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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- 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
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- C12Q1/6804—Nucleic acid analysis using immunogens
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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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| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |