WO2000029849A1 - An immunoassay for the determination of transmissible spongiform encephalopathies in mammals - Google Patents
An immunoassay for the determination of transmissible spongiform encephalopathies in mammals Download PDFInfo
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- WO2000029849A1 WO2000029849A1 PCT/FI1999/000896 FI9900896W WO0029849A1 WO 2000029849 A1 WO2000029849 A1 WO 2000029849A1 FI 9900896 W FI9900896 W FI 9900896W WO 0029849 A1 WO0029849 A1 WO 0029849A1
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- 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 an immunoassay for the determination of transmissible spongiform encephalopathies in mammals other than bovine animals. More particularly, the invention concerns a novel method for the detection of the prion protein, a form of which is a diagnostic marker for the transmissible spongiform encephalopathies (TSE).
- TSE transmissible spongiform encephalopathies
- Scrapie Creutzfeldt-Jakob disease (CJD), Gerstmann-Slraussler-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 post-mortem 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 2 .
- the infected animal is clinically no ⁇ nal 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 progiessive 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
- 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 10 .
- 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 Sc ) 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.
- arnino acid sequence of mouse, human and bovine PrP are given below using the IUPAC single letter code for amino-acids :
- PH GGGWGQ amino-acid repetitive sequences
- -C C- cystine bridge
- -GPI C-terminal phospho-inositol glycolipid membrane anchor
- 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 e ⁇ ly in the development of disease. It is therefore correct to say that the total concentration of PrP is greater in affected tissues compared to healthy tissues.
- the 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 proteolytic 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 em area of current investigation.
- 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 26 in the (apparent) absence of infectivity (and PrP Sc ) in milk 27 , blood, placenta and other peripheral tissues of the BSE-infected cow .
- FHl 1 and 3F4 are mapped out below (Birkett et al., unpublished; 29 ).
- the use of FHl 1 for the immunocytochemical detection of PrP in sheep brain tissue sections has been reported 30 ; this report included its N-terminal specificity and the use of trypsin to enhance the detection of PrP Sc .
- this invention concerns an immunometric method for the determination of prion protein (PrP) in a body tissue or body fluid sample from a non-bovine mammal.
- the method comprises the steps of
- this invention concerns a method for diagnosing a transmissible spongiform encephalopathy (TSE) in a non-bovine mammal, said method comprising the dete ⁇ nination of prion protein (PrP) in a body tissue or body fluid sample from said mammal, by the novel immunometric method of this invention.
- TSE transmissible spongiform encephalopathy
- PrP prion protein
- this invention concerns a method for screening blood samples, particularly blood samples obtained from human blood donors, to investigate whether a sample is infected by a transmissible spongiform encephalopathy (TSE), said method comprising the determination of prion protein (PrP) in a sample of whole blood or blood fraction from said blood donor, by the novel immunometric method of this invention.
- TSE transmissible spongiform encephalopathy
- PrP prion protein
- Figure 2 shows a calibration curve for bovine PrP.
- Figure 3 demonstrates an assay of PrP in hamster brain tissue where detected PrP concentration is plotted versus Proteinase K concentration in the experiment (filled bars: scrapie, and striped bars: normal).
- Figure 4 demonstrates an assay of PrP in bovine brain tissue where detected PrP concentration is plotted versus Proteinase K concentration in the experiment (filled bars: BSE, and striped bars: normal).
- Figure 5 demonstrates an assay of PrP in ovine brain tissue where detected PrP concentration is plotted versus Proteinase K concentration in the experiment (filled bars: scrapie, and striped bars: normal).
- This invention relates to a novel two-site immunometric assay for the measurement of prion protein (PrP).
- the method involves the use of a capture antibody that recognises an epitope, preferably an epitope in the N-terminal region of the molecule.
- the detecting antibody recognises preferably an epitope in the protease resistant core of the PrP molecule that is occluded when the PrP is in an aggregated state.
- the use of a limited concentration of proteolytic enzyme facilitates the disaggregation of the PrP. This leads to an increase in the binding of the detector antibody.
- N-terminal region shall particularly mean the region defined by aa 50 to 95 in the PrP molecule. It shall, however, be noted that either or both of the capture and detecting antibodies may be directed to epitopes in the N-terminal region, the protease resistant core, or the C-terminal region of the PrP molecule.
- the treatment of the PrP may include, for example, any of the following: (i) limited proteolysis; (ii) addition of detergents; (iii) addition of chaotropic agents and/or solvents; (iv) variation of temperature; and (v) sonication. These treatments can be used individually or in combination.
- the "protease resistant core of the PrP molecule” is the part of the PrP molecule that is not degraded by proteolysis.
- the normal metabolic cleavage of the molecule is in the amino acid region 109 - 112.
- the cleavage caused by, for example, a proteolytic enzyme depends on the tissue, cell type, mammal etc..
- the cleavage takes place in the region of position 90 for 263K strain in LVG hamsters.
- the capture antibody as well as the detecting antibody are preferably both monoclonal antibodies.
- the detecting antibody can be labelled with any detectable label.
- the label is a lanthanide chelate, wherein the detection is based on time-resolved fluorescence.
- the sample can be, for example, brain tissue, spinal cord, lymphoid tissue, spleen, tonsil, whole blood or a blood fraction.
- brain tissue is a particularly prefe ⁇ ed sample.
- body fluid samples can be mentioned whole blood or fractions thereof.
- the mammal can be any mammal, i.e. a primate (a human individual or monkeys) or a non-primate.
- the use of the antibody FHl 1 as a capture reagent is new and nonobvious in any assay system for the detection of PrP sc using ProteinaseK (PK).
- PK ProteinaseK
- Conventional assay systems e.g. Western Blot analysis
- PK ProteinaseK
- the 3F4 antibody has been widely used to detect PrP in hamster and human tissue samples. However, it has never been shown to cross-react with ovine or bovine PrP in Western Blot analysis. Consequently, its use in an assay for the measurement of PrP in ovine or bovine tissue is new and nonobvious. It has proved to be a very useful reagent because of the increased sensitivity obtainable in DELFIA which allows for the exploitation of the low cross-reaction of 3F4 for the ovine and bovine epitope.
- the other novel aspect of the use of this antibody is the finding that the 3F4 epitope is in the 'core' region of the molecule which is involved in the aggregation process.
- PrP When PrP is in an aggregated state, the 3F4 epitope is occluded and is not available for antibody binding.
- PrP When PrP is disaggregated by limited PK digestion, treatment with a chaotropic agent (e.g. > 2M guanidinium chloride) and/or detergent addition, the 3F4 epitope becomes exposed, the antibody binds and the signal increases.
- a chaotropic agent e.g. > 2M guanidinium chloride
- PK at low dose may facilitate the measurement of highly aggregated PrP by:
- the PK effect may be achieved by the addition of limiting concentrations of PK. Increased concentrations of the enzyme will result in the cleavage of PrP resulting in the ultimate loss of the primary FHl 1 epitope.
- 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
- DELFIA (trademark) reagents were obtained from: EG & G Wallac Ltd, 20
- wash solution was prepared by diluting wash concentrate 25-fold (i.e. 40 mL concentrate diluted to 1 litre) with distilled water.
- Triton X-100 Ready for use with Triton X-100, acetic acid and chelators. Stored between 2-25 degrees Celsius until expiry date. Direct sunlight avoided.
- kits contains 0.2 mg labelling reagent plus Eu-standard, enhancement solution, stabiliser (purified BSA) for increasing the stability of the labelled protein, an uncoated microtitration strip plate, assay buffer and wash concentrate.
- the kit contents were sufficient for a labelling of up to 1 mg of protein. Stored between 2-8 degrees Celsius until expiry date.
- DELFIA plates were in strip format 8 x 12 and manufactured by NUNC from a plastic with a low fluorescent background. They had a high immunoglobulin binding surface (MAXISORB).
- ImmunoPure IgG binding buffer 1000 mL; pH 8.0
- the buffer contains EDTA as a preservative.
- ImmunoPure IgG elution buffer 500 ml; pH 2.8). The buffer has been purged with nitrogen to exclude oxygen. The absence of oxygen and low pH allowed good reagent stability without the use of a preservative.
- Microcon concentrators employed Amicon's low binding, anisotropic, hydrophilic YM membrane.
- the Microcon-30 used a membrane with a molecular weight cut-off of 30,000 KDa.
- the devices were usable in any Eppendorf centrifuge (e.g. MSE Micro-Centaur Microfuge) and offer a simple, efficient, means of concentrating, desalting and purifying proteins before and after labelling with lanthanide chelate (or biotin).
- Sodium bicarbonate buffered saline 50 mmol/L was prepared by dissolving 4.2 g NaHC0 3 (Sigma S-8875) and 9.0 g NaCl (Sigma S-9625) in 1 litre of ultra pure water (Milli-Q or equivalent). The pH of the resulting solution is adjusted with NaOH (2N; Aldrich 22, 146-5) to between 8.5 and 9.0. The solution was stored between 2-8 degrees Celsius.
- Phosphate Buffered Saline (Sigma 1000-3) prepared by dissolving sachet contents in 1 litre of distilled water producing a solution containing NaCl (120 mmol/L), KCl (2.7 mmol/L) and phosphate buffer (10 mmol/L), pH 7.4 at 25 degrees Celsius.
- NaCl 120 mmol/L
- KCl 2.7 mmol/L
- phosphate buffer 10 mmol/L
- pH 7.4 pH 7.4
- sodium azide (Sigma S-2002; 0.1%) may be added. The solution was stored between 2-8 degrees Celsius.
- Phosphate Buffered Saline (Sigma 1000-3) with addition of sodium azide (Sigma S-2002; 0.1%) and bovine serum albumin (Sigma A-7888; 2%). Stored between 2-8 degrees Celsius.
- the stock solution was estimated at 0.48 mg/mL and was stored in chaotropic buffer containing 4M Guanidinium hydrochloride at - 20 degrees Celsius.
- Stock PrP-GST fusion protein was serially diluted ten-fold in assay buffer to give a series of standards as follows: (i) buffer blank; (ii) 0.48 ng/mL; (iii) 4.8 ng/mL; (iv) 48 ng/mL; (v) 480 ng/mL; and, (vi) 4800 ng/mL.
- Subsequent recombinant Hamster PrP protein comprised extracts of a histidine-tagged PrP protein in chaotropic buffer solution.
- the protein concentration of these extracts ranged from 0.78 to 8 mg/mL.
- the stock solutions are stored at -20 degrees Celsius.
- Recombinant bovine PrP (4 mg/mL) stock solution was stored at -20 degrees Celsius.
- the stock solution was serially diluted in assay buffer to give a series of standards as follows: (i) buffer blank; (ii) 4 ng/mL; (iii) 40 ng/mL; (iv) 400 ng/mL; (v) 4000 ng/mL; and, (vi) 40,000 ng/mL.
- the standards were prepared fresh prior to each experiment. Purification of antibodies using immunopure (A) IGG kit
- the specific IgG was eluted, following application of elution buffer.
- the presence of protein in the eluate was detected by the monitor and the buffer collected in a glass vial.
- the concentration of the eluted protein was estimated by the measurement of absorbance at 280 nM using a spectrophotometer.
- the column was regenerated by washing with 4 column volumes of 0.1 M citric acid, adjusted to pH 3.0 with 6 N NaOH. For storage, the column is washed with an additional 5 ml of water containing 0.02 % sodium azide.
- the bound label can be removed by gel filtration on a Sephadex G-50 column. Elution can be carried out with TBS (as above) and the column decontaminated by washing with phthalate buffer, 10 mmol/L, pH 4, containing 0.001 % DTPA. Possible aggregates present in the protein fraction or formed during labelling sometimes cause elevated backgrounds in solid phase assays. These aggregates can be removed by using suitable gel filtration media (e.g.Sepharose 6B or Sephacryl S400). Characterization of the labelled protein
- the europium content of the labelled antibody was determined by diluting the labelled product in enhancement solution (1 : 10,000 v/v) and measuring the signals obtained in the time-resolved fluorometer. The counts are compared with the signal generated from a 1: 100 dilution of europium standard in enhancement solution (equivalent to 1 nmol/L Eu3 + ).
- the protein concentrations of the labelled antibody was determined using a spectrophotometer at 280 nm. This concentration must be adjusted by co ⁇ ecting for the absorbance generated by the thiourea bond (0.008A 1 ⁇ mol/L of Eu at levels of less than 20 Eu/IgG).
- the labelled antibodies were stored at high concentration and in the absence of competing metals (or chelators) in the buffer.
- a concentrated solution (0.050 mg/mL) was stored at +4 degrees Celsius.
- the stability can be increased by adding small amounts (about 0.1%) of stabiliser contained in the labelling kit which consists of BSA which has been purified free from heavy metal contaminants.
- microtitration plates On the day prior to the DELFIA assay, the required number of microtitration plates were coated with purified monoclonal FHl 1 diluted in coating buffer (1 : 1000 v/v). Two hundred (200) ⁇ L of diluted antibody solution were added to each well using a multichannel pipette. The plates were covered and incubated overnight at 4 degrees Celsius. Next day, the plates were washed three times using the WALLAC plate washer.
- Brain tissue was carefully weighed and transfe ⁇ ed to sterile Dounce homogeniser. 1.5 mL of homogenisation buffer was added to each gram of brain tissue.
- Homogenisation buffer A PBS (without detergent) or B: lMGdnCL in 50mM Tris-HCl pH 7.5 containing 0.5% sulphabetaine (3-14)
- the brain tissue was homogenised with 10 - 15 passes using the 'loose' pestle.
- Proteinase K (Sigma or Boehringer) was carefully weighed and dissolved in PBS to provide an appropriate stock solution. An aliquot of the PK solution was added to the emulsion in the polycarbonate tubes to give a final concentration of PK within the range 0 to 100 ⁇ g/mL. The tubes were sealed with PVC tape and incubated at 37°C for 30 minutes with gentle agitation. The digestion was stopped by the addition of 10 ⁇ L Pefabloc SC (0.5 M solution in PBS).
- the polycarbonate tubes were transfe ⁇ ed to a Beckman TL100.3 rotor and centrifuged @ 100,000 rpm for 8 minutes at 22°C in TL100 ultracentrifuge. The supernatant was removed and retained (primary supernatant). Extraction buffer was added equal to the volume of supernatant removed. Extraction buffer: 6MGdnHCl in 50mM Tris-HCl pH 7.5 containing 0.5% sulphabetaine (3-14) The pellet was resuspended by trituration with a plastic disposable pasteur pipette. The samples were incubated for 5 minutes at room temperature.
- the polycarbonate tubes were centrifuged @ 100,000 rpm for a further 8 minutes at 22°C in TL100 ultracentrifuge. The supernatant in designated the secondary extract.
- the time-resolved fluorescence immunoassay of PrP in brain tissue extracts from scrapie-infected and normal hamsters is shown in Figure 3.
- the time-resolved fluorescence immunoassay of PrP in brain tissue extracts from BSE-infected and normal cattle is shown in Figure 4
- the time-resolved fluorescence immunoassay of PrP in brain tissue extracts from scrapie-infected and normal sheep is shown in Figure 5.
- a typical PK titration experiment involved the production 10 mL of total brain homogenate. This was equivalent to 2.5 grams of brain tissue (approximately 2.5 brains). 10 aliquots (0.8 mL) were tr.ansfe ⁇ ed to separate polycarbonate tubes and added the same volume of PK at 10 different concentrations to ensure that each tube was comparable. This full experiment was repeated 4 times with normal and scrapie hamster brains and gave essentially identical results both in terms of concentration and PK titration profile. This approach was adopted for bovine and ovine brain tissue and similar results were obtained.
- PK has a dual effect. It not only digests PrPc but facilitates the measurement of PrP in the primary and secondary extracts.
- PK releases' up to 100 times more PrP in hamster brain tissue than can be measured in samples processed without PK or in the presence of 100 ⁇ g/mL PK.
- Bovine spongiform encephalopathy - epidemiological-studies. Vet. Rec. 123, 638-644 (1988).
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EP99954029A EP1135687A1 (en) | 1998-11-17 | 1999-10-27 | An immunoassay for the determination of transmissible spongiform encephalopathies in mammals |
JP2000582801A JP2002530649A (en) | 1998-11-17 | 1999-10-27 | Immunoassay for determining infectious spongiform encephalopathy in mammals |
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FI982480A FI982480A0 (en) | 1998-11-17 | 1998-11-17 | Immunoassay for the detection of infectious spongiform encephalopathy in mammals |
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US7429463B2 (en) | 1999-11-12 | 2008-09-30 | Commissariat A L'energie Atomique | Method for diagnosing a transmissible spongiform subacute encephalopathy caused by an unconventional transmissible agent strain in a biological sample |
WO2002033420A3 (en) * | 2000-10-22 | 2003-01-03 | Hadasit Med Res Service | A urine test for the diagnosis of prion diseases |
US8110391B2 (en) | 2001-01-08 | 2012-02-07 | Health Protection Agency | Degradation and detection of TSE infectivity |
US7303907B2 (en) | 2001-01-08 | 2007-12-04 | Health Protection Agency | Degradation and detection of TSE infectivity |
WO2002057793A3 (en) * | 2001-01-19 | 2003-03-06 | Baxter Int | Method of detecting prp protein and kits therefor |
WO2002097443A3 (en) * | 2001-05-31 | 2003-07-31 | Sec Dep For Environment Food & | Method for diagnosing transmissable spongiform encephalopathy |
GB2391624B (en) * | 2001-05-31 | 2005-07-20 | Sec Dep For Environment Food & | Method for typing a strain of transmissible spongiform encephalopathy |
GB2391624A (en) * | 2001-05-31 | 2004-02-11 | Sec Dep For Environment Food & | Method for diagnosing transmissable spongiform encephalopathy |
WO2003040685A3 (en) * | 2001-11-09 | 2003-12-31 | King S College London | Diagnosis demyelinating or spongiform disease |
US7439041B2 (en) | 2003-08-13 | 2008-10-21 | Novartis Vaccines And Diagnostics, Inc. | Prion-specific peptide reagents |
WO2005114214A1 (en) * | 2004-05-14 | 2005-12-01 | Prionics Ag | Method for the detection of disease-related prion |
EP1596199A1 (en) * | 2004-05-14 | 2005-11-16 | Prionics AG | Method for the detection of disease-related prion |
US7834144B2 (en) | 2005-09-09 | 2010-11-16 | Novartis Ag | Prion-specific peptoid reagents |
Also Published As
Publication number | Publication date |
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EP1135687A1 (en) | 2001-09-26 |
JP2002530649A (en) | 2002-09-17 |
FI982480A0 (en) | 1998-11-17 |
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