WO2001038880A1 - Procede de determination d'encephalies spongiformes transmissibles chez les mammiferes - Google Patents

Procede de determination d'encephalies spongiformes transmissibles chez les mammiferes Download PDF

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Publication number
WO2001038880A1
WO2001038880A1 PCT/FI2000/001000 FI0001000W WO0138880A1 WO 2001038880 A1 WO2001038880 A1 WO 2001038880A1 FI 0001000 W FI0001000 W FI 0001000W WO 0138880 A1 WO0138880 A1 WO 0138880A1
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prp
supernatant
tse
solution
tissue
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PCT/FI2000/001000
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English (en)
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Geoffrey John Russell Barnard
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Wallac Oy
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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

  • This invention relates to a method for the determination of transmissible spongiform encephalopathies in mammals. More particularly, the invention concerns a novel method for the determination of the percentage of disease related prion protein in a sample by differential extraction using two solutions of a chaotropic agent. Moreover, the invention concerns the use of this percentage as a diagnostic marker for the transmissible spongiform encephalopathies (TSE).
  • TSE transmissible spongiform encephalopathies
  • Scrapie, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Sheinker (GSS) syndrome and related diseases of mink (transmissible mink encephalopathy), mule deer and elk (chronic wasting disease) are classified as the transmissible degenerative (or spongiform) encephalopathies (TSE's).
  • New species have been affected in recent years including cattle (bovine spongiform encephalopathy), cats (feline spongiform encephalopathy) and a variety of captive zoo felines and antelope and a new form of CJD in man has recently emerged. Iatrogenic transmission of CJD in man occurs and these diseases can be transmitted from affected to healthy animals by inoculation or by feeding diseased tissues. The following text describing these diseases is taken from a review by Hope, 1998 (see reference 31).
  • Scrapie of sheep has been known in Europe for centuries and has spread to most parts of the world, excluding Australasia and Argentina, with the migrations of man and his livestock. It is characterised by altered behaviour, hypersensitivity to sound or touch, loss of condition, pruritus and associated fleece loss and skin abrasions and in-coordination of the hind limbs. Diagnosis is confirmed postmortem by examination of brain tissue for a triad of histopathological signs - vacuolation, loss of neurones and gliosis 1 .
  • Scrapie has been reported in most breeds of sheep and, within a flock, it appears to occur in related animals.
  • the natural clinical disease has a median peak incidence in flock animals of 3.5 years, with a range of 2.5 to 4.5 years covering the vast majority of cases . For most of this period, the infected animal is clinically normal and indistinguishable from its un-infected flockmates.
  • the within-flock incidence of clinical disease is usually 1-2 cases per 100 sheep per year but there have been several instances of 40-50% of animals of a flock succumbing to the disease within a year.
  • a number of genetic markers have recently been identified as risk factors and the introduction of gene typing has greatly facilitated interpretation of field studies on the incidence of natural and experimental disease .
  • Creutzfeldt-Jakob disease is a progressive dementia with clinical signs suggesting dysfunction of the cerebellum, basal ganglia and lower motor neurones. It is associated with gradual mental deterioration leading to dementia and confusion, and a progressive impairment of motor function. Most patients die within six months of onset of clinical signs and there are no verified cases of recovery. Pathologically the lesions of the brain included variable vacuolation of the neuropil, astrocytosis and, in about 10% of CJD cases, amyloid plaques. Gerstman-Straussler syndrome is a familial variant of CJD with an extended, clinical time course.
  • CJD-related disease in man is remarkably constant at 0.5-1 cases per million of population per year throughout the world and so is not linked to the incidence of any of the animal diseases. This low incidence casts doubt on the role of infection in its propagation within the population (but see below). About one in seven of cases are familial and linked to mutations in the open reading frame of the PrP (prion protein) gene. There has been a large amount of clinical and pathological studies on human cases of neurological disease which seem to be associated with these rare mutations of the PrP gene (for a review, see 4 ). In some families, there is complete penetrance of the phenotype and so the mutation is regarded as the cause of the disease.
  • FPI Fatal familial insomnia
  • nvCJD CJD
  • Bovine spongiform encephalopathy Its co-incidence with a novel bovine TSE (BSE) which is largely restricted to the UK has led to speculation, as yet unproven, that this new form of CJD represents a cross-species transmission of infection from cattle.
  • Bovine spongiform encephalopathy
  • Bovine spongiform encephalopathy (BSE) has affected the UK cattle industry for the past decade 8 . From isolated cases first reported in 1986 and some retrospectively identified in May 1985, a major epidemic was underway by 1988 which has to date claimed over 180 000 cattle within the British Isles. Some other countires have also confirmed cases : Switzerland (450+), Ireland (700+), Portugal (300+), France and Germany with one or two cases in Italy, Denmark, Canada, the Netherlands, Oman and the Falkland Islands.
  • the disease produces a progressive degeneration of the central nervous system and was named because of the sponge-like appearance of BSE-brain tissue when seen under the light microscope 9 .
  • Warning signs of the illness include changes in the behaviour and temperament of the cattle.
  • the affected animal becomes increasingly apprehensive and has problems of movement and posture, especially of its hindlimbs.
  • the cow or bull
  • This clinical phase of BSE lasts from a fortnight to over six months.
  • this neurological disease can occur in either sex with a modal age of onset of 4-4.5 years (range 1.8 - 18 years).
  • Most cases of BSE have occurred in cattle between the ages of 3 and 5 years and for most of its development time the disease gives no tell-tale sign of its presence .
  • BSE-affected cow brains are virtually identical to those found in scrapie-affected sheep and include the spongiform change which gives BSE its name. From its clinical and neuropathological signs, BSE was immediately suspected to belong to the scrapie family of transmissible spongiform encephalopathies. This has been confirmed by biochemical studies 11 and by experimental transmission of BSE to mice 12 , sheep and goats 13 amongst other species.
  • the prion protein (PrP) The prion protein (PrP)
  • PrP Sc The conversion of a normal membrane glycoprotein, the cellular prion protein or PrP c , to an aggregated, insoluble isoform, PrP Sc , is a key process in the pathogenesis of BSE, scrapie and other transmissible spongiform encephalopathies (TSE's).
  • TSE's transmissible spongiform encephalopathies
  • the specific detection of PrP Sc forms the basis for biochemical diagnosis of these diseases.
  • Folding differences in the abnormal isoform of the prion protein (PrP c ) can be investigated by probing the conformation of the protein in diseased tissues by proteolysis under conditions where the normal protein is either destroyed and drastically reduced in amount prior to detection by SDS-PAGE/immunoblotting or ELISA techniques.
  • the prion protein is expressed in many different cells but is found in greatest abundance associated with the neurones of the central nervous system. Consequently, the abnormal form of PrP accumulates predominantly in the brain although it can also be detected in extra-neural tissues such as the tonsil and spleen early in the development of disease.
  • PrP protein In cell culture, the PrP protein is cycled to and from the cell surface via the endosome-lysosome system; during this process the protein appears to undergo proteolytie cleavage between residues 109 and 112. To what extent this cleavage occurs in vivo is unknown although C-terminal fragments of PrP similar to those expected to result from the lysis of this peptide bond have been seen in deposits of mouse and human PrP Sc . The exact site of cleavage may be related to the phenotype of disease or strain of infectious agent; this is an area of current investigation. Previous work on detection of PrP
  • PrP prion protein
  • Improving the sensitivity of the assay system may shed light on the scientific conundrum of why lateral or maternal transmission of BSE occurs in the (apparent) absence of infectivity (and PrP Sc ) in milk 27 , blood, placenta and other peripheral tissues of the BSE-infected cow 28 .
  • the immuno-diagnosis of a TSE invariably involves the identification of deposits of prion protein (PrP) using specific antibodies (e.g. Senetek 3F4) in tissue preparations (Immunohistochemistry) or Western Blot analysis.
  • specific antibodies e.g. Senetek 3F4
  • prior proteolysis of the sample with an appropriate concentration of enzyme e.g. proteinase K
  • PrP c normal non-aggregated PrP
  • PrP c normal non-aggregated PrP
  • the residual core of the PrP molecule designated PrP res
  • PrP aggregated (i.e. deposited) PrP is more resistant to proteolysis but this resistance will be directly related to the concentration, specificity and potency of the proteolytie enzyme employed in the experiment.
  • proteinase K PK
  • PK proteinase K
  • PrP res The concentration of PrP res , however, is not an absolute.
  • the extent of proteolysis of the deposited prion protein will be related to the concentration and activity of the enzyme that has been added experimentally to the extracted protein. The more enzyme added the more proteolysis and vice versa. Consequently, the presence of PrP res is totally dependent on the experimental conditions employed. In other words, PrP res is an experimental artefact.
  • pre-clinical disease must be categorised by little deposited and/or aggregated PrP. In this situation, less PK will be required to digest the material since the enzyme is no respector of protein. Consequently, the measurement of p r pres following PK digestion will always be technically difficult. Furthermore, it may turn out to be an inappropriate parameter to measure in order to diagnose a TSE from a wide variety of tissues taken from animals at different stages of the disease.
  • the present invention concerns a method for determining the solvent resistence of prion protein (PrP) in a tissue or body fluid sample of a mammal, comprising the steps of
  • this invention concerns a method for diagnosing a transmissible spongiform encephalopathy (TSE) in a mammal, said method comprising the determination of prion protein (PrP) in a body fluid or tissue sample from said mammal, by a method for determining the solvent resistence of prion protein (PrP) according to this invention, wherein the percentage of sparingly soluble PrP (solvent resistent PrP) calculated as
  • TSE is used as indication of TSE in said mammal.
  • Figure 2 shows the percentage of solvent resistent PrP, using the differential extraction method described in the Example, in paired samples of bovine brain tissue taken from the caudal and rostal regions of the brain stem from 9 clinically positive animals,
  • Figure 3 shows the percentage of solvent resistent PrP, using the differential extraction method described in the Example, in paired samples of bovine brain tissue taken from the caudal and rostal regions of the brain stem from 9 clinically negative animals,
  • Figure 4 shows the “within assay variation”
  • Figure 5 shows the “between assay variation”
  • Figure 6 shows the solubility of normal PrP and disease related PrP in an aqueous solution of guanidine hydrochloride versus molarity
  • Figure 7 shows the solubility of normal PrP and disease related PrP in an aqueous solution of urea versus molarity.
  • a method has been developed that quantifies disease- associated deposited and aggregated PrP as a percentage of the total PrP on the premise that aggregated and deposited PrP (PrPSc) characteristic of a TSE, is much less soluble in low molarity chaotropic agent (e.g. 1M guanidine hydrochloride) than non-aggregated PrP.
  • low molarity chaotropic agent e.g. 1M guanidine hydrochloride
  • a measure of total PrP can be obtained by using high molarity chaotrope (e.g. 6M guanidine hydrochloride) to extract the residual aggregated prion from the tissue homogenate. Consequently, by the use of differential extraction using low and then high molarity chaotrope, a measure of solvent resistance can be established which is both independent of protein concentration and directly related to the concentration of disease-associated aggregated and deposited PrP.
  • chaotropic agent means generally a reagent that denatures another substance.
  • an aggregated polymer e.g. an aggregated protein
  • the chaotrope causes both disaggregation and unfolding (i.e. loss of both quaternary and tertiary structure).
  • chaotropic agents to be used in the present invention can be mentioned highly soluble guanidine salts such as guadinine hydrocloride; urea; and certain detergents such as sodium dodecyl sulphate (SDS).
  • the solvent for the chaotropic agent is preferably water, but not limited thereto. Also organic solvents may be useful.
  • the solution of a chaotropic agent can also contain a chaotropic agent dissolved in another chaotropic agent, or two or more chaotropic agents dissoved in another solvent.
  • low molarity and “high molarity” solutions of chaotropic agents will need some, but not undue, experimentation to define, because the ranges of "low” and “high” molarity will depend i.a. on the chaotropic agent and the solvent used.
  • a low molarity solution is defined as a solution which does not substantially affect (i.e. dissolve) the disease related prion protein.
  • the upper limit of "low molarity" for an aqueous solution of guanidine hydrochloride is below 2 M.
  • the preferable upper value for "low molarity” is about 1 M.
  • the preferable upper range for "low molarity” is about 2 M.
  • the lower limit for the "low molarity” solution of the chaotrope is the concentration where the normal, non-disease related PrP is essentially soluble. For many chaotropes, this point is rather close to zero.
  • the low molarity chaotrope solution is an aqueous solution of less than 2 M guanidine hydrochloride
  • the high molarity chaotrope solution is a is an aqueous solution of at least 3 M guanidine hydrochloride.
  • the method be carried out on a body fluid, e.g. a blood sample, a or tissue sample.
  • a body fluid e.g. a blood sample, a or tissue sample.
  • the sample can thus be, for example, brain tissue, spinal cord, lymphoid tissue, spleen, tonsil, whole blood or a blood fraction.
  • brain tissue is a particularly preferred sample.
  • body fluid samples can be mentioned whole blood or fractions thereof.
  • the results of the immunometric method according to this invention can be used for many different purposes. They can be used for diagnosing a transmissible spongiform encephalopathy (TSE) in the mammal, wherein the mammal can be a living individual or a deceased individual.
  • TSE transmissible spongiform encephalopathy
  • TSE transmissible spongiform encephalopathy
  • the 20% homogenate is vortexed and 50 microliter is pipetted into a 1.5 milliliter Eppendorf tube.
  • Fifty (50) microliter of freshly prepared 2M GdnHCl (guadinine hydrochloride) is added and the tube stoppered and vortexed.
  • 2M GdnHCl guadinine hydrochloride
  • 900 microliter of Wallae assay buffer added, tube stoppered and vortexed and eentrifuged at 13,000 g for 10 minutes.
  • the Wallae assay buffer (Catalogue No.
  • the supernatant (designated supernatant 1) is carefully removed and transferred to a separate test tube taking great care not to dislodge the pellet.
  • a filter paper wick is placed into the Eppendorf to remove the remaining supernatant. After a few moments, the filter paper wick is removed and discarded.
  • One hundred (100) microliter of freshly prepared 6M GdnHCl in water is added to pellet.
  • the tube is stoppered and the pellet carefully vortexed into solution.
  • a further 900 microliter of Wallae assay buffer is added, the tube stoppered, vortexed and eentrifuged at 13,000 g for 5 minutes. This supernatant is designated supernatant 2.
  • the lyophilised human platelet enriched plasma standard is reconstituted by the addition 1 milliliter distilled water.
  • Six standards (including a blank) are prepared by serially diluting this stock material 1 :5 (v/v) with Wallae assay buffer.
  • the plate is incubated on shaker at 4°C for 60 minutes (Note: alternatively, plate may be sealed and placed at 4°C overnight).
  • the plate is washed three times and 200 microliter of europium- labelled 3F4 (diluted at 1 : 1000 v/v in Wallae assay buffer) is added to each well.
  • the plate is incubated on shaker in fridge for 60 minutes and then washed six times.
  • Two hundred (200) microliter of Wallae enhancement solution is added using the dispenser, the plate is shaken for 5 minutes at room temperature and the fluorescence measured.
  • Wallae enhancement solution Catalogue No. 1244-105, is ready for use with Triton X-100, acetic acid and chelators.
  • % PrP retained in pellet i.e. % solvent resistance
  • Transmissible spongiform encephalopathies eds. Bradley, R. & Marchant, B. 301-313 (Commission of the European Communities, Brussels, Belgium, 1993).

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Abstract

L'invention concerne un procédé permettant de déterminer des encéphalies spongiformes transmissibles chez les mammifères. Plus précisément, l'invention concerne un nouveau procédé de détermination du pourcentage de protéines prions apparentées à la maladie dans un échantillon par extraction différentielle utilisant deux solutions d'un agent chaotrope. L'invention concerne en outre l'utilisation de ce pourcentage comme marqueur diagnostique pour encéphalies spongiformes transmissibles.
PCT/FI2000/001000 1999-11-23 2000-11-16 Procede de determination d'encephalies spongiformes transmissibles chez les mammiferes WO2001038880A1 (fr)

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FI992488 1999-11-23
FI19992488 1999-11-23

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999041280A1 (fr) * 1998-02-16 1999-08-19 Commissariat A L'energie Atomique PROCEDE DE PURIFICATION DE LA PrPres A PARTIR D'UN ECHANTILLON BIOLOGIQUE ET SES APPLICATIONS
WO2000048003A1 (fr) * 1999-02-11 2000-08-17 Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. Test du prion

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999041280A1 (fr) * 1998-02-16 1999-08-19 Commissariat A L'energie Atomique PROCEDE DE PURIFICATION DE LA PrPres A PARTIR D'UN ECHANTILLON BIOLOGIQUE ET SES APPLICATIONS
WO2000048003A1 (fr) * 1999-02-11 2000-08-17 Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. Test du prion

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS PREVIEWS [online] BARNARD GEOFF A. ET AL.: "The measurement of prion protein in bovine brain tissue using differential extraction and DELFIA(R) as a diagnostic test for BSE", retrieved from 12897669 accession no. dialog Database accession no. 200100104818 *
LUMINESCENCE, vol. 15, no. 6, November 2000 (2000-11-01) - December 2000 (2000-12-01), CHICHESTER, pages 357 - 362 *

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