METHOD FOR DETECTION OF THE beta -AMYLOID PROTEIN USING UNSPECIFIC BINDING OF OLIGONUCLEOTIDES

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: Method for detection of the β-amyloid protein using unspecific binding of oligonucleotides 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 plaques 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 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 β-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 β-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 corresponding 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 β- amyloid gene located in the same chromosome (St. George- Hyslop et al. 1978, Tanzi et al. 1987). The β-amyloid precursor is a receptor-like, transmembranal glycosylated protein, in which two thirds are located in the extracellular state (Kang et al. 1987) . The β-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 β-amyloid gene codes for at least three transcripts acting as models for polypeptides 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 β-amyloid have been presented: 1) it may prevent the cumulation of amyloid by inhibiting proteases which release β-peptide from the precursor, or 2 ) the protease inhibitor may advance the cumulation of amyloid by preventing specific proteinases from purifying the tissue from β-amyloid. The function of the protease inhibitor sequence of the β-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 β-amyloid protein or its precursor. The β-amyloid protein or its precursor thus binds DNA. The binding occurs especially in the β-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 β- a yloid 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 different areas of the β-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 β-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 sense/antisen¬ 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 β-amyloid (e.g. erb cancer gene or 18 and 16 of human papilloma virus (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 β-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 β-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 β-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 β-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: Claims 1. A method for detecting the β-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 β-amyloid protein or its precursor from a sample.

Inventors: HEINONEN OUTI, RIEKKINEN PAAVO, SYRJAENEN KARI, SYRJAENEN STINA, HEINONEN, OUTI, RIEKKINEN, PAAVO, SYRJAENEN, KARI, SYRJAENEN, STINA
Applicants: LOCUS GENEX OY, LOCUS-GENEX OY
Application year: 1991
Main classification: C12Q1
IPC codes: C12Q1, G01N33