen that the PrP Sc only in Proteinase K-treated samples can be safely detected.
  • Example 4 Determination of the optimal composition of the resolubilization buffer
  • the need for the heat step was then checked. For this purpose, 4 BSE-positive and 4 BSE-negative CSF samples, as well as 2 BSE-positive and 2 BSE-negative eye chamber water samples were prepared as described above. After adding the resolubilization buffer, the samples were divided. One part was subjected to the heat step (5 min at 100 ° C.) and then analyzed, while the other part was analyzed immediately. Figure 2 illustrates the need for the heat step to completely resolubilize the PrP Sc . Without heating, approximately 80% of the signal is lost. Table 3: Comparison of different resolubilization buffers

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Abstract

L'invention concerne un procédé de préparation de liquides corporels pour la détection de protéines du prion (PrP<sc>) à modification pathologique dans des organismes vivants.
PCT/DE2004/002563 2003-11-20 2004-11-19 Procede de detection de proteines du prion (prpsc) a modification pathologique Ceased WO2005052585A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2003154207 DE10354207B8 (de) 2003-11-20 2003-11-20 Verfahren zum Nachweis von pathologisch veränderten Prion-Proteinen (PrPSc) und Kit zur Durchführung des Verfahrens
DE10354207.8 2003-11-20

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WO2005052585A1 true WO2005052585A1 (fr) 2005-06-09

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AR (1) AR047042A1 (fr)
DE (1) DE10354207B8 (fr)
WO (1) WO2005052585A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008122274A1 (fr) * 2007-04-04 2008-10-16 Priontype Gmbh & Co. Kg Méthode de décèlement de la protéine prion pathologiquement modifiée (prpsc)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033420A2 (fr) * 2000-10-22 2002-04-25 Hadasit Medical Research Services And Development Ltd. Analyse d'urine servant a diagnostiquer des maladies a prion
WO2002057789A2 (fr) * 2001-01-19 2002-07-25 Enfer Technology Limited Test pour encephalopathies spongiformes transmissibles
WO2005024432A1 (fr) * 2003-09-04 2005-03-17 Cedi Diagnostics B.V. Procedes et kits de detection des maladies a prions

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US6150172A (en) * 1999-01-08 2000-11-21 The United States Of America As Represented By The Secretary Of Agriculture Method and kit for extracting prion protein
WO2003001211A1 (fr) * 2001-06-22 2003-01-03 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede de determination de la presence de proteines prions dans le tissu et echantillons de culture
ES2564293T5 (es) * 2002-02-28 2019-04-04 Microsens Biophage Ltd Enlazamiento de formas patológicas de proteínas priónicas

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033420A2 (fr) * 2000-10-22 2002-04-25 Hadasit Medical Research Services And Development Ltd. Analyse d'urine servant a diagnostiquer des maladies a prion
WO2002057789A2 (fr) * 2001-01-19 2002-07-25 Enfer Technology Limited Test pour encephalopathies spongiformes transmissibles
WO2005024432A1 (fr) * 2003-09-04 2005-03-17 Cedi Diagnostics B.V. Procedes et kits de detection des maladies a prions

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
TZABAN SALIT ET AL: "Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes", BIOCHEMISTRY, vol. 41, no. 42, October 2002 (2002-10-01), pages 12868 - 12875, XP002324410, ISSN: 0006-2960 *
WONG B -S ET AL: "Mapping the antigenicity of copper-treated cellular prion protein with the scrapie isoform.", CMLS CELLULAR AND MOLECULAR LIFE SCIENCES, vol. 60, no. 6, June 2003 (2003-06-01), pages 1224 - 1234, XP008045531, ISSN: 1420-682X *
XIONG LIANG-WEN ET AL: "Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein", JOURNAL OF NEUROCHEMISTRY, vol. 79, no. 3, November 2001 (2001-11-01), pages 669 - 678, XP002252786, ISSN: 0022-3042 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008122274A1 (fr) * 2007-04-04 2008-10-16 Priontype Gmbh & Co. Kg Méthode de décèlement de la protéine prion pathologiquement modifiée (prpsc)

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DE10354207B8 (de) 2006-06-14
DE10354207B3 (de) 2005-08-18

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