CA2237739A1 - A specific and sensitive method to detect prpsc in peripheral blood using a capture-immunosorbent assay-polymerase chain reaction (prp-immuno-pcr) - Google Patents
A specific and sensitive method to detect prpsc in peripheral blood using a capture-immunosorbent assay-polymerase chain reaction (prp-immuno-pcr) Download PDFInfo
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- CA2237739A1 CA2237739A1 CA 2237739 CA2237739A CA2237739A1 CA 2237739 A1 CA2237739 A1 CA 2237739A1 CA 2237739 CA2237739 CA 2237739 CA 2237739 A CA2237739 A CA 2237739A CA 2237739 A1 CA2237739 A1 CA 2237739A1
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- 238000000034 method Methods 0.000 title claims abstract description 14
- 238000003752 polymerase chain reaction Methods 0.000 title abstract description 3
- 239000003547 immunosorbent Substances 0.000 title 1
- 210000005259 peripheral blood Anatomy 0.000 title 1
- 239000011886 peripheral blood Substances 0.000 title 1
- 102000029797 Prion Human genes 0.000 claims abstract description 17
- 108091000054 Prion Proteins 0.000 claims abstract description 17
- 210000004369 blood Anatomy 0.000 claims abstract description 8
- 239000008280 blood Substances 0.000 claims abstract description 8
- 208000018756 Variant Creutzfeldt-Jakob disease Diseases 0.000 claims abstract 6
- 208000005881 bovine spongiform encephalopathy Diseases 0.000 claims abstract 6
- 208000020406 Creutzfeldt Jacob disease Diseases 0.000 claims description 8
- 208000010859 Creutzfeldt-Jakob disease Diseases 0.000 claims description 8
- 208000003407 Creutzfeldt-Jakob Syndrome Diseases 0.000 claims 4
- 230000015572 biosynthetic process Effects 0.000 claims 2
- 239000012530 fluid Substances 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 abstract description 9
- 230000002458 infectious effect Effects 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 238000003556 assay Methods 0.000 abstract description 3
- 230000001413 cellular effect Effects 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 8
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- 239000013504 Triton X-100 Substances 0.000 description 5
- 229920004890 Triton X-100 Polymers 0.000 description 5
- 239000003599 detergent Substances 0.000 description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 102100034452 Alternative prion protein Human genes 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 108010067770 Endopeptidase K Proteins 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 208000003736 Gerstmann-Straussler-Scheinker Disease Diseases 0.000 description 2
- 206010072075 Gerstmann-Straussler-Scheinker syndrome Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 102100025818 Major prion protein Human genes 0.000 description 2
- 101710138751 Major prion protein Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 201000006061 fatal familial insomnia Diseases 0.000 description 2
- 102000009091 Amyloidogenic Proteins Human genes 0.000 description 1
- 108010048112 Amyloidogenic Proteins Proteins 0.000 description 1
- 101710095339 Apolipoprotein E Proteins 0.000 description 1
- 102100029470 Apolipoprotein E Human genes 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 208000024556 Mendelian disease Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 231100000875 loss of motor control Toxicity 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- Proteomics, Peptides & Aminoacids (AREA)
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- Food Science & Technology (AREA)
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Abstract
Currently, there is no method to identify with high specificity and sensitivity, the prion proteins (PrP sc) that lead to the development of the human diseases, Cruetzfeldt-Jakob Disease (CJD) and new variant CJD (nvCJD), also known as bovine spongiform encephalopathy (BSE).
The invention is such a diagnostic assay and hereforth defined as PrP-Immuno-PCR. The method involves the combination of two well-described techniques to detect as little as one infectious unit from whole blood using monoclonal antibodies specific to the disease-causing prion proteins and the polymerase chain reaction to amplify such signal using a novel antibody-DNA complex. The method also differentiates the infectious prion PrP sc from a non-infectious cellular prion protein (PrP c) which is found in all healthy individuals.
The invention is such a diagnostic assay and hereforth defined as PrP-Immuno-PCR. The method involves the combination of two well-described techniques to detect as little as one infectious unit from whole blood using monoclonal antibodies specific to the disease-causing prion proteins and the polymerase chain reaction to amplify such signal using a novel antibody-DNA complex. The method also differentiates the infectious prion PrP sc from a non-infectious cellular prion protein (PrP c) which is found in all healthy individuals.
Description
SPECIFICATION
The invention allows for the identification of singular infectious units of the prion protein that is known to cause CJD, nvCJD and other hereditary diseases such as Gerstmann-Straussler-Scheinker syndrome (GSS) and Fatal-Familial Insomnia (FFI).
Until recently, diagnosis of CJD has been twofold: clinical identification of CJD-like symptoms, which include loss of motor control, dementia, paralysis wasting, and post-mortem histological identification of aggregates or plaques of amyloid protein, which have become insoluble, in the brain. The change in diagnostics came when Pruisner determined that the infectious agent was merely a protein that had somehow crossed the brain-blood barrier and passively caused an aggregation of the surrounding protein. His theories eventually led to the identification of the disease-causing prion protein (PrP~').
Several different antibodies have been developed to detect both the cellular and the infectious, aggregate forms of the prion protein. Of these, one monoclonal antibody, 3F4, is specific for the 109-112 epitope of PrPg° post proteinase K digestion.
Another antibody, known as anti-C, is specific for the epitope spanning residues 220-231. Both have shown the ability to recognize the same prion protein by Western Blot analysis. Several other antibodies have been developed to recognize PrPs° but their use has been limited in the literature.
Diagnosis by western blotting of the prion protein in brain and CSF has been shown for both humans and animals. More recent unpublished results show that western blots can identify as little as 100 infectious units in these tissue samples and in plasma.
However, in the context of blood, when compared to the fact that as little as one infectious unit may be needed to develop CJD, the validity of this test potentially drops significantly. The other problem is that western blots cannot determine an actual amount of protein with respect to a single aggregate. Only estimations can be made in terms of actual protein quantities.
Recently, other assays have been developed to identify proteins that may or may not be associated with CJD. Of these, apolipoprotein E, 14-3-3, and S 100 serum protein seem to be possible candidates for detection. However, their presence does not directly confer CJD infection as the prion protein is not targeted.
The invention (PrP-Immuno-PCR), overcomes the sensitivity barrier by incorporating a highly-sensitive technique, known as polymerase chain reaction, which amplifies signals of DNA.
In order to create an amplified signal, the DNA must be somehow linked to the prion protein.
This is accomplished by primarily performing a modification of the 'capture' enzyme-linked immunosorbent assay (ELISA), in which the secondary antibody is covalently linked to streptavidin The antibody is then subjected to a piece of pBR322 DNA
previously amplified and labeled with biotin using either enzymatic or light activation. The attached DNA is then amplified using PCR and the resultant product is resolved by agarose gel electrophoresis. The entire reaction is easily performed in a single well of a 96 well plate and should be at least 1000 times more sensitive than associated ELISA techniques alone.
The actual protocol for the technique is as follows and is illustrated in part in Figure 1, where possible:
1) The night before the technique is to be performed, the wells of a 96-well plate are coated with rabbit serum, which contains polyclonal antibodies against a C-terminal region of the prion protein, more specifically, amino acids 220-223.
CONFIDENTIAL AND PROPRIETARY INFORMATION
~ Jason A. Tetro, B. Sc., 1998 2) The following morning, the plate is washed 5 times with a solution of phosphate-buffered-saline in which 0.5% of a detergent (either Tween-20 or Triton X-100) has been added.
The invention allows for the identification of singular infectious units of the prion protein that is known to cause CJD, nvCJD and other hereditary diseases such as Gerstmann-Straussler-Scheinker syndrome (GSS) and Fatal-Familial Insomnia (FFI).
Until recently, diagnosis of CJD has been twofold: clinical identification of CJD-like symptoms, which include loss of motor control, dementia, paralysis wasting, and post-mortem histological identification of aggregates or plaques of amyloid protein, which have become insoluble, in the brain. The change in diagnostics came when Pruisner determined that the infectious agent was merely a protein that had somehow crossed the brain-blood barrier and passively caused an aggregation of the surrounding protein. His theories eventually led to the identification of the disease-causing prion protein (PrP~').
Several different antibodies have been developed to detect both the cellular and the infectious, aggregate forms of the prion protein. Of these, one monoclonal antibody, 3F4, is specific for the 109-112 epitope of PrPg° post proteinase K digestion.
Another antibody, known as anti-C, is specific for the epitope spanning residues 220-231. Both have shown the ability to recognize the same prion protein by Western Blot analysis. Several other antibodies have been developed to recognize PrPs° but their use has been limited in the literature.
Diagnosis by western blotting of the prion protein in brain and CSF has been shown for both humans and animals. More recent unpublished results show that western blots can identify as little as 100 infectious units in these tissue samples and in plasma.
However, in the context of blood, when compared to the fact that as little as one infectious unit may be needed to develop CJD, the validity of this test potentially drops significantly. The other problem is that western blots cannot determine an actual amount of protein with respect to a single aggregate. Only estimations can be made in terms of actual protein quantities.
Recently, other assays have been developed to identify proteins that may or may not be associated with CJD. Of these, apolipoprotein E, 14-3-3, and S 100 serum protein seem to be possible candidates for detection. However, their presence does not directly confer CJD infection as the prion protein is not targeted.
The invention (PrP-Immuno-PCR), overcomes the sensitivity barrier by incorporating a highly-sensitive technique, known as polymerase chain reaction, which amplifies signals of DNA.
In order to create an amplified signal, the DNA must be somehow linked to the prion protein.
This is accomplished by primarily performing a modification of the 'capture' enzyme-linked immunosorbent assay (ELISA), in which the secondary antibody is covalently linked to streptavidin The antibody is then subjected to a piece of pBR322 DNA
previously amplified and labeled with biotin using either enzymatic or light activation. The attached DNA is then amplified using PCR and the resultant product is resolved by agarose gel electrophoresis. The entire reaction is easily performed in a single well of a 96 well plate and should be at least 1000 times more sensitive than associated ELISA techniques alone.
The actual protocol for the technique is as follows and is illustrated in part in Figure 1, where possible:
1) The night before the technique is to be performed, the wells of a 96-well plate are coated with rabbit serum, which contains polyclonal antibodies against a C-terminal region of the prion protein, more specifically, amino acids 220-223.
CONFIDENTIAL AND PROPRIETARY INFORMATION
~ Jason A. Tetro, B. Sc., 1998 2) The following morning, the plate is washed 5 times with a solution of phosphate-buffered-saline in which 0.5% of a detergent (either Tween-20 or Triton X-100) has been added.
3) The plate is then coated for three hours with a 1.0% skim milk powder solution, also known as Blotto.
3a) While the plate is sitting, 10 ml of blood is spinned down (a) and the plasma and white blood cells are taken off and placed into separate 0.5 ml vials (b). The mixture is treated with Proteinase K at a concentration of 50 ~g/ml for a period of 1 hour at 37°C. After 1 hour, the mixture is denatured at either 56°C for 20 minutes (half) or 95°C for 5 minutes (other half) (c).
3a) While the plate is sitting, 10 ml of blood is spinned down (a) and the plasma and white blood cells are taken off and placed into separate 0.5 ml vials (b). The mixture is treated with Proteinase K at a concentration of 50 ~g/ml for a period of 1 hour at 37°C. After 1 hour, the mixture is denatured at either 56°C for 20 minutes (half) or 95°C for 5 minutes (other half) (c).
4) The plate is washed 5 times with a solution of phosphate-buffered-saline in which 0.5% of a detergent (either Tween-20 or Triton X-100) has been added.
5) The denatured blood mixture is then added to the wells in amounts of 100 ~1 to each well and allowed to sit for 2 hours at 37°C (d).
6) The plate is washed 5 times with a solution of phosphate-buffered-saline in which 0.5% of a detergent (either Tween-20 or Triton X-100) has been added.
7) A solution containing a streptavidin-linked, monoclonal antibody, namely 3F4, which recognizes the prion protein at amino acids 109-112, is then added and incubated for 1 hour at 37°C (e).
8) The plate is washed 5 times with a solution of phosphate-buffered-saline in which 0.5% of a detergent (either Tween-20 or Triton X-100) has been added.
9) A solution containing a concentration of DNA previously PCR-amplified from pBR
322 (334-715) and biotinylated with either biotinylated primers or by post-PCR
phot-active biotinylation is then added to the plate and incubated for 30 minutes at 37°C (f).
322 (334-715) and biotinylated with either biotinylated primers or by post-PCR
phot-active biotinylation is then added to the plate and incubated for 30 minutes at 37°C (f).
10) The plate is washed 5 times with a solution of phosphate-buffered-saline in which 0.5% of a detergent (either Tween-20 or Triton X-100) has been added.
11) A PCR mixture containing 1X PCR buffer (containing potassium chloride, Tris buffer, magnesium chloride), primers which span the 334-71 S region of the pBR322 plasmid and Taq DNA polymerase is added to the plate and a 45 cycle PCR run (g) is performed using the following profile:
~ 95°C for 1 minute 56°C for 30 seconds 72°C for 30 seconds 12) When the run is finished, the product is run on an 0.8% agarose gel for electrophoresis. A product approximately 280 base pairs should be seen on the gel after ethidium bromide staining (h).
The test is run with two controls: a negative control consisting of uninfected human whole blood and a positive control consisting of a spiked blood sample with 1000 infectious units.
CONFIDENTIAL AND PROPRIETARY INFORMATION
~ Jason A. Tetro, B. Sc., 1998 OTHER APPLICATIONS APPLICABLE TO THIS INVENTION
This technique can also be used to detect prions in cordal blood as in the described claim, and also in tissues, using a technique well described in scientific literature to pre-process samples prior to proteinase K digestion and subsequent assay using the described claim.
CONFIDENTIAL AND PROPRIETARY INFORMATION
~ Jason A. Tetro, B. Sc., 1998
~ 95°C for 1 minute 56°C for 30 seconds 72°C for 30 seconds 12) When the run is finished, the product is run on an 0.8% agarose gel for electrophoresis. A product approximately 280 base pairs should be seen on the gel after ethidium bromide staining (h).
The test is run with two controls: a negative control consisting of uninfected human whole blood and a positive control consisting of a spiked blood sample with 1000 infectious units.
CONFIDENTIAL AND PROPRIETARY INFORMATION
~ Jason A. Tetro, B. Sc., 1998 OTHER APPLICATIONS APPLICABLE TO THIS INVENTION
This technique can also be used to detect prions in cordal blood as in the described claim, and also in tissues, using a technique well described in scientific literature to pre-process samples prior to proteinase K digestion and subsequent assay using the described claim.
CONFIDENTIAL AND PROPRIETARY INFORMATION
~ Jason A. Tetro, B. Sc., 1998
Claims (2)
1) The PrP-Immuno-PCR technique therein defined may be used to detect, from whole blood, prion proteins which lead to the formation of Creutzfeldt-Jakob disease as well as the new variant Creutzfeldt-Jakob disease, also known as Bovine Spongiform Encephalopathy (BSE).
2) The PrP-Immuno-PCR technique therein defined may be used to detect, in spinal fluid, prion proteins which lead to the formation of Creutzfeldt-Jakob disease as well as the new variant Creutzfeldt-Jakob disease, also known as Bovine Spongiform Encephalopathy (BSE).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2237739 CA2237739A1 (en) | 1998-06-23 | 1998-06-23 | A specific and sensitive method to detect prpsc in peripheral blood using a capture-immunosorbent assay-polymerase chain reaction (prp-immuno-pcr) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2237739 CA2237739A1 (en) | 1998-06-23 | 1998-06-23 | A specific and sensitive method to detect prpsc in peripheral blood using a capture-immunosorbent assay-polymerase chain reaction (prp-immuno-pcr) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2237739A1 true CA2237739A1 (en) | 1999-12-23 |
Family
ID=29275724
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2237739 Abandoned CA2237739A1 (en) | 1998-06-23 | 1998-06-23 | A specific and sensitive method to detect prpsc in peripheral blood using a capture-immunosorbent assay-polymerase chain reaction (prp-immuno-pcr) |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2237739A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1596199A1 (en) * | 2004-05-14 | 2005-11-16 | Prionics AG | Method for the detection of disease-related prion |
-
1998
- 1998-06-23 CA CA 2237739 patent/CA2237739A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1596199A1 (en) * | 2004-05-14 | 2005-11-16 | Prionics AG | Method for the detection of disease-related prion |
| WO2005114214A1 (en) * | 2004-05-14 | 2005-12-01 | Prionics Ag | Method for the detection of disease-related prion |
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