WO2018212261A1 - Specific binding reagent for detecting qualitative difference of 4-repeat tau, and detection method, detection kit, and medication screening method using same - Google Patents

Abstract

It is found that, in an Alzheimer's disease patient, an asparagine at position 279 of a tau protein is deamidated and modified by an aspartic acid. The Alzheimer's disease can be identified and diagnosed by using a reagent for specifically recognizing a peptide in which the asparagine at position 279 of the tau protein is modified by the aspartic acid.

Description

Specific binding reagent for detecting qualitative difference of 4-repeat tau, test method using the same, test kit, and drug screening method

The present invention relates to a test method for distinguishing neurodegenerative diseases in which accumulation of phosphorylated tau, which is collectively called tauopathy, is observed. In particular, Alzheimer's disease (hereinafter sometimes referred to as AD) in which 4 repeat tau accumulates and progressive supranuclear palsy (hereinafter sometimes referred to as PSP) / The present invention relates to a specific binding reagent for 4-repeat tau capable of distinguishing corticobasal degeneration (hereinafter sometimes referred to as CBD), and a test method using the same. Moreover, it is related with the screening method of the pharmaceutical using this specific binding reagent.

It is said that the number of patients with dementia has increased with the increase in the elderly population, and the number of patients with dementia in 2012 has reached 4.62 million (Cabinet Office, 2016 White Paper on Aging Society (Summary)). As the number of elderly people increases in the future, the number of patients with dementia is considered to increase more and more, which is a big social problem.

However, it has been clarified that even if it is dementia, there are various causative diseases that exhibit similar symptoms. For example, for diseases exhibiting a clinical image of corticobasal syndrome (CBS), in addition to CBD, AD, and PSP, Pick's disease (hereinafter sometimes referred to as PiD) may also be used. It has been clarified that various diseases including the above are included (Non-patent Document 1).

In recent years, with the advancement of diagnostic imaging, diagnostic imaging such as CT and MRI has become indispensable for the diagnosis of dementia. However, although image diagnosis enables confirmation of localization changes and tracking of changes over time, it is not sufficient to capture qualitative changes, and there is a limit to reflecting it in objective diagnostic criteria.

Therefore, it is said that dementia is still difficult to make an accurate prenatal diagnosis only by examination findings and clinical findings by image diagnosis using MRI, etc., and a pathological diagnosis is made after the patient's death. It is the current situation that can be done. Therefore, it is believed that many patients are not receiving appropriate treatment due to inaccurate clinical diagnosis before birth. Therefore, establishment of a test method with high diagnostic accuracy and specificity using biomarkers in body fluids such as cerebrospinal fluid and blood is desired.

Furthermore, markers that can accurately diagnose neurodegenerative diseases not only contribute to the selection of treatment methods but also greatly contribute to the development of new drugs by definitive diagnosis of patients. For example, at present, only a clinical trial of a new drug is being conducted for “a group of patients with a high probability of Alzheimer's disease”. As a result, it is highly likely that patients with other neurodegenerative diseases are included in the group targeted for clinical trials. Therefore, the effect of the drug on a specific neurodegenerative disease such as Alzheimer's disease cannot be accurately determined. If the accuracy of diagnosis can be improved not only by the doctor's judgment based on clinical symptoms but also by objective test results, it will be possible to conduct clinical trials on appropriate patient groups and make a correct evaluation of the effects of candidate drugs. Can do.

The causative disease of dementia in Japan has been said to be the most cerebrovascular, but according to recent epidemiological studies, Alzheimer's disease is said to be the most common. Therefore, clinically distinguishing Alzheimer's disease from other neurodegenerative diseases is very important for the development of future therapeutic agents.

As the cause of Alzheimer's disease, there is a position to focus on amyloid β, which constitutes senile plaque, which is characterized by pathological features, and tau that constitutes neurofibrils, but the cause is not clear is the current situation. For the diagnosis of Alzheimer's disease, methods of detecting amyloid β and tau protein in body fluids and methods of detecting with tau imaging probes have been proposed, but they have not yet reached the accuracy that can influence clinical diagnosis. (Patent Documents 1 and 2).

Tau is a protein that contributes to the polymerization and stabilization of microtubules and is also present in the normal brain. Tau accumulates abnormally in AD, PSP, CBD, PiD, etc. and is observed as neurofibrillary tangles and other inclusions. These diseases in which phosphorylated tau accumulates abnormally are collectively called “tauopathy” (Non-patent Document 2).

It is known that tauopathy increases and accumulates phosphorylation that occurs in normal brain tau. Non-Patent Document 3 reports that the subtype of frontotemporal dementia (FTLD) can be classified based on the ratio of phosphorylated tau to total tau protein in cerebrospinal fluid. For example, it is shown that the amount ratio of phosphorylated tau is different in the tau and amyloid β accumulated AD as well as tau phosphorylation in subtypes such as FTLD-TDP with ALS (Amyotropic lateral sclerosis). Statistically, however, the amount of phosphorylated tau varies greatly among healthy subjects and subtype groups, but there is a large overlap between groups. No discrimination has been made based on quantitative differences.

Tau has six isoforms due to alternative splicing of exons 2, 3, and 10 (see FIG. 1 (A)). Among these, the number of repeating structures of the microtubule-binding region existing on the C-terminal side is called 3 as 3 repeat tau, and 4 as 4 repeat tau. Tau isoforms are known to be regulated during development and all six isoforms of tau are expressed in the adult brain.

Analysis of insoluble tau in inclusion bodies of brain inclusions in patients with degenerative neurological diseases of tauopathy shows that tauopathy has only 3 repeat tau (PiD), 4 repeat tau only (PSP / CBD), and both It has been clarified that it is broadly classified as accumulated (AD) (Non-patent Document 4). If the amount of 3 repeat tau and 4 repeat tau in the cerebrospinal fluid can be distinguished and quantified, it is expected to provide a clue to distinguish these diseases qualitatively. However, even if the amount of 3 repeat tau and 4 repeat tau in the cerebrospinal fluid is measured by an immuno PCR method, these diseases are not distinguished (Non-patent Document 5). Since these isoforms are also expressed in the normal brain, there is no accuracy that can distinguish normal cases, AD, and other cases of dementia by the amount of tau isoforms.

International Publication No. 2013/111578 International Publication No. 2012/057312 JP 2005-512550 Gazette Special table 2003-521713 gazette Special table 2003-504015 gazette

Takahiko Tokuda, 2013, BRAIN and NERVE, Vol.65 (1), pp.55-64. Yoshida Mari, 2013, BRAIN and NERVE, Vol.65 (12), pp.1445-1458. Pijnenburg, Y. A. L. et al., 2015, Alzheimer ’s Dementia, Vol. 1, pp.505-512. de Sliva, R. et al., 2003, Neuropathol. Appl. Neurobiol., Vol.29, pp.288-302. Luk, C. et al., 2012, J, Neurochem., Vol.123, pp.396-405. Dan, A. et al., 2013, Acta Neuropathologica Commun., 1:54, doi: 10.1186 / 2051-5960-1-54 Oumi Shinobu, 2014, Anti-Peptide Antibody Basic, Gakken Medical Shujunsha

As mentioned above, there is currently no effective biomarker for differential diagnosis of diseases that cause lesions in the brain and cause dementia, etc., so it currently depends on probabilistic inference based on clinical symptoms and image diagnosis It is. In neurodegenerative diseases such as Alzheimer's disease, the distribution of lesions varies from case to case even within the same disease, and the clinical symptoms and image findings in individual cases are also large. For this reason, it is said that image diagnosis, evaluation based on clinical symptoms only, and diagnosis methods vary greatly depending on the judgment of a doctor. In order to perform appropriate treatment, a highly reliable diagnosis by a highly objective method is necessary. In addition, in order to develop a drug effective for each disease, it is necessary to conduct a clinical trial on a target group for which an accurate diagnosis has been made, and to analyze and evaluate it. To that end, accurate diagnosis of each disease is an essential requirement. An object of the present invention is to provide a highly reliable test method, an antibody necessary for the test, a test kit, and a pharmaceutical screening method using the same.

The present invention relates to a test method for clinically distinguishing Alzheimer's disease from other neurodegenerative diseases, a test reagent used for this, a test kit, and a pharmaceutical screening method.
(1) Alzheimer's disease and progressive supranuclear paralysis / basal ganglia degeneration with a specific binding reagent that specifically binds to a peptide in which asparagine at position 279 of the tau protein in the sample is substituted with aspartic acid Inspection method to differentiate.
(2) The test method according to (1), wherein the specific binding reagent is an antibody.
(3) The examination method according to (1) or (2), wherein the specimen is cerebrospinal fluid, blood, cranial nerve tissue, or biopsy tissue.
(4) The inspection method according to (2) or (3), wherein detection is performed by ELISA or immunohistochemical staining.
(5) A specific binding reagent that distinguishes Alzheimer's disease from progressive supranuclear palsy / basal ganglia degeneration, for peptides in which the asparagine at position 279 of tau protein is substituted with aspartic acid A reagent characterized by binding specifically.
(6) The specific binding reagent according to (5), wherein the specific binding reagent is an antibody.
(7) The specific binding reagent according to (6), wherein the antibody is a monoclonal antibody.
(8) The specific binding reagent according to (6), wherein the antibody is obtained by purifying a polyclonal antibody.
(9) A test kit comprising the specific binding reagent according to any one of (5) to (8) and a reagent necessary for detection.
(10) The test kit according to (9), wherein the detection is performed by ELISA or immunostaining.
(11) A method for screening a drug, comprising screening using deamidation at position 279 of tau protein as an index.
(12) When a specific binding reagent that specifically binds to a peptide in which asparagine at position 279 of tau protein is substituted with aspartic acid recognizes tau protein in a sample obtained from a patient, Alzheimer's disease How to diagnose that.
(13) The diagnostic method according to (12), wherein the specific binding reagent is an antibody.
(14) The diagnostic method according to (12) or (13), wherein the specimen is cerebrospinal fluid, blood, cranial nerve tissue, or biopsy tissue.
(15) The diagnostic method according to (13) or (14), which is detected by ELISA or immunohistochemical staining.

FIG. 1A schematically shows a tau isoform. FIG. 1 (B) shows pathologically stained images of AD and PSP cases with commercially available antibodies. An antibody that specifically detects aspartic acid at position 279 indicates that tau accumulated in AD cases is detected. The figure which analyzed the reactivity with respect to the peptide of the antibody which detects the aspartic acid of the 279th position specifically by ELISA.

As described above, tauopathy can be roughly classified according to the difference in tau isoform accumulated in inclusion bodies. 3 repeat tau can be detected by an RD3 antibody that recognizes a peptide (KHQPGGGGVIVYKPV: SEQ ID NO: 1) in a region adjacent to the C-terminal microtubule-binding regions I and III, and 4 repeat tau can be detected by a microtubule-binding region. It can be detected by an RD4 antibody that recognizes a peptide containing II (VQIINKKLDLSNVQSKC: SEQ ID NO: 2) (see FIG. 1 (A)). However, tau protein in cerebrospinal fluid cannot be quantified by these antibodies, and tauopathy has not been differentially diagnosed. The present inventor has found that in AD and PSP pathological specimens, antibodies that recognize 4 repeat tau have different reactivity due to deamidation, and have completed the present invention.

4 Repeat Tau is translated as asparagine at 279th place. It has been reported that some of them are deamidated and converted to aspartic acid (Non-patent Document 6). Until now, it was thought that the tau protein in which asparagine at position 279 was deamidated was present regardless of diseases such as AD, PSP and CBD.

This is because when a pathological specimen is stained with a commercially available antibody, an antibody (RD4 antibody) prepared against a tau peptide (SEQ ID NO: 2) in which position 279 is asparagine is a peptide in which position 279 is substituted with aspartic acid. This is because accumulation of 4 repeat tau was detected in the same manner even in an antibody (4R antibody) prepared against VQII D KKLDLSNVQSKC (SEQ ID NO: 3, the underlined portion indicates the amino acid at position 279). 1 (B).)

The present inventor has prepared a commercially available polyclonal antibody (4R antibody) prepared against the peptide of SEQ ID NO: 3 with an antibody that recognizes the peptide represented by SEQ ID NO: 2 and an antibody that recognizes the peptide represented by SEQ ID NO: 3. I found it mixed. As a result of analysis using the purified antibody, it was revealed that tau deamidated at position 279 exists only in AD. That is, an antibody specific for a peptide deamidated at position 279 detects only tau accumulated in AD.

The present inventor has found for the first time that tau deamidated asparagine at position 279 is specifically detected by AD among neurodegenerative diseases collectively referred to as tauopathy. Here, deamidated tau is detected by an antibody. However, any reagent can be used as long as it is a reagent that can detect tau in which asparagine at position 279 is deamidated. . Therefore, the specific binding reagent in the present specification is not limited to an antibody, and may include a reagent that specifically binds to a specific molecule such as an aptamer. Nucleic acid aptamers and peptide aptamers may be prepared by known methods, and those that can specifically bind to tau in which the 279th position is substituted with aspartic acid may be selected. Furthermore, the specific binding reagent of the present invention includes a modified form. Specific examples include those that can be labeled with radioactive substances and used as PET diagnostic agents, and those that are labeled with fluorescent substances and can be detected without using secondary antibodies.

In addition, when referring to an antibody in the present invention, Fab, Fab ′, F (ab ′) ₂ , single chain antibody (scFv), disulfide-stabilized V region fragment (dsFv), or a functional antibody such as a peptide containing CDR Fragments are included. A functional fragment of an antibody can be obtained by a known method such as digestion with an enzyme such as pepsin or papain.

Further, in the present invention, detection of a specific binding reagent including an antibody may be performed by directly labeling the specific binding reagent, an indirect method using a secondary antibody or a tertiary antibody, or avidin- Known detection techniques such as detection using biotin can be used.

Hereinafter, the present invention will be described, but it goes without saying that not only the following method but also a known method can be used.

1. Antibody Purification Method 4 Antibodies that recognize repeat tau include monoclonal antibodies (RD4 antibody, manufactured by Merck & Co., Inc.) using the peptide shown in SEQ ID NO: 2 as an immunogen, and polyclonal antibodies using the peptide shown in SEQ ID NO: 3 as an immunogen. (4R antibody, manufactured by Cosmo Bio Inc.) is commercially available.

The commercially available antibody obtained by immunizing the peptide shown in SEQ ID NO: 3 is found to contain an antibody in which the amino acid at position 279 reacts with both an asparagine peptide and an aspartic acid peptide. It became. An antibody that specifically recognizes tau in which the amino acid at position 279 is deamidated to aspartic acid (hereinafter referred to as D279 antibody) is obtained from a commercially available antibody or an animal immunized with the peptide shown in SEQ ID NO: 3. The obtained antibody can be used after being purified by a peptide column. The antibody may be purified by utilizing specific binding between the antibody and the peptide, and may be purified by combining known anti-peptide antibody purification methods. Specifically, for example, it can be purified as follows.

Purification of D279 antibody that reacts only with a peptide in which the amino acid at position 279 reacts with asparagine or with both aspartic acid and reacts only with a peptide whose position 279 is aspartic acid is performed, for example, as follows. be able to. The antibody in which both are mixed is adsorbed onto a column to which the peptide of SEQ ID NO: 3 (a peptide in which the position 279 is substituted with aspartic acid, hereinafter sometimes referred to as D279 peptide) is bound, and the adsorbed fraction is eluted. Alternatively, the peptide of SEQ ID NO: 2 (a peptide in which the position 279 is substituted with asparagine, hereinafter sometimes referred to as N279 peptide) is adsorbed to a coupled column and purified by collecting a fraction that does not adsorb. Can do. Further, the fraction that is not adsorbed on the N279 peptide column can be further adsorbed on the D279 peptide column, and the adsorbed fraction can be eluted and purified. Further, when the antibody is mixed with the N279 peptide, the antibody that binds to the N279 peptide is masked, so that only the antibody fraction highly reactive with the D279 peptide is used.

2. Antibody production method
The antibody can be prepared with reference to a known method (Non-patent Document 7). Polyclonal antibodies and monoclonal antibodies can be prepared by immunizing rabbits and mice with an antibody specific for the peptide shown in SEQ ID NO: 2 or 3.

3. Testing Method When the D279 antibody is used for testing, body fluids such as cerebrospinal fluid, blood, urine, and biopsy specimens can be used as samples. As a biopsy specimen, nasal mucosa, skin, salivary gland, gastrointestinal mucosa and the like in which detection of tau and α-synuclein is recognized can be suitably used. Cerebrospinal fluid is preferably used from the viewpoint of sensitivity. Specific examples of immunostaining and ELISA are given below, but any method may be used as long as it is a method capable of detecting a specific reaction.

(1) Immunostaining method For tissue staining, formalin-fixed and paraffin-embedded tissue is sliced by a conventional method, and stained using a primary antibody specific to the above tau peptide and a labeled secondary antibody. Can be detected. The detection method can be performed using a known method such as coloring by an enzyme label or a method of detecting a fluorescent label.

(2) ELISA
In order to confirm the reactivity of the obtained antibody, the peptide of SEQ ID NO: 2 or 3 is placed in a 96-well ELISA plate, left standing for a certain period of time, and then immobilized. The solid-phased plate may be washed and blocked and used for inspection. Specifically, the prepared antibody or purified antibody is put into an ELISA plate on which the peptide is immobilized, and then an antibody that binds to the antibody of the animal from which the antibody was prepared, such as an anti-mouse antibody or anti-rabbit antibody, is used And may be detected by a conventional method. Detection may be performed using an antibody in which the antibody of the animal from which the antibody is produced is enzyme-labeled, or using an enzyme-labeled antibody as a secondary antibody for detection. Enzyme substrate is added, the reaction is stopped for a certain period of time, the reaction is stopped, and the enzyme product is measured using a microplate reader.

When examining a patient specimen, for example, the specimen is put on a plate on which an antibody that binds to tau protein regardless of 4 repeat tau or 3 repeat tau is immobilized, and then reacted with D279 antibody. After the enzyme-labeled secondary antibody that binds to is added and bound, the enzyme substrate is added, the reaction is stopped for a certain period of time, and the enzyme product is measured using a microplate reader. AD, PSP and PiD can be distinguished by performing detection using RD3 antibody and RD4 antibody in addition to D279 antibody. As patient specimens, patient body fluids such as cerebrospinal fluid and blood are preferably used, but cerebrospinal fluid is more preferably used because tau protein is present in a higher concentration in the cerebrospinal fluid.

Further, the above ELISA method is an example, and it goes without saying that detection can be performed using an anti-tau protein antibody after binding a specimen using a plate on which a D279 antibody is immobilized. Furthermore, detection can be performed by a detection method usually performed using an antibody or the like, such as detection by RIA or fluorescently labeled antibody instead of ELISA.

4). Medicinal Screening Method Since the D279 antibody specifically recognizes AD lesions, it can be used to screen a medicament for treating Alzheimer's disease.

(1) Screening method using cultured cells A compound capable of suppressing the deamidation of tau protein to aspartic acid at position 279 is highly likely to be effective as a drug for Alzheimer's disease. A compound that is effective against Alzheimer's disease can be selected by screening a compound using cultured cells as an index of deamidation of aspartic acid at position 279.

As the cultured cells to be used, any cells may be used as long as deamidation at position 279 of tau is observed. As suitable culture cells, neuronal cells established from Alzheimer's disease patients or model animals, and neuronal cells derived from iPS cells obtained from Alzheimer's disease patients, 4 repeat tau is forcibly expressed, and deamidation occurs. Examples of such cells have been confirmed.

After adding a candidate compound to the culture solution, the deamidation at the 279 position of tau is quantified by a known method such as ELISA using D279 antibody or a specific binding reagent having equivalent reactivity to suppress deamidation. Can be screened for.

Although it is necessary to wait for future analysis, if deamidation is one of the causes of AD, compounds known to suppress deamidation may be potential drug candidates. There is.

(2) Screening method using model animal The compound which showed the effect by the screening system using a cultured cell can further be examined using a model animal. As the model animal, an aging model such as an aged normal animal or an aging promoting model mouse can be used. In addition, transgenic animals that modify the tau gene described in Patent Documents 3 to 5 and cause Alzheimer-like lesions may be used.

Regardless of which model animal is used, a drug can be screened by administering a candidate compound to the animal, using inhibition of deamidation at position 279 of tau protein as an index.

[Example 1] Purification of antibody Hereinafter, the test method of the present invention will be described in detail while showing data. The affinity column for purifying the antibody was used by binding the peptide of SEQ ID NO: 3 to the formyl group of Cellufine Formyl (manufactured by JNC Corporation). Specifically, after washing Serfine Formyl with distilled water, the suspension was suspended in a coupling buffer (50 mM carbonate-bicarbonate buffer, pH 8.5) together with the D279 peptide represented by SEQ ID NO: 3 at room temperature. Shake for 30 minutes. Next, sodium cyanoborohydride is added and shaken at 4 ° C. overnight to reduce excess formyl group. After washing with a blocking buffer (0.1 M monoethanolamine, 50 mM Tris-HCl, pH 8.0), sodium cyanoborohydride is added again to perform complete reduction. Thereafter, the resin bound with the peptide is packed in a column, and distilled water, elution buffer (0.1 M Gly-HCl, pH 2.5), distilled water, washing buffer (1 M NaCl, 1% Triton-X 100, 20 mM Tris). -Hcl, pH 7.5), and then washed with distilled water in this order to produce an affinity column.

Commercially available polyclonal antibody (4R antibody: an antibody in which an antibody that reacts with a tau peptide whose amino acid at position 279 is asparagine and an antibody that reacts with a tau peptide that is aspartic acid are mixed, hereinafter sometimes referred to as DN279 antibody ) And the N279 peptide represented by SEQ ID NO: 2 and placed in an affinity column equilibrated with TBS (0.15 M NaCl, 20 mM Tris-HCl, pH 7.5), mixed by inversion, and mixed at 4 ° C. for 8 hours. Adsorb to the column. After the unadsorbed antibody is allowed to flow out, it is washed with TBS, a washing buffer, and 0.15 M NaCl, and then the antibody adsorbed on the column is eluted with an elution buffer. The eluted antibody was confirmed to bind specifically to the D279 peptide shown in SEQ ID NO: 3 by ELISA or the like, and used in subsequent experiments.

[Example 2] Tissue staining Using a pathological specimen, tissue staining was performed using an unpurified polyclonal antibody (DN279 antibody) prepared using the peptide shown in SEQ ID NO: 3 or a D279 antibody purified using a peptide column. went.

The samples used are pathological specimens of the olfactory intestine of a patient diagnosed with AD by definitive diagnosis and the lower olive nucleus of a patient diagnosed with PSP. Formalin-fixed, paraffin-embedded pathological specimens are sliced to 5-6 μm, deparaffinized, heated in an autoclave at 0.01 ° C. for 10 minutes in 0.01 M citrate buffer, and then immersed in 100% formic acid for 10 minutes. Then, antigenic activation treatment was performed. Endogenous peroxidase was inactivated with 1% hydrogen peroxide, blocked with PBS containing 5% serum, and then purified as RD4 antibody (1000-fold dilution), 4R antibody (DN279 antibody, 30,000 dilution), or purified as a primary antibody. D279 antibody (diluted 20 times) was reacted at 4 ° C. for 2 days or more, washed, and then used as a secondary antibody, biotinylated anti-rabbit IgG (Vector, diluted 1000 times), or biotinylated anti-mouse IgG (Vector, 1000 times dilution) and at room temperature for 2 hours. The secondary antibody was washed, reacted with Avidin biotin-peroxidase complex (manufactured by Vector, ABC Elite, diluted 1000 times) for 1 hour, and then developed with diaminobenzidine for detection (FIG. 2).

The DN279 antibody, in which an antibody that recognizes both aspartic acid and asparagine at the 279th amino acid of tau protein is mixed, recognizes tau of both AD and PSP samples, but D279 antibody recognizes tau of AD but PSP Can't recognize tau. Although the pathological tissue is shown here, the detection can be similarly performed using patient cells mixed in cerebrospinal fluid.

[Example 3] ELISA
Tau protein present in body fluids such as cerebrospinal fluid can also be detected by ELISA. FIG. 3 shows the results of analyzing the reactivity of the D279 antibody by coating the microtiter plate at different concentrations of the peptide represented by SEQ ID NO: 2 (N279 peptide) and the peptide represented by SEQ ID NO: 3 (D279 peptide).

Peptide immobilization was carried out at 4 ° C for 16 hours at a concentration of 1.0 × 10 ⁻¹⁶ to 1.0 × 10 ⁻⁶ μM / μl on a 96-well ELISA plate (Sumitomo Bakelite Co., Ltd.). Added. After washing with ELISA washing buffer (0.05% Tween-20 / PBS), blocking with ELISA blocking buffer (10% BSA / 0.01 × PBS), washing again, and then adding purified D279 antibody And left at room temperature for 1.5 hours. After washing, HRP-labeled goat anti-rabbit IgG (Pierce) was added as a secondary antibody and allowed to stand at room temperature for 1 hour. After washing, color was developed using O-phenylenediamine, and measurement was performed with a microplate reader (Chameleon, manufactured by Hidex).

The D279 antibody can recognize the D279 peptide even at a very low concentration of 1 × 10 ⁻¹⁶ μmol / μl. It is also clear from this result that the D279 antibody is very specific for the D279 peptide. Therefore, tau containing D279 peptide, that is, AD can be detected with very high specificity.

As described above, the D279 antibody can diagnose Alzheimer's disease among tauopathy. Until now, it is possible to perform a test with good sensitivity and accuracy for the diagnosis of tauopathy for which no objective index has been obtained.

Claims (11)

Distinguish Alzheimer's disease from progressive supranuclear palsy / basal ganglia degeneration with a specific binding reagent that specifically binds to a peptide in which asparagine at position 279 of tau protein in the sample is substituted with aspartic acid Inspection method. The test method according to claim 1, wherein the specific binding reagent is an antibody. The specimen is
The examination method according to claim 1 or 2, which is cerebrospinal fluid, blood, cranial nerve tissue, or biopsy tissue. 4. The inspection method according to claim 2, wherein the detection is performed by ELISA or immunohistochemical staining. A specific binding reagent that differentiates Alzheimer's disease from progressive supranuclear palsy / basal ganglia degeneration,
A reagent which specifically binds to a peptide in which aspartic acid at position 279 of tau protein is substituted with aspartic acid. The specific binding reagent according to claim 5, wherein the specific binding reagent is an antibody. The specific binding reagent according to claim 6, wherein the antibody is a monoclonal antibody. The specific binding reagent according to claim 6, wherein the antibody is obtained by purifying a polyclonal antibody. A test kit comprising the specific binding reagent according to any one of claims 5 to 8 and a reagent necessary for detection. 10. The test kit according to claim 9, wherein the detection is performed by ELISA or immunostaining. A method for screening a pharmaceutical, comprising screening using deamidation at position 279 of tau protein as an index.

Images