CA2069731A1 - Method for detection of the beta-amyloid protein using unspecific binding of oligonucleotides - Google Patents
Method for detection of the beta-amyloid protein using unspecific binding of oligonucleotidesInfo
- Publication number
- CA2069731A1 CA2069731A1 CA 2069731 CA2069731A CA2069731A1 CA 2069731 A1 CA2069731 A1 CA 2069731A1 CA 2069731 CA2069731 CA 2069731 CA 2069731 A CA2069731 A CA 2069731A CA 2069731 A1 CA2069731 A1 CA 2069731A1
- Authority
- CA
- Canada
- Prior art keywords
- amyloid
- oligonucleotides
- beta
- amyloid protein
- precursor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- 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
- G01N2800/2821—Alzheimer
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method for detecting the .beta.-amyloid protein from a sample by reacting the sample with an oligonucleotide. The method is especially suitable to be used for detecting an amyloidosis related to e.g. Alzheimer's disease.
Description
~092/06376 PCT/FI91/00300 .`
2~73 Method for detection of the ~-amvloid protein usina unspecific bindina of oli~onucleotides Technical field The invention relates to the detection of proteins by means of oligonucleotides. The invention may be utilized especially in diagnostics.
Background Alzheimer's disease is a disease resulting in a difficult dementia, in which the risk of falling ill increases along with aging. Symptoms of Alzheimer's disease are the progressive deterioration of memory, perception and other higher brain functions, which finally results in the patient's complete feeblemindedness.
Although the reasons for the outbreak of Alzheimer's disease are not known, the pathological changes caused by it are fairly well-known. The most typical changes are the extracellular accumulation of amyloid protein on the walls of the cerebral membranes and blood vessels as well as as senile plaques. In addition, the cyto-skelenton components of neurons aggregate intracellularly as so-called neurofi-brillary tangles. In both cases, the filamentary proteins may completely change the structure of the neuron, which finally results in the dystrophy and cell death of the neurons. The number of amyloid plaques correlates fairly well with the deterioration of cognitive functions, the neuron loss and the decrease in neurotransmitters (Tanzi et al. 1989).
Senile or neuritic plagues consist of an amyloid core, which is surrounded by a periphery comprised of dystrophic neurites and extracellular material. The plaques may be dense, so-called classic plaques or diffuse concentrations : - . , -, ~: , -: , : :- ' : :,' :
- - :- - . :. . : . , - . -....
W O 92/06376 P ~ tF191/00300 206~731 2 formed of loosely interconnected material. Diffuse plaques have in fact been assumed to be pre-stages of denser formations, which may form during several years without first causing neurologic symptons (Joachim et al. 1989, Tanzi 1989).
The amyloid in the senile plaques forms of 6-10 nm stranded ~-amyloid protein, whose identical subunits are arranged antiparallelly into a beta-structure. This hydrofobic protein of ca. 4.2-4.5 kDa is synthesized most likely in neurons as part of a longer ~-amyloid precursor protein. The gene coding for the precursor protein is localized in the chromosome 21 (Goldgaber et al. 1978, ~-Tanzi 1987).
One reason for the cumulation of amyloid has been assumed to be a defect in the gene locus, since a correspondiny plaque formation also occurs on nearly all Down's syndrome patients of over 40 years (trisomy in chromosome 21), whereby the overproduction of amyloid can be explained by means of an extra gene. An autosomally dominant gene defect in the chromosome 21 has been observed in patients with the hereditary Alzheimer's disease, but it has not been possible to prove a connection between it and the ~-amyloid gene located in the same chromosome (St. George-Hyslop et al. 1978, Tanzi et al. 1987).
The ~-amyloid precursor is a receptor-like, transmembranal glycosylated protein, in which two thirds are located in the extracellular state (Kang et al. 1987). The ~-amyloid dipeptide is in turn located partly in the transmembranal area so that its proteolytic release from the precursor occurs either before the precursor's loosening onto or after loosening from cellular membrane (Tanzi 1989).
It has been observed that the ~-amyloid gene codes for at least three transcripts acting as models for polypeptides ,' '. '~ - . ' : ,. . . : .
- .' - '.'' : , : , : . ' '' , .-.- . . .
-YYO 92/06376 P ~ /FI91/~0300 ~ ` 3 2~973~
with 695, 751 and 770 amino acids. The two longer polypep-tides contain an extra sequence with 56 the amino acid, which is 50% homogenous with the enzymes belonging to the so-called Kuniz's serine protease inhibitor family. Two contradictory hypotheses corcerning the possible share of the protease inhibitor sequence in the aggregation of the ~-amyloid have been presented: 1) it may prevent the cumulation of amyloid by inhibiting proteases which release ~-peptide from the precursor, or 2) the protease inhibitor may advance the cumulation of amyloid by preventing specific proteinases from purifying the tissue from ~-amyloid. The function of the protease inhibitor sequence of the ~-amyloid precursor protein and also its release from the precursor into an active enzyme are thus not known for certain.
General description of invention The invention and certain preferred applications thereof are defined in the patent claims.
The invention is based on the surprising discovery that oligonucleotides bind to the ~-amyloid protein or its precursor. The ~-amyloid protein or its precursor thus binds DNA. The binding occurs especially in the ~-amyloid protein or its precursor present in a peripheral tissue (e.g. skin, mucous membrane). The phenomenon may be utilized especially in diagnostic tests (e.g. Alzheimer's disease, Down's syndrome, dementias, old persons).
Detailed description of invention The object of our examination was the expression of ~-amyloid on the frontal cortex and hippocampus of Alzheimer patients. As a research method was used the RNA in situ hybridization and as a specific tester was a cocktail formed by three short oligonucleotides corresponding to . . .
~ ~ ; -, -: - . .
- - . ..
. :'- ' . . : ' : :
- . - . . : ~ :
W092/06376 :2 ~ ~ ~7 31 PCT/F191/00300 different areas of the ~-amyloid sequence. The oligonuc-leotides were labelled at the 3'-end with biotin. For specific antisense-RNA-oligonucleotides were synthesized complementary sense-RNA-oligonucleotides, which were labelled in the same way as the antisense probes. The in situ hybridization was performed by using the routine methods of our laboratory (a hybridized biotinylated intron is allowed to bind to a streptavidine-alkaline phosphatase complex, which is in turn detected by means of a substrate reaction). The brain samples of 3 different Alzheimer patients as well as two normal samples were examined. As a result could be observed strong signals in the area of the cortex and hippocampus corresponding both by their location and shape to senile plaques. In additi-on, a positive colouring was seen in the walls of blood vessels, as described in connection with the ~-amyloid protein. When we used the sense probe, we could to our surprise see the same result as when using the antisense probe. (When a specific mRNA expression is examined, the sense probe should produce a negative result.) A negative result was by contrast obtained, when water was used instead of the gene probe; the reactions were otherwise performed in the same way as when using the senselantisen-se probe. All colouring results were negative, when the normal samples were subjected to examination.
The in situ hybridization was repeated several times to preclude a possible false positive reaction caused by endogenic biotin or alkaline phosphatase.
Furthermore, the in situ hybridization was repeated by using a radioactive label or other enzymes for the detection of a biotinylized hybrid (e.g. peroxidase).
However, the results were always the same. Owing to the results, also other oligonucleotides were tested, which deviated from the published sequence of ~-amyloid (e.g.
erb cancer gene or 18 and 16 of human papilloma virus .. . - . - . , : .
- - . : .
. . . . . .
. ' -W092/06376 ~ PCT/FI9l/00300 5 20~973~
(HPV). The results remained the same.
The location of senile plaques and amyloid was additional-ly ensured by using conventional stainings: kongo, Bielschowski. The samples were additionally stained also immunohistochemically by using as a primary antibody an antibody made against a commercial ~-amyloid protein.
These stainings gave considerably weaker signals, but the positive areas were the same.
Our results indicated that oligonucleotides (independently of the sequence) bind to the ~-amyloid protein or its precursor.
It has previously been postulated (Pepys and Butler, 1987 Breathnach et al. 1989) that serum's amyloid P binds DNA.
This binding is dependent on calcium. It has generally been believed that serum's P amyloid cannot be seen in the amyloid cumulations in connection with Alzheimer's disease. However, it has recently been shown that serum P's antibodies could provide reactivity also in the brain area (Kalaria and Grahovac, 1990).
Joachim et al., 1989, indicated that ~-amyloid is also found in the skin samples of Alzheimer patients (the method used was immunohistology). We have been able to show that oligonucleotides bind to ~-amyloid or its precursor also in the skin sample of an Alzheimer patient.
Our results show that the cumulation in amyloid is a secondary phenomenon and not only related to the brain area.
. ~ . . ,. ,.. , . . . - ... . . .
, - : . : - .: ' -: - .- . - . . '. -.. : . , , . :
Background Alzheimer's disease is a disease resulting in a difficult dementia, in which the risk of falling ill increases along with aging. Symptoms of Alzheimer's disease are the progressive deterioration of memory, perception and other higher brain functions, which finally results in the patient's complete feeblemindedness.
Although the reasons for the outbreak of Alzheimer's disease are not known, the pathological changes caused by it are fairly well-known. The most typical changes are the extracellular accumulation of amyloid protein on the walls of the cerebral membranes and blood vessels as well as as senile plaques. In addition, the cyto-skelenton components of neurons aggregate intracellularly as so-called neurofi-brillary tangles. In both cases, the filamentary proteins may completely change the structure of the neuron, which finally results in the dystrophy and cell death of the neurons. The number of amyloid plaques correlates fairly well with the deterioration of cognitive functions, the neuron loss and the decrease in neurotransmitters (Tanzi et al. 1989).
Senile or neuritic plagues consist of an amyloid core, which is surrounded by a periphery comprised of dystrophic neurites and extracellular material. The plaques may be dense, so-called classic plaques or diffuse concentrations : - . , -, ~: , -: , : :- ' : :,' :
- - :- - . :. . : . , - . -....
W O 92/06376 P ~ tF191/00300 206~731 2 formed of loosely interconnected material. Diffuse plaques have in fact been assumed to be pre-stages of denser formations, which may form during several years without first causing neurologic symptons (Joachim et al. 1989, Tanzi 1989).
The amyloid in the senile plaques forms of 6-10 nm stranded ~-amyloid protein, whose identical subunits are arranged antiparallelly into a beta-structure. This hydrofobic protein of ca. 4.2-4.5 kDa is synthesized most likely in neurons as part of a longer ~-amyloid precursor protein. The gene coding for the precursor protein is localized in the chromosome 21 (Goldgaber et al. 1978, ~-Tanzi 1987).
One reason for the cumulation of amyloid has been assumed to be a defect in the gene locus, since a correspondiny plaque formation also occurs on nearly all Down's syndrome patients of over 40 years (trisomy in chromosome 21), whereby the overproduction of amyloid can be explained by means of an extra gene. An autosomally dominant gene defect in the chromosome 21 has been observed in patients with the hereditary Alzheimer's disease, but it has not been possible to prove a connection between it and the ~-amyloid gene located in the same chromosome (St. George-Hyslop et al. 1978, Tanzi et al. 1987).
The ~-amyloid precursor is a receptor-like, transmembranal glycosylated protein, in which two thirds are located in the extracellular state (Kang et al. 1987). The ~-amyloid dipeptide is in turn located partly in the transmembranal area so that its proteolytic release from the precursor occurs either before the precursor's loosening onto or after loosening from cellular membrane (Tanzi 1989).
It has been observed that the ~-amyloid gene codes for at least three transcripts acting as models for polypeptides ,' '. '~ - . ' : ,. . . : .
- .' - '.'' : , : , : . ' '' , .-.- . . .
-YYO 92/06376 P ~ /FI91/~0300 ~ ` 3 2~973~
with 695, 751 and 770 amino acids. The two longer polypep-tides contain an extra sequence with 56 the amino acid, which is 50% homogenous with the enzymes belonging to the so-called Kuniz's serine protease inhibitor family. Two contradictory hypotheses corcerning the possible share of the protease inhibitor sequence in the aggregation of the ~-amyloid have been presented: 1) it may prevent the cumulation of amyloid by inhibiting proteases which release ~-peptide from the precursor, or 2) the protease inhibitor may advance the cumulation of amyloid by preventing specific proteinases from purifying the tissue from ~-amyloid. The function of the protease inhibitor sequence of the ~-amyloid precursor protein and also its release from the precursor into an active enzyme are thus not known for certain.
General description of invention The invention and certain preferred applications thereof are defined in the patent claims.
The invention is based on the surprising discovery that oligonucleotides bind to the ~-amyloid protein or its precursor. The ~-amyloid protein or its precursor thus binds DNA. The binding occurs especially in the ~-amyloid protein or its precursor present in a peripheral tissue (e.g. skin, mucous membrane). The phenomenon may be utilized especially in diagnostic tests (e.g. Alzheimer's disease, Down's syndrome, dementias, old persons).
Detailed description of invention The object of our examination was the expression of ~-amyloid on the frontal cortex and hippocampus of Alzheimer patients. As a research method was used the RNA in situ hybridization and as a specific tester was a cocktail formed by three short oligonucleotides corresponding to . . .
~ ~ ; -, -: - . .
- - . ..
. :'- ' . . : ' : :
- . - . . : ~ :
W092/06376 :2 ~ ~ ~7 31 PCT/F191/00300 different areas of the ~-amyloid sequence. The oligonuc-leotides were labelled at the 3'-end with biotin. For specific antisense-RNA-oligonucleotides were synthesized complementary sense-RNA-oligonucleotides, which were labelled in the same way as the antisense probes. The in situ hybridization was performed by using the routine methods of our laboratory (a hybridized biotinylated intron is allowed to bind to a streptavidine-alkaline phosphatase complex, which is in turn detected by means of a substrate reaction). The brain samples of 3 different Alzheimer patients as well as two normal samples were examined. As a result could be observed strong signals in the area of the cortex and hippocampus corresponding both by their location and shape to senile plaques. In additi-on, a positive colouring was seen in the walls of blood vessels, as described in connection with the ~-amyloid protein. When we used the sense probe, we could to our surprise see the same result as when using the antisense probe. (When a specific mRNA expression is examined, the sense probe should produce a negative result.) A negative result was by contrast obtained, when water was used instead of the gene probe; the reactions were otherwise performed in the same way as when using the senselantisen-se probe. All colouring results were negative, when the normal samples were subjected to examination.
The in situ hybridization was repeated several times to preclude a possible false positive reaction caused by endogenic biotin or alkaline phosphatase.
Furthermore, the in situ hybridization was repeated by using a radioactive label or other enzymes for the detection of a biotinylized hybrid (e.g. peroxidase).
However, the results were always the same. Owing to the results, also other oligonucleotides were tested, which deviated from the published sequence of ~-amyloid (e.g.
erb cancer gene or 18 and 16 of human papilloma virus .. . - . - . , : .
- - . : .
. . . . . .
. ' -W092/06376 ~ PCT/FI9l/00300 5 20~973~
(HPV). The results remained the same.
The location of senile plaques and amyloid was additional-ly ensured by using conventional stainings: kongo, Bielschowski. The samples were additionally stained also immunohistochemically by using as a primary antibody an antibody made against a commercial ~-amyloid protein.
These stainings gave considerably weaker signals, but the positive areas were the same.
Our results indicated that oligonucleotides (independently of the sequence) bind to the ~-amyloid protein or its precursor.
It has previously been postulated (Pepys and Butler, 1987 Breathnach et al. 1989) that serum's amyloid P binds DNA.
This binding is dependent on calcium. It has generally been believed that serum's P amyloid cannot be seen in the amyloid cumulations in connection with Alzheimer's disease. However, it has recently been shown that serum P's antibodies could provide reactivity also in the brain area (Kalaria and Grahovac, 1990).
Joachim et al., 1989, indicated that ~-amyloid is also found in the skin samples of Alzheimer patients (the method used was immunohistology). We have been able to show that oligonucleotides bind to ~-amyloid or its precursor also in the skin sample of an Alzheimer patient.
Our results show that the cumulation in amyloid is a secondary phenomenon and not only related to the brain area.
. ~ . . ,. ,.. , . . . - ... . . .
, - : . : - .: ' -: - .- . - . . '. -.. : . , , . :
Claims (5)
1. A method for detecting the .beta.-amyloid protein or its precursor from a sample, characterized in that the sample is reacted with an oligonucleotide.
2. A method according to Claim 1 to be used in a diagnos-tic test.
3. A method according to Claim 2 for detecting an amy-loidosis.
4. A method according to Claim 3 for detecting an amy-loidosis from a peripheral tissue sample, such as from a skin or mucous-membrane sample.
5. An oligonucleotide probe, characterized in that it is intended to be used for detecting the .beta.-amyloid protein or its precursor from a sample.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI904766A FI904766A (en) | 1990-09-27 | 1990-09-27 | FOERFARANDE FOER DETEKTERING AV PROTEIN. |
| FI904766 | 1990-09-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2069731A1 true CA2069731A1 (en) | 1992-03-28 |
Family
ID=8531126
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2069731 Abandoned CA2069731A1 (en) | 1990-09-27 | 1991-09-27 | Method for detection of the beta-amyloid protein using unspecific binding of oligonucleotides |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0503022A1 (en) |
| JP (1) | JPH05502512A (en) |
| CA (1) | CA2069731A1 (en) |
| FI (1) | FI904766A (en) |
| WO (1) | WO1992006376A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ242955A (en) * | 1991-06-13 | 1995-01-27 | Ici Plc | Dna containing gene sequences corresponding to sequences causing alzheimers disease, detection, recombinant yeast with such code |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0274826B1 (en) * | 1986-11-17 | 1998-08-12 | Scios Inc. | Recombinant Alzheimer's amyloid protein |
-
1990
- 1990-09-27 FI FI904766A patent/FI904766A/en not_active Application Discontinuation
-
1991
- 1991-09-27 JP JP51494291A patent/JPH05502512A/en active Pending
- 1991-09-27 CA CA 2069731 patent/CA2069731A1/en not_active Abandoned
- 1991-09-27 WO PCT/FI1991/000300 patent/WO1992006376A1/en not_active Application Discontinuation
- 1991-09-27 EP EP19910916158 patent/EP0503022A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| EP0503022A1 (en) | 1992-09-16 |
| JPH05502512A (en) | 1993-04-28 |
| WO1992006376A1 (en) | 1992-04-16 |
| FI904766A0 (en) | 1990-09-27 |
| FI904766A (en) | 1992-03-28 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 19950327 |