WO2013056841A1 - Procédé de diagnostic de l'encéphalopathie spongiforme transmissible - Google Patents

Procédé de diagnostic de l'encéphalopathie spongiforme transmissible Download PDF

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WO2013056841A1
WO2013056841A1 PCT/EP2012/004377 EP2012004377W WO2013056841A1 WO 2013056841 A1 WO2013056841 A1 WO 2013056841A1 EP 2012004377 W EP2012004377 W EP 2012004377W WO 2013056841 A1 WO2013056841 A1 WO 2013056841A1
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prp
protein
reaction mixture
tse
caprinae
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PCT/EP2012/004377
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Olivier ANDREOLETTI
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Inra (Institut National De La Recherche Agronomique)
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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
    • 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 Transmissible Spongiform Encephalopathies (TSE), more specifically to the diagnosis of a TSE that is not scrapie.
  • TSE Transmissible Spongiform Encephalopathies
  • Prion diseases which are also called Transmissible Spongiform Encephalopathies (TSEs)
  • TSEs Transmissible Spongiform Encephalopathies
  • CJD Creutzfeldt- Jakob disease
  • GSS Gerstmann-Strauss!er Sheinker syndrome
  • FFI fatal familial insomnia
  • sFI sporadic fatal insomnia
  • BSE bovine spongiform encephalopathy
  • CWD chronic wasting disease
  • PrP Sc a misfolded protein
  • PrP c an host normal protein
  • PrP c is soluble in non-denaturing detergents, PrP Sc is insoluble; PrP c is readily digested by proteases, while PrP Sc is partially resistant, resulting in the formation of a N-terminally truncated fragment.
  • PrP Sc is not only the most likely cause of the disease, but also is the best known marker. Detection of PrP Sc in tissues and cells correlates widely with the disease and with the presence of TSE infectivity.
  • the present invention is based on the discovery by the present inventors that
  • the present invention relates to a method for diagnosing a Transmissible Spongiform encephalopathy (TSE) in a subject, wherein said subject is not a caprinae and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), and wherein said method comprises the steps of:
  • said caprinae PrP protein isoform having a glutamine amino acid residue at its position 171 is an ovine or a caprine PrP c protein isoform having a glutamine amino acid residue at its position 171.
  • the ovine PrP c protein isoform having a glutamine amino acid residue at its position 171 is selected in the group comprising the ARQ Ovis aries PrP c protein isoform having the sequence SEQ id n° l (Accession number CAE00186), the VRQ Ovis aries PrP c protein isoform having the sequence SEQ id n°2 (Accession number CAE00190) and the AHQ Ovis aries PrP c protein isoform having the sequence SEQ id n°3 (Accession number ABG78479).
  • said ovine PrP c protein isoform is the Ovis aries PrP c protein isoform having the sequence SEQ id n°l (Accession number CAE00186).
  • said biological sample is a blood sample or any product derived thereof, such as a buffy coat or a White blood cell sample.
  • the caprinae PrP c protein isoform derivative or fragment is from a tissue homogenate, cell lysate or preferably from a source overexpressing said protein isoform derivative or fragment thereof, and most preferably said caprinae PrP protein isoform, derivative or fragment is from a brain homogenate.
  • the step b) of incubating the reaction mixture in conditions allowing the amplification of the PrP Sc protein present in the biological sample is done by maintaining the reaction mixture at a temperature comprised between 36 and 45°C, preferably between 38 and 41 °C, and most preferably between 39 and 40°C.
  • the step c) of disrupting the reaction mixture is done by sonication.
  • the steps b) and c) are repeated at least 10 times, preferably at least 50 times and most preferably at least 90 times.
  • said subject is a human
  • said TSE is CJD such as sporadic (sCJD), familial (fCJD), iatrogenic (iCJD), and variant form of CJD (vCJD), preferably said CJD is vCJD.
  • said subject is a bovine
  • said TSE is BSE
  • the resent invention relates to a kit for diagnosing a Transmissible Spongiform Encephalopathy (TSE) in a subject, wherein said subject is not a caprinae and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), wherein said kit comprises a caprinae PrP c protein isoform having a glutamine amino acid residue at its position 171 , derivative or fragment thereof.
  • TSE Transmissible Spongiform Encephalopathy
  • BSE bovine spongiform encephalopathy
  • CJD Creutzfeldt-Jakob disease
  • Figure 1 shows v-CJD amplification using Wild typegoat PrP c , ovine ARQ PrP c , or ovine AHQ PrP as substrate.
  • Figure 2 shows the vCJD/BSE agent amplification by PMCA using brain from transgenic mice expressing different species PrP sequence as substrate.
  • Figure 3 shows the PrP res detection in PMCA reactions seeded with white blood cells (WBC) from BSE infected and healthy sheep.
  • Figure 4 shows the vCJD agent detection in the blood of experimentally infected primates.
  • Figure 5 shows PrP res in PMCA reactions seeded with blood samples from vCJD infected and healthy primates.
  • Figure 6 shows PrP res in PMCA reactions seeded with WBC from a vCJD affected patient and healthy controls.
  • Figure 7 shows the results in White blood cells prepared from one v-CJD affected patients after 2 rounds of amplification
  • the first aspect of the invention relates to a method for diagnosing a Transmissible Spongiform Encephalopathy (TSE) in a subject, wherein said subject is not a caprinae and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), and wherein said method comprises the steps of:
  • the term subject refers to a mammal except caprinae, preferably except ovine.
  • the method provides sensitivity and reproducibility that is sufficiently high to detect or diagnose disease prior to the onset of clinical symptoms.
  • the subject may be healthy, but the method of the invention is particularly useful for testing a subject thought to develop TSE.
  • said TSE is BSE (classical, 1-Type, H-type) and said subject is a bovine.
  • said TSE is CJD and said subject is a human.
  • CJD sporadic
  • fCJD familial
  • iCJD iatrogenic
  • vCJD a new variant form of CJD
  • vCJD affects young patients with an average age of 27 years old, and is a relatively long illness (14 months compared with 4.5 months for sCJD). Because of insufficient information available about the incubation time and the levels of exposure to contaminated cattle food products it is impossible to make well-founded predictions about the potential future incidence of vCJD.
  • said CJD is selected among sporadic (sCJD), familial (fCJD), iatrogenic (iCJD), and variant form of CJD (vCJD).
  • sCJD The clinical diagnosis of sCJD is actually based on a combination of rapidly progressive multifocal dementia with pyramidal and extrapyramidal signs, myoclonus, and visual or cerebellar signs, associated with a characteristic periodic electroencephalogram (EEG).
  • EEG periodic electroencephalogram
  • Variant CJD appears initially as a progressive neuropsychiatric disorder characterized by symptoms of anxiety, depression, apathy, withdrawal and delusions. This is combined with persistent painful sensory symptoms and is followed by ataxia, myoclonus, and dementia. Variant CJD is differentiated from sCJD by the duration of illness (usually longer than 6 months) and EEG analysis (vCJD does not show the atypical pattern observed in sCJD). A high bilateral pulvinar signal noted during MR! is usually used to help diagnose vCJD.
  • a tonsil biopsy may be used to help diagnose vCJD, based on a number of cases of vCJD have been shown to test positive for PrP Sc staining in lymphoid tissue (such as tonsil and appendix).
  • lymphoid tissue such as tonsil and appendix.
  • said CJD is vCJD.
  • biological sample refers to a sample from tissues including, but not limited to, blood, lymph nodes, brain, spinal cord, tonsils, spleen, skin, muscles, appendix, olfactory epithelium, cerebrospinal fluid, urine, milk, intestines, tears and/or saliva.
  • said biological sample refers to a blood sample or any product derived thereof.
  • said blood sample comprises a volume of 100 ⁇ or less from one subject, preferably 50 ⁇ or less, and still preferably 10 ⁇ or less from one subject.
  • the blood sample refer to a plasma sample
  • said sample corresponds to a plasma volume of 5ml or less from one subject, preferably a volume of 2.5 ml or less and more preferably of 1 ml or less.
  • a product derived from a blood sample refers to a product comprising blood components such as plasma or plasma derived products, red cells, platelets, or leukocytes, etc.
  • said product derived from a blood sample refers to a buffy coat or a white blood cell sample.
  • said buffy coat sample is obtained as disclosed in the examples.
  • said buffy coat sample is diluted more than ten folds, preferably more than twenty folds, and preferably at least fifty folds.
  • the inventors have established that a minimum dilution of this buffy coat sample fosters PrP Sc protein amplification.
  • said White blood cell sample is obtained as disclosed in the examples.
  • said product derived from a blood sample refers to a plasma sample.
  • Said plasma sample may be further processed, as an example, ultracentrifuged (i.e. about lOO OOOg and for at least 10 minutes, preferably one hour) so as to purified microvesicles for said plasma.
  • the method of the invention may further comprise a previous step of concentration of the PrP Sc protein of the biological sample.
  • protein may be concentrated by phosphotungstic acid (PTA) precipitation, or binding to ligands, shown to interact specifically to PrP Sc , such as conformational antibodies, certain nucleic acids, plasminogen or various short peptides (SOTO & CASTILLA, Nature Med., vol.10, p:S63-S67, 2004).
  • samples may be fractionated. For example, the fraction that is insoluble in mild detergent could be harvested, a procedure that would increase the concentration of prion within the sample (WO 02/04954).
  • the Ovis aries PrP c protein isoform having the sequence SEQ id n° l corresponds to the ARQ isoform (Alal 36-Argl 54-Gln l 71 ) of sheep PrP (Accession number: CAE00186)
  • the Ovis aries PrP c protein isoform having the sequence SEQ id n°2 corresponds to the VRQ isoform (Val l 36-Argl 54-Glnl 71 ) of sheep PrP (Accession number: CAE00190)
  • Ovis aries PrP c protein isoform having the sequence SEQ id n°3 corresponds to the AHQ isoform (Alal36-Hisl 54-Glnl71) of sheep PrP (Accession number: ABG78479).
  • said ovine PrP c protein isoform is the Ovis aries PrP c protein isoform having the sequence SEQ id n° 1.
  • the term "derivative” refers to a polypeptide having an identity of at least 85%, preferably at least 95% and most preferably at least 95% with the complete sequence of a mammalian PrP c protein isoform and an identity of at least 95%, preferably at least 99% and most preferably 100% of identity with the amino acids 160 to 180 of a caprinae PrP c protein isoform having a glutamine amino acid residue at its position 171 such as SEQ id n° l , SEQ id n°2 or SEQ id n°3.
  • percentage of identity between two amino acids sequences, means the percentage of identical amino-acids, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the amino acids sequences.
  • best alignment or “optimal alignment” means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two amino acids sequences are usually realized by comparing these sequences that have been previously align according to the best alignment; this comparison is realized on segments of comparison in order to identify and compared the local regions of similarity.
  • the identity percentage between two sequences of amino acids is determined by comparing these two sequences optimally aligned, the amino acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences.
  • the percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.
  • fragment refers to a polypeptide comprising the amino acids 25 to 233 of a caprinae PrP c protein isoform having a glutamine amino acid residue at its position 171 such as SEQ id n°l , 2 or 3 corresponding to the full length maturated PrP protein, but also to a polypeptide comprising the amino acids 90-23 1 of a caprinae PrP c protein isoform having a glutamine amino acid residue at its position 171 such as SEQ id n° 1 , 2 or 3, such as 80-231 or 70-231 , preferably 60-23 1 , such as 50-231 or 40-231 , and more preferably 30-231 of a caprinae PrP protein isoform having a glutamine amino acid residue at its position 171 such as SEQ id n° l , 2 or 3.
  • PrP c protein isoform, derivative or fragment thereof for use in the method of the invention preferably said source is a cell lysate.
  • the protein may be the endogenous protein isoform expressed in caprinae cells such as ovine cells, and these cells used to make a lysate that provides the corresponding protein isoform.
  • the lysate may be from tissue culture cells, or extracted from whole organisms, organs or tissues.
  • the source of the protein isoform may be from ovine brain homogenates.
  • the source of said PrP c protein isoform, derivative or fragment thereof may be from cells made or engineered to over-express it.
  • cells may be transformed with a nucleic acid vector that expresses said PrP protein isoform, derivative or fragment thereof.
  • These cells may comprise mammalian cells, bacterial cells, yeast cell, insect cells, whole organisms, such as transgenic mice, or other cells that may be a useful source of the non-pathogenic protein.
  • the protein isoform's source may be a brain homogenate.
  • any of the wide variety of vectors known to those of skill in the art could be used to over express the PrP c protein isoform, derivative or fragment thereof.
  • plasmids or viral vectors may be used. It is well understood to these of skill in the art that these vectors may be introduced into cells by a variety of methods including, but not limited to, transfection (e.g., by liposome, calcium phosphate, electroporation, particle bombardment, etc.), transformation, and viral transduction.
  • the source of said PrP c protein isoform, derivative or fragment thereof is a transgenic mammal such as mouse over-expressing it, such as the mouse Tg ARQ disclosed in the example, preferably a brain homogenate obtained from said transgenic mammal.
  • Such a brain homogenate can be simply obtained by methods well known from the skilled person.
  • such brain homogenate can be obtained by (i) low speed homogenization (e.g. using ULTRATURAX) of brain tissue in 4°C PBS pH 7-7.65 + 0.1% TRITON® X100+ 150 mM NaCl (10% Weight/vol) , (ii) lying on ice for 15 to 45 min, (iii) filtration through a 50 ⁇ mesh disposable cell strainer or low speed centrifugation 100 to 500 g for 1 min. The obtained homogenate is then aliquoted and snap frozen at -80°C before use.
  • low speed homogenization e.g. using ULTRATURAX
  • filtration through a 50 ⁇ mesh disposable cell strainer or low speed centrifugation 100 to 500 g for 1 min The obtained homogenate is then aliquoted and snap frozen at -80°C before use.
  • PrP c For instance in the case of PrP c , it has been shown that the majority of the protein localizes to the membrane in structures known as "lipid-rafts.” Thus, partial purification of PrP c can be achieved by enriching the lysate for lipid-rafts. Methods for this enrichment typically rely on the resistance of lipid-raft structures to mild detergent, such as ice-cold TRITON® X-100, and are well known to those in the art.
  • mild detergent such as ice-cold TRITON® X-100
  • purified will refer to a caprinae ovine PrP c protein isoform composition that has been subjected to fractionation or isolation to remove various other protein or peptide components, and which composition substantially retains caprinae PrP c protein isoform, as may be assessed, for example, by Western blot to detect said protein isoform.
  • compositions will be subjected to fractionation to remove various other components from the composition.
  • Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PTA, PEG, antibodies and the like, or by heat denaturation followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.
  • caprinae PrP c protein isoform, derivative or fragment thereof be deglycosylated.
  • caprinae PrP c protein isoform, derivative or fragment thereof may be treated with peptide N- glycosidase F (NEW ENGLAND BIOLABS) according to the manufacturer's instructions. In this case, incubation for about 2h at 37°C results in significant deglycosylation of the protein.
  • the recombinant protein be fused with additional amino acid sequence.
  • additional amino acid sequence For example over expressed protein may be tagged for purification or to facilitate detection of the protein in a sample.
  • Some possible fusion proteins that could be generated include histidine tags, Glutathione S-transferase (GST), Maltose binding protein (MBP)), green fluorescent protein (GFP), Flag and myc tagged PrP.
  • GST Glutathione S-transferase
  • MBP Maltose binding protein
  • GFP green fluorescent protein
  • Flag myc tagged PrP.
  • coding sequence for a specific protease cleavage site may be inserted between the non-pathogenic protein coding sequence and the purification tag coding sequence.
  • One example for such a sequence is the cleavage site for thrombin.
  • fusion proteins may be cleaved with the protease to free the non-pathogenic protein from the purification tag.
  • the reaction mixture may further comprise additional factors or cell lysate to provide secondary factors important for conversion.
  • additional factors or cell lysate may be ideal or polyanions such as RNA or synthetic polyanions (DELEAULT et al, Proc. Natl. Acad. Sci. USA., vol.l04(23), p:9741 -6, 2007; and International Patent Application PCT WO 2007/082173).
  • reaction mixture refers to a composition minimally comprising a biological sample and the caprinae PrP c protein isoform, derivative or fragment thereof.
  • the reaction mixture further comprises a "conversion buffer” that is favorable for prion replication.
  • said conversion buffer comprises a salt solution and detergents such as the buffer comprising l x phosphate buffered saline (PBS) with 150 mM additional NaCl, 0.5% TRITONX®-100 and a protease inhibitor cocktail.
  • the conversion buffer may further comprise a metal chelator. This is of particular advantage since Cu 2+ and to some extent Zn 2+ interferes with the amplification PrP Sc .
  • the metal chelator is EDTA.
  • the reaction mixture may also comprise additional elements, for example, one or more buffers, salts, detergents (e.g. digitonin, saponin or SDS), lipids, or proteins or polypeptides (e.g. Plasminogen).
  • reaction mixture be kept in a sealed environment to prevent evaporation and cross contamination.
  • amplification may be carried out while reaction mixture is maintained in a sealed plastic enclosure.
  • the step (b) of incubating the reaction mixture in conditions allowing the amplification of the PrP Sc protein present in the biological sample is done by maintaining the reaction mixture at a temperature comprised between 36 and 45°C, preferably between 38 and 41°C, and most preferably between 39 and 40°C
  • Said temperature enable the conversion of the caprinae PrP c protein isoform or fragment thereof by the PrP Sc protein present in the biological sample.
  • the temperature of the reaction mixture is monitored and/or controlled by a programmable thermostat.
  • the sample may be placed in an automated thermocycler thus allowing the temperature of the reaction mixture to be programmed over the course of amplification.
  • incubation of the reaction mixture could be performed over a range of time periods.
  • the reaction mixture may be incubated at step (b) for about one minute to about 10 hours, preferably for about 10 minutes to 1 hour and most preferably for about 30 minutes.
  • the incubation time may be varied through out the amplification.
  • the incubation time may be increased or decreased by an increment of time after each amplification step.
  • the term "disrupting" refers to any method by which molecular complexes in reaction mixture may be break out.
  • Exemplary disaggregation methods include treatment with solvents, modification of pH, temperature, ionic strength, or by physical method such as sonication or homogenization.
  • the step c) of disrupting the reaction mixture is done by high energy agitation of microbeads contained in the reaction mixture, which high energy agitation is realized by sonication.
  • the sonication apparatus not come in direct contact with the samples.
  • this step of sonication may be performed with a commercially available sonication apparatus such as MISON1X 3000 or MISONIX 4000.
  • the sonication apparatus may be automated and capable of programmed operation thus allowing high throughput sample amplification.
  • microbeads which can be used at this step and which have to be introduced in the reaction mixture, one can cite iron, glass and silica microbeads, preferably said microbeads have diameters ranging from 0.3 to 2.5 mm, most preferably silica beads displaying 0.8 to 1.2 mm diameter.
  • said sonication could comprise a pulse or a serial of pulses of about 5 to 60 seconds, preferably 10 to 50 seconds, as an example of 30 seconds.
  • said sonication could comprise one or several pulses at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% potency.
  • the steps b) and c) could be repeated at least 10 times, preferably at least 50 times and most preferably at least 90 times.
  • the method of the invention may include the step d') of mixing, in an alternative step a), the obtained amplified reaction mixture with a caprinae PrP protein isoform having a glutamine amino acid residue at its position 171 , a derivative or a fragment thereof to obtain a new reaction mixture, which new reaction mixture is subjected to new steps b), c) and d).
  • said step d') is repeated one or more times.
  • step d' may be repeated one time, two times, three times or , four times
  • the total duration of the steps a) to d) is of three days or less, preferably of two days or less, and more preferably of one day or less. This may be preferable since in some cases the PrP c protein isoform or other cofactors may have a limited stability and extended incubation may result in an eventual fall-off of the conversion rate. In particular it has been shown that PrP conversion rates drop after about 48 hours of incubation.
  • the step e) of detecting the PrP Sc protein in the amplified reaction mixture can be done by both direct and indirect assays known to those of skill in the art such as Western Blot, Immunoassays like ELISA, animal bioassays, cellular infectivity assays and spectroscopic assays.
  • discrimination between the newly-formed PrP Sc from remaining PrP c usually is required. This typically is accomplished based on the different natures of PrP Sc versus PrP c . For instance, PrP Sc typically is highly insoluble and resistant to protease treatment. Therefore, in the case of PrP Sc and PrP c , separation can be by, for instance, protease treatment.
  • reaction mixtures are incubated with, for example, Proteinase (PK).
  • PK Proteinase
  • An exemplary protease treatment includes digestion of the PrP c protein in the reaction mixture with 1 -20 ⁇ g/ml of PK for about 1 hour at 37° C. Reactions with PK can be stopped prior to assessment of prion levels by addition of PMSF or electrophoresis sample buffer. Depending on the nature of the sample, incubation at 37° C with 1 -50 ⁇ g/ml of PK generally is sufficient to remove PrP c protein.
  • PrP Sc also can be separated from the PrP c protein by the use of ligands that specifically bind and precipitate the misfolded form of the protein, including conformational antibodies, certain nucleic acids, plasminogen, PTA and/or various peptide fragments.
  • PrP Sc protein can be then directly detected by Western blot or by immunoassays such as ELISA using anti-PrP antibody, for example the 3F4 monoclonal antibody. If an ELISA assay is used, this assay may be a two-site immunometric sandwich ELISA.
  • Typical Western blot procedures begin with separating proteins by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The proteins are then electroblotted onto a membrane, such as nitrocellulose or PVDF and probed, under conditions effective to allow immune complex (antigen/antibody) formation, with an anti-PrP protein antibody.
  • SDS-PAGE sodium dodecyl sulfate- polyacrylamide gel electrophoresis
  • Exemplary antibodies for detection of prion protein include the 3F4 monoclonal antibody, the monoclonal antibody D13 (directed against residues 96-106 (PERETZ et al, Nature, vol.412, p: 739-743, 2001), the polyclonal antibodies Rl 8 (directed against residues 142-154), and R20 (directed against C-terminal residues 218-232) (CAUGHEY et al, J Virol, vol.65, p:6597-6603, 1991).
  • An exemplary washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.
  • the immunoreactive bands are visualized by a variety of assays known to those in the art.
  • the enhanced chemoluminesence assay AMERSHAM
  • PrP Sc protein concentration can be estimated by Western blot followed by densitometric analysis, and comparison to Western blots of samples for which the concentration of PrP protein is known. For example, this can be accomplished by scanning data into a computer followed by analysis with quantitation software. To obtain a reliable and robust quantification, several different dilutions of the sample generally are analyzed in the same gel.
  • immunoassays in their most simple and direct sense are binding assays.
  • Specific non-limiting immunoassays of use include various types of enzyme linked immunosorbent assays (ELISAs), immunochromatographic strip assays, radioimmunoassays (RIA), and specifically conformation-dependent immunoassays.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • anti-PrP antibodies are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a reaction mixture suspected of containing prion protein antigen is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound prion protein can be detected.
  • Detection generally is achieved by the addition of another anti-PrP antibody that is linked to a detectable label.
  • This type of ELISA is a simple "sandwich ELISA.”
  • Detection also can be achieved by the addition of a second anti- PrP antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the reaction mixture suspected of containing the prion protein antigen is immobilized onto the well surface and then contacted with the anti-PrP antibodies. After binding and washing to remove non-specifically bound immune complexes, the bound anti-prion antibodies are detected.
  • the immune complexes can be detected directly. Again, the immune complexes can be detected using a second antibody that has binding affinity for the first anti-PrP antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which protein of the reaction mix is immobilized involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against PrP protein are added to the wells, allowed to bind, and detected by means of their label. The amount of PrP Sc protein antigen in a given reaction mix is then determined by mixing it with the labeled antibodies against PrP before or during incubation with coated wells.
  • PrP Sc protein acts to reduce the amount of antibody against prion available for binding to the well and thus reduces the ultimate signal.
  • the amount of PrP Sc in the sample can be quantified.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below. In coating a plate with either antigen or antibody, one generally incubates the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate are then washed to remove incompletely adsorbed material.
  • any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antibodies.
  • a nonspecific protein that is antigenically neutral with regard to the test antibodies.
  • these include bovine serum albumin, casein, and solutions of milk powder.
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, and thus reduces the background caused by nonspecific binding of antibodies onto the surface.
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin, milk proteins, and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • Suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about lto 2 to 4 hours, at temperatures preferably on the order of 25° C to 27° C, or can be overnight at about 4° C or so.
  • the contacted surface is washed so as to remove non-complexed material.
  • An exemplary washing procedure includes washing with a solution such as PBS/Tween or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes can be determined.
  • the second or third antibody generally will have an associated label to allow detection. In some examples, this is an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • the first or second immune complex is contacted and incubated with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (for instance, incubation for two hours at room temperature in a PBS-containing solution such as PBS-Tween).
  • a urease for instance, incubation for two hours at room temperature in a PBS-containing solution such as PBS-Tween.
  • PBS-containing solution such as PBS-Tween
  • the amount of label is quantified, for instance, by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid) and H 2 0 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, for instance, using a visible spectra spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid) and H 2 0 2 , in the case of peroxidase as the enzyme label.
  • Quantification is then achieved by measuring the degree of color generation, for instance, using a visible spectra spectrophotometer.
  • the caprinae PrP c protein isoform, derivative or fragment thereof can be labeled to enable high sensitivity of direct detection of protein that is converted into PrP Sc .
  • the caprinae PrP c protein isoform, derivative or fragment thereof can be radioactively labeled, epitope tagged, or fluorescently labeled.
  • the label can be detected directly or indirectly. Radioactive labels include, but are not limited to ,25 1, 32 P, 33 P, and 35 S.
  • the amplified PrP Sc protein may be also directly detected by animal bioassays, wherein test animal are inoculated with the reaction mixture and assessed for clinical symptoms.
  • Moderate behavioral problems including tremor of the head, ataxia, wobbling gait, head bobbing, irritability and aggressiveness; [000125] 4. Severe behavioral abnormalities including all of the above plus jerks of the head and body and spontaneous backrolls;
  • Brains and other tissues are extracted and analyzed histologically by methods that are well known in the art. For instance one hemisphere is fixed in 10% formaldehyde solution, cut in sections and embedded in paraffin. Serial sections ( ⁇ 6 ⁇ thick) from each block are stained with hematoxylin-eosin, using standard protocols or incubated with antibodies recognizing PrP, in some cases incubation with an antibody to the glial fibrillary acidic protein may be used as a control. Immunoreactions are developed, for example using the peroxidase-antiperoxidase methods. In this case antibody specificity is verified by absorption. In some cases biochemical examination for PrP Sc using Western blot analysis may also be used, in some case both histologic and biochemical analyses may be undertaken, by using one brain hemisphere for each.
  • Amplified PrP Sc protein may be also be detected by functional assays, such as by their ability to infect certain mammalian cells in culture (cellular infectivity assay; KLOHN et al., Proc. Natl. Acad. Sci. USA, vol.100 (20), p: l 1666-1 1671 , 2003).
  • functional assays such as by their ability to infect certain mammalian cells in culture (cellular infectivity assay; KLOHN et al., Proc. Natl. Acad. Sci. USA, vol.100 (20), p: l 1666-1 1671 , 2003).
  • susceptible mouse neuroblastoma N2a cells are exposed to prion-containing samples for 3 days, grown to confluence, and split three times.
  • the proportion of PrP Sc -containing cells is determined with automated counting equipment.
  • the number of prion containing cells may also be determined by flow cytometry.
  • the dose-response to infection is linear over two logs
  • amplified PrP Sc protein may be also detected by indirect methods such as spectroscopic assays, including multispectral ultraviolet fluoroscopy, confocal dual-color fluorescence correlation spectroscopy, Fourier-transformed infrared spectroscopy or capillary electrophoresis, and Fluorescence Resonance Energy Transfer (FRET).
  • spectroscopic assays including multispectral ultraviolet fluoroscopy, confocal dual-color fluorescence correlation spectroscopy, Fourier-transformed infrared spectroscopy or capillary electrophoresis, and Fluorescence Resonance Energy Transfer (FRET).
  • ET Fluorescence Resonance Energy Transfer
  • ET dyes include a complex molecular structure consisting of a donor fluorophore and an acceptor fluorophore as well as a labeling function to allow their conjugation to biomolecules of interests.
  • FRET Fluorescence Resonance Energy Transfer
  • Different acceptors can be used with a single donor to form a set of ET dyes so that when the set is excited at one single donor frequency, various emissions can be observed depending on the choice of the acceptors. Upon quantification of these different emissions, changes in the folding of a labeled protein may be rapidly determined.
  • Some exemplary dyes that may be used comprise BODIPY FL, fluorescein, tetmethylrhodamine, IAEDANS, EDANS or DABCYL.
  • Other dyes have also been used for FRET for examples dyes disclosed in U.S. Patents 5,688,648, 6, 150, 107, 6,008,373 and 5,863,727 and in PCT publications WO 00/13026, and WO 01/19841.
  • the method of the invention further comprises a step f of inactivating residual PrP Sc protein.
  • Said residual PrP Sc protein may be inactivated by various methods known to those in the art, such as treatment with a concentrated base or treatment at high temperature, for example, treatment with 2N NaOH for 1 hour and/or autoclaving at 134°C for 18 min. This would eliminate the danger of prion as biohazardous waste and also help to minimize contamination that could occur when testing multiple samples.
  • the method of the invention is using no-powdered gloves.
  • the inventors established that the talc or the corn starch inhibit the PrP Sc protein amplification.
  • the present invention relates to a kit for the method described previously of diagnosing a Transmissible Spongiform Encephalopathy (TSE) in a subject, wherein said subject is not a caprinae , preferably not an ovine or a caprine, and said TSE is not scrapie, preferably said TSE being bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD) wherein said kit comprises a caprinae PrP c protein isoform having a glutamine amino acid residue at its position 171 , derivative or fragment thereof.
  • TSE Transmissible Spongiform Encephalopathy
  • BSE bovine spongiform encephalopathy
  • CJD Creutzfeldt-Jakob disease
  • the kit is made up of instructions for carrying out the method described herein for diagnosing a TSE in a subject.
  • the instructions can be provided in any intelligible form through a tangible medium, such as printed on paper, computer readable media, or the like.
  • Kits for implementing methods of the invention described herein are specifically contemplated.
  • a kit can comprise, in suitable container means, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more of the following: 1) a conversion buffer; 2) PrP c protein; 3) decontamination solution; 4) a positive control, PrP Sc containing sample; 5) a negative control sample, not containing PrP Sc ; or 6) reagents for detection of PrP Sc .
  • the kit may further comprise reagents for expressing or purifying the caprinae PrP c protein isoform, derivative or fragment thereof.
  • the kit may also comprise reagents that may be used to label the said protein isoform, with for example, radio isotopes or fluorophors.
  • Reagents for the detection of PrP Sc can comprise one or more of the following: pre coated microtiter plates for ELISA and/or CDI detection of prion; tissue culture cells in which PrP Sc can replicate; or antibodies for use in ELISA, CDI or Western blot detection methods.
  • kits of the invention may contain one or more of the following: protease free water; copper salts for inhibiting PrP Sc replication; EDTA solutions for enhancing PrP Sc replication; Proteinase K for the separation of PrP Sc from PrP c protein; fractionation buffers for the separation of PrP Sc from PrP c , modified, or labeled proteins (increase sensitivity of detection); or conversion factors (enhance efficiency of amplification).
  • the conversion buffer may be supplied in a "ready for amplification format" where it is allocated in a microtiter plate such that the sample and PrP c protein isoform may be added to first well, and subjected to amplification.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, plate, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present invention also will typically include a means for containing proteins, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • the liquid solution is typically an aqueous solution that is sterile and proteinase free.
  • proteinatious compositions may be lyophilized to prevent degradation and/or the kit or components thereof may be stored at a low temperature (i.e. less than about 4°C).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • transgenic mice expressing sheep isoforms were established following the construction method described for the transgenic mice expressing the VRQ sheep isoform in VILOTTE et al. (J. Virol, vol.75(13), p:5977-5984, 2001 ).
  • mice expressing bovine PrP isoform were established following the method disclosed in CASTILLA et al. ⁇ Arch. Virol, vol.148, p:677-691 , 2003).
  • mice expressing human PrP (Meti 29 ) and murine PrP isoform were established following the method disclosed in PADILLA et al. (PloS Pathog., vol.7 (3), p :el001319, 201 1).
  • TSE free sheep were produced in the DEFRA 'TSE free flock' which is a unique source able to provide animals that can be considered free from classical scrapie (SIMMONS et al , BMC Vet. Res., vol.5, p:8, 2009) .
  • the animals included in our experiments were imported into France and housed, in a dedicated scrapie free facilities before their use in experiments.
  • PrP genotype was obtained by sequencing the Exon 3 of the Prnp gene.
  • WBC White blood cells
  • the other primates were intravenously inoculated with clarified supernatants (obtained by centrifugation at 1 ,500 g for 10 minutes after extensive sonication) derived from 10 or 100 mg of brains from BSE- or vCJD-infected primates.
  • Primate blood samples were drawn into sodium citrate and fractionated by centrifugation at 2,000 g for 13 minutes according to the techniques classically applied in human transfusion.
  • WBCs were obtained by osmotic lysis of buffy coat (one volume) with EASY- LYSE (DAKO, 9 volumes) for 10 minutes RT. WBCs were washed three times with 50 mL of PBS. All samples were encoded before dispatch and tested blind. Animals were handled under anesthesia to limit stress, and euthanasia was performed for ethical reasons when animals lost autonomy
  • Blood is collected on anticoagulant (like Citrate sodium/EDTA/Heparinate). 5 ml whole blood is centrifuged at 3600 rpm 10 min room temperature. Plasma is removed and buffy coat is collected using a disposable hard bulb pipette. Buffy coat are then mixed volume/volume with ACK solution (NH4CL 0, 15 M, KHC03 I mM, Na2EDTA 0, 1 mM, pH 7.4) for 5 min RT or with other red cell lysis solution . The obtained white blood cells are washed twice with PBS. Cell numeration performed using Mallasez's cell or automatic cell counter.
  • anticoagulant like Citrate sodium/EDTA/Heparinate
  • Desired amount of cells (Typically 10 7 cells) are then resuspended in 200 ⁇ of 4°C PBS pH 7.4 + 150mMoL NaCl+ 0.1 TRITON®X100 and homogenized at high speed (For example using Precess 48). Samples are then spin down 15000g -20 sec. Sample can be stored at -80°C or used freshly.
  • PrP 5 ' amplification usins PMC A
  • Desired amount of WBC or Buffy coat were re suspended in 4°C PBS pH 7.4 + 150mMoL NaCl+ 0.1 TRITON XI 00 and homogenized at high speed. Samples were then spin down 15000g for 20 seconds and then stored at -80°C or used freshly. 7 ⁇ 1 of the blood sample are mixed with 63 ⁇ of substrate in 0.2mL ultrathin wall PCR tube or 96 well PGR plate. In each tube or well 5 to 15 silica microbeads (1.00 mm diameter) were added beforehand. The tubes are then sealed hermetically with appropriate caps or film.
  • Tubes- Plate are placed on MISONIX 4000 microplate horn containing 200-240 mL of deionized water (water level in the horn is at the same level than the sample level in the reaction tubes). The horn is then sealed with hood to avoid evaporation. The horn is at 39.5°C in a way ensuring that after energy burst the temperature in the horn returns to nominal level within 20-40 seconds.
  • the samples are then submitted to 96 cycles of 30 sec sonication at potency 70% - followed by 29 min 30 incubation period. After 96 cycles, 7 ⁇ , of the amplicon are collected and mixed with fresh substrate and new amplification round (96 cycles) can be performed. The amplification is typically stopped after 3 to 6 rounds and PrPSc detection is carried out on amplicons.
  • Amplification of human CJD was tested by PCMA using WT goat (source Transgenic mice expressing goat WT PrP c : "TgGo WT substrate”), ovine AHQ (source Transgenic mice over-expressing AHQ ovine PrP c : "Tg AHQ substrate”), or ovine ARQ
  • PrP source Transgenic mice over-expressing ARQ ovine PrP : "Tg ARQ substrate" as substrates.
  • the Figure 1 shows v-CJD amplification (dilution series of human brain in PBS) using caprinae WT or ovine ARQ or AHQ PrP c as substrate.
  • T+ positive control
  • 0 negative Human brain
  • Amplification was performed under the same run.
  • the figure 2 shows the vCJD/BSE agent amplification by PMCA using brain from transgenic mice expressing different species PrP sequence as substrate.
  • PMCA reactions were seeded with ten-fold dilution series of vCJD/BSE brain material ( 10 " to 10 "9 ) from different species (human, cynomologus macaque, bovine, sheep and porcine).
  • PMCA substrates were prepared using brain from transgenic mice over-expressing either human (methionine 129 variant- ⁇ ), bovine (V), murine (o) or sheep (VRQ variant:A,
  • ARQ variant A) Prion protein.
  • WBCs were then homogenized in PMCA buffer and homogenates were used to seed PMCA reactions in which the brain of transgenic mice that expressed the ARQ variant of the ovine PrP was used as substrate. Each sample was used to seed 4 independent reactions (two different runs onto 2 different sonicators). Five successive PMCA amplification rounds (R) were applied. The resulting PMCA products were analysed by Western Blot (WB) for the presence of abnormal P resistant PrP (PrP res -antibody Sha31 epitope YEDRYYRE, SEQ id n°4). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control).
  • WB control Western Blot
  • the table I shows the PrP res detection in Protein Misfolding Cyclic Amplification (PMCA) reactions seeded with white blood cells from ARQ/ARQ sheep orally inoculated with BSE agent, collected at different time points of the incubation period. After each amplification round, the number of PrP res positive replicates (as assessed by Western Blot) is indicated in the table. (-) indicated that all 4 replicates were negative.
  • PMCA Protein Misfolding Cyclic Amplification
  • the Figure 3 A shows the Western Blot of PMCA products (3 rounds) from WBC prepared from BSE orally challenged sheep at different time points (indicated as month post inoculation: mpi).
  • the figure 3B shows the Western Blot of PMCA products (6 rounds) from WBC from BSE affected sheep (3 different individuals) and from healthy controls.
  • the WBC or BC samples were used (as homogenates 1/100 diluted in PMCA buffer except Table 2, wherein dilution 1/10, 1/50 or 1 /100 were used) to seed PMCA reactions in which brain homogenate from ovine PrP transgenic mouse (ARQ variant) was used as substrate.
  • Each sample was submitted to successive rounds of amplification (i.e. up to 6) each constituted with 96 cycles (30s sonication-30 minutes incubation at 39.5°C) in a MISONIX 4000 sonicator.
  • PMCA products were analysed by Western Blot (WB) for the presence of abnormal PK resistant PrP (PrP res -antibody Sha31 epitope YEDRYY E, SEQ id n°4).
  • WB control classical scrapie isolate
  • Samples were received encoded and tested blind.
  • the figure 4 indicates for each macaque, the clinical onset (din) and time to euthanasia of the animals are indicated (upper label on arrows) as months post inoculation (mpi). The time point corresponding to blood samples (months post inoculation) that were tested and the results of PrP res WB detection in PMCA reactions are indicated (under arrow). No positive WB result was observed before the third PMCA round. No additional positive result was observed after 5 PMCA rounds
  • FIG 5 A shows the Western Blot of buffy coat BC samples (collected between 2005 and 2010) at different time points of the incubation period (indicated as mpi) in a vCJD inoculated primate (intravenous route, macaque 6 in figure 4).
  • the animal developed clinical signs at 46 mpi and was euthanized at 58 mpi.
  • the figure 5 B shows the Western Blot of buffy coat from four different unchallenged cynomolgus macaques (cont) and 5 different vCJD affected primates (see figure 4)
  • the figure 5 C shows the Western Blot of White blood cells and of Buffy Coat prepared from the same blood sample collected in a clinically affected primate (macaque 6, 38 mpi). WBC and BC (both 1/50 diluted) were used to seed PMCA reactions.
  • the table II shows the PrP res detection results in PMCA reactions seeded with White blood cells (WBC) or Buffy coat (BC) from Cynomologus macaques intravenously challenged with (i) blood from a vCJD affected macaque (macaque 6) or with (ii) human vCJD brain homogenate (macaque 8).
  • WBC and BC were homogenized and then diluted 1/10, 1/50 and 1/100 in PMCA buffer before seeding PMCA reactions for 5 successive rounds (R) of amplification.
  • the figure 6 A shows the Western Blot of PMCA reaction unseeded (no seed), or seeded with WBC from the vCJD affected patient (vCJD-WBC) or with WBC from 6 different (among the 135 tested) healthy human control (H-WBC).
  • the figure 7 shows the results of a two rounds amplification in White blood cells prepared from one v-CJD affected patients (BC+) and a dilution series of v-CJD brain material.
  • 1 WB positive control
  • 2 negative Human brain
  • 10 v CJD WBC cells equivalent to 30 ⁇ , of whole blood
  • the figure 6 B shows the Western Blot of ten-fold dilution series of WBC homogenate from the vCJD affected patient (Hu vCJD WBC), which were submitted to three PMCA amplification rounds. The equivalent whole blood amount used to seed the reactions is indicated in the figure. Each dilution was tested in duplicate. WBCs from healthy patients (H-WBC) were used as amplification specificity control After three amplification rounds, PrP res could be detected in one out two replicates seeded with WBC material equivalent to 0.05 ⁇ , of starting whole blood.
  • WBC from the vCJD patient were mixed with WBC from either eleven (8 different pools), twenty-three (4 different pools), forty-seven (2 different pools) or ninety-five (1 pool) healthy donors and subjected to 3 rounds of PMCA reactions.
  • the PMCA products were then tested as previously.
  • the WBC homogenates used to prepare pools were equivalent to 50 ⁇ of starting whole blood. Reactions seeded with WBC from healthy controls (H-WBC) were included as controls
  • FIG. 6 C shows the Western Blot obtained of WBC homogenate from the vCJD affected patient (Hu vCJD WBC) alone, or after pooling with WBC from 1 1 (pi 2), 23 (p24), 47 (p48) or 95 (p96) healthy controls.

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Abstract

L'invention concerne un procédé de diagnostic de l'encéphalopathie spongiforme transmissible (EST) chez un sujet, ledit sujet n'étant pas un caprin et ladite EST n'étant pas la tremblante du mouton, mais de préférence une encéphalopathie spongiforme bovine (ESB) ou maladie de Creutzfeldt-Jacob. Ledit procédé consiste a) à mélanger un échantillon biologique prélevé sur ledit sujet avec un isoforme de protéine PrPc comportant un résidu d'acide aminé glutamine à la position 171, ou un dérivé ou un fragment de cet isoforme, pour obtenir un mélange réactionnel ; à faire incuber ledit mélange réactionnel dans des conditions permettant l'amplification de la protéine PrPSc présente dans l'échantillon biologique ; c) à interrompre le mélange réactionnel ; d) à répéter les opérations 2 et 3 une nouvelle fois ou à plusieurs reprises ; et e) à détecter la protéine PrPSc dans le mélange réactionnel amplifié. L'invention concerne également une trousse correspondante.
PCT/EP2012/004377 2011-10-19 2012-10-19 Procédé de diagnostic de l'encéphalopathie spongiforme transmissible WO2013056841A1 (fr)

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US11598783B1 (en) 2019-10-23 2023-03-07 Colorado State University Research Foundation In vitro detection of prions

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