WO1994010205A1

WO1994010205A1 - Protein specific for alzheimer's disease and method of diagnosing alzheimer's disease through detection of the protein

Info

Publication number: WO1994010205A1
Application number: PCT/JP1993/001503
Authority: WO
Inventors: Kiminobu Sugaya; Hiroshi Marusawa
Original assignee: Fujisawa Pharmaceutical Co., Ltd.
Priority date: 1992-10-23
Filing date: 1993-10-19
Publication date: 1994-05-11
Also published as: AU5157493A

Abstract

The invention provides a method of diagnosing Alzheimer's disease and a kit therefor by finding out a protein that can be detected from the patients with Alzheimer's disease but not from the normal subjects and using the protein or an antibody recognizing the fragments of the same. The protein has the following characteristics: (1) molecular weight: 30 kD as determined according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (2) isoelectric point (pI): 6.9; and (3) amino acid sequence at the N-terminus (α) wherein Xaa represents an unidentified amino acid residue.

Description

Specification

TECHNICAL FIELD The present invention relates to a specific protein relating to Alheimer's disease and a method for diagnosing Alheimer's disease by detecting the protein.

The present invention relates to a protein, an antibody recognizing the protein and a fragment of the protein, and a method for diagnosing Alzheimer's disease using the antibody or the protein, which is useful for the study of diagnosis, cause and treatment of Alzheimer's disease. It is related to kits used in the medical field.

Background art

Alzheimer's disease mainly develops in the elderly and elderly, with progressive dementia as the main symptom, as well as mental symptoms and problem behaviors associated with dementia, such as excitement, violence, wandering, hallucinations, delusional insomnia, delirium, etc. It is symptomatic and pathologically characterized by general brain atrophy, extensive neuronal loss, senile plaques, amyloid protein deposition, and the presence of neurofibrillary tangles. In today's aging society, Alzheimer's disease is one of the social problems. No cure has been found for Alzheimer's disease at present, but drugs such as neurotransmitters and related substances, brain stimulants, and cerebral circulation improvers are being developed to halt and improve the progress of the disease.

However, the progression of the disease is usually very slow and there is no subjective symptom, so it is often too late to treat when the surrounding people notice the dementia symptoms of the patient.

An important issue is the early diagnosis of Alzheimer's disease. Methods such as the conventional positron emission tomography (PET) method for injecting radioisotopes into the body and various biological markers have been proposed. Have been.

Since the above-mentioned PET method uses radioisotopes, there are problems that the risk to the subject is high and that early diagnosis cannot be performed sufficiently.

Examples of various biological markers include, for example, a tau protein (Lee VL, eta1: Science, 251, and A68 with a molecular weight of 68,000) extracted from the brain of Alzheimer's disease patients. 6 7 5-6 7 8, 1 9 9 1) Insoluble /? 4 protein from brain-derived amyloid from patients with D. Alzheimer's disease (Masters CL, eta 1: Proc. Natl) ... a ca d S ci USA, 8 2, 4 2 4 5 - 4 0 2 4 9, 1 9 8 5) include, antibodies that react to these (Wo 1 ozin BL, eta 1 : S cience, 2 3 2,6 4 8-6 5 0, 1 9 8 6), (A 11 sop D, etal: N eurosci Lett, 68, 25 2-2 56, 1 9 8 6) Have been. However, it has not been sufficiently confirmed that these proteins are always present only in patients with Alzheimer's disease and not present in persons without Alzheimer's disease ⁵ (hereinafter referred to as “normal persons”). Furthermore, it has been reported that antibodies obtained from these proteins recognize not only proteins of patients with Alzheimer's disease but also proteins of normal individuals, and cannot clearly discriminate between the two (P. D elaereeta 1 .:? A 4 andtaui mmu nocytoch em i0 stryinthebrainof 2 0 dementedandnon— dementedcentenarian s. T hird I nternational Conferenceon A 1 zheimer's D iseaseand R eluted D isorders, July 1992, Italy) 5 Can not. Definitive diagnosis of Alzheimer's disease in the brain It is desired to establish a simple and early method for early diagnosis of Alzheimer's disease using the method and samples obtained from the periphery of the subject, such as blood, skin, hair, and nasal mucosa. Disclosure of the invention

The inventors of the present invention have studied proteins expressed in the brains of Alzheimer's disease patients and normal subjects, and as a result, found proteins that are detected in the brains of Alzheimer's disease patients and are not detected in the brains of normal subjects. Was. Furthermore, since this protein has a relatively small molecular weight and is water-soluble, it has been found that there is a high possibility that it is specifically detected in patients with Alzheimer's disease in the periphery as well as in the brain.

By studying the nature and origin of this protein, it has been found that it has great potential to provide a means of investigating the causes of Alheimer's disease and treating it.

An antibody recognizing this protein or a fragment of the protein is prepared using the protein or a peptide synthesized from the N-terminus of the protein obtained by sequence analysis in accordance with the amino acid sequence as an antigen, and the antibody and Alzheimer's disease are prepared. By reacting the protein expressed in the brain and the like of the patient with the above, and by detecting this reaction, it was found that a definite diagnosis of Alzheimer's disease can be made relatively easily.

Furthermore, by analyzing samples collected from subjects by two-dimensional electrophoresis, etc., it was found that diagnosis of Alzheimer's disease can be made reliably by examining the presence and amount of these proteins.

The present invention was completed by producing a kit used for this diagnosis. This invention is divided into the following four inventions.

[I] A protein obtained from at least the brain of a patient with Alzheimer's disease and having the following properties.

(1) Molecular weight: sodium dodecyl sulfate-polyacrylamide gel 30 kD as measured by electrophoresis

(2) Isoelectric point: p I = 6.9

(3) Amino acid sequence from the N-terminal to the 15th residue:

(1) (5)

X a a -X a a -V a 1 -P ro -X a a -G ly -A 1-(1 0) (1 5)

Xaa—Thr—Gly—I1e—Tyr—G1u—Xaa—Leu (where Xaa represents an unidentified amino acid)

[II] An antibody that recognizes the protein of [I] or a fragment of the protein.

[III-1] reacting a sample collected from a subject with at least one antibody that recognizes the protein of [I] or a fragment of the protein, and detects the degree or presence or absence of the reaction. To diagnose Alzheimer's disease.

[I I I 1-2] A method for diagnosing Alzheimer's disease, comprising analyzing a sample collected from a subject to check for the presence or amount of the protein of [I].

[IV] As a component, the composition includes at least one antibody recognizing the protein of [I] or a fragment of the protein, and means for detecting a reaction between the antibody and a sample collected from a living body. A diagnostic kit for Alzheimer's disease.

Hereinafter, each invention will be described in detail. [I] Protein specific to Alzheimer's disease patients

The protein of the present invention can be obtained from at least the brain of Alzheimer's disease patients, and can be obtained, for example, by extracting from the brain of a dead Alzheimer's disease patient.

As the solvent used for citrus, an aqueous solvent, for example, various buffers (HEPES buffer, etc.) is preferable. This protein can be extracted by collecting, for example, the cerebral cortex from the brain of a dead Alzheimer's disease patient, adding the aqueous solvent described above, homogenizing, and then centrifuging under cooling to obtain a supernatant.

Separation of this protein from the supernatant can be performed by two-dimensional electrophoresis in a conventional manner.

This protein of interest is determined as a spot of the protein detected in Alzheimer's disease patients by comparing it with the electrophoresis pattern obtained from the brain of a normal person through the same method. The properties of this protein are determined by the method described in the examples below. Then, the target protein can be extracted from the two-dimensional electrophoresis gel by subjecting it directly to coomassie-prilian-blue staining. This protein is a protein having the following properties.

(2) Isoelectric point: p I = 6.9

(3) Amino acid sequence from the N-terminal to the 15th residue:

(1) (δ)

X aa -X aa -V a 1 — Pro — X aa— G ly-A 1 a-(1 0) (15) X aa— T hr— G ly— I le -T yr -G lu -X aa-L eu (expression Xaa is an unidentified amino acid)

The protein of the present invention is not limited to the protein obtained from the brain of an Alzheimer's disease patient, but also includes the protein obtained from a part other than the brain of a living body or by a genetic recombination technique.

[I I] Antibody

The antibody used in the method for diagnosing Alzheimer's disease of the present invention recognizes the protein or the fragment of the protein detected from the Alzheimer's disease patient of the above [I].

A fragment of the protein includes a part of the amino acid sequence contained in the protein, a sugar chain that modifies the protein, and the like.

This antibody can be either polyclonal or monoclonal and can be prepared by methods well known in the art, but monoclonal antibodies are preferred.

The polyclonal antibody against the protein of the present invention is obtained by administering an animal such as a heron to a peptide synthesized by the Fm0c solid phase method or the like according to the amino acid sequence from the N-terminal of the protein or the protein obtained by sequence analysis. After immunization, it can be obtained by subjecting the resulting antiserum to isolation and purification by subjecting it to conventional procedures such as salting out ammonium sulfate or the like, centrifugation, dialysis, and column chromatography.

On the other hand, a monoclonal antibody can be used in an animal such as a mouse in accordance with, for example, the method of Cola and Milstein (Kohler & MiIstein: Nature, 256, 495-497, 19775). Alternatively, a peptide synthesized from the N-terminus of the protein obtained by sequence analysis according to the amino acid sequence such as the Fm0c solid phase method and the like is administered and immunized, and then spleen cells are obtained from the spleen. Cell fusion with mouse myeloma cells, etc. To get Hypri-Dorma. Next, a hybridoma producing a monoclonal antibody specific to the protein of the present invention is screened, and an antibody most suitable for discriminating between Alzheimer's disease patients and normal subjects is screened out of the hybridomas. Subcloning a hybridoma that produces an antibody that does not react with the protein of interest. Then, it can be obtained by isolating and purifying from the subcloned culture supernatant or the like in the same manner as the above-mentioned polyclonal antibody. .

[I I I-1] Method for diagnosing Alzheimer's disease

The method for diagnosing Alzheimer's disease according to the present invention comprises the steps of: using a sample collected from the subject's brain or a ^Q organism other than the brain, such as cerebrospinal fluid, blood, skin, hair, etc. It is characterized by reacting at least one kind of antibody that recognizes a fragment of the protein and detecting the degree or presence or absence of the reaction.

I.e. detecting the presence or absence of proteins with a fragment of protein or The protein of only detected the invention said antibody specifically reactive with an antigen, ^five Mari Alzheimer's disease patients expressing these samples, the amount of It determines Alzheimer's disease with some or no presence.

Specifically, for example, a case where this protein or a protein having a fragment of the protein is detected by the antibody of the present invention in a sample of a subject is a case where Alzheimer's disease or a sign thereof is present or not detected. A normal person can be diagnosed. In addition, it is considered that the amount of this protein gradually increases in the process of developing Alzheimer's disease in normal individuals.

Therefore, if the protein or a protein having a fragment of the protein is detected in the sample of the subject by the antibody of the present invention in a predetermined amount or more, Alheimer's disease or a sign thereof is diagnosed. You can also.

In this diagnostic method, one kind or two or more kinds of the antibodies of the present invention of the above [II] are used.

When the subject's brain is used as a sample, the sample is homogenized in a buffer, centrifuged, and the supernatant is collected. The supernatant is reacted with the antibody of the present invention. When using a subject's cerebrospinal fluid or blood as a sample, react with the antibody of the present invention directly or after pretreatment (such as centrifugation).

When the skin or hair of a subject is used as a sample, for example, the sample is immobilized in 10% neutral buffered formalin and then reacted with the antibody of the present invention.

Methods for detecting the reaction include Enzymlink Immunosorbent Assay (ELISA), Enzyme Imuno Assay (EIA), Fluorescent Imuno Assay (FIA), Radioimino Assay (RIA), and Chemiluminescence immunoassay. (CLIA), chemiluminescent enzyme immunoassay (CLEIA), latex agglutination, and the like.

[I I I 1 2] Diagnosis of Alzheimer's disease

In another method for diagnosing Alzheimer's disease of the present invention, the presence or amount of the protein [I] is analyzed by analyzing a sample of a subject.

Examples of the analysis method at this time include an electrophoresis method (for example, a two-dimensional electrophoresis method), various chromatography methods (for example, a high-performance liquid chromatography method), a bioassay method, and the like. Specifically, for example, a sample of a subject is extracted by the same method as in the above [I], then subjected to two-dimensional electrophoresis, and the obtained gel is subjected to silver staining or the like according to a conventional method, or To the ロッ stamp lot, incubate the blotting membrane and the antibody of [II] above at room temperature, and then add the labeled secondary antibody. In addition, if the spots of the protein of [I] are detected to some extent or more than a predetermined amount from the electrophoresis pattern, Alzheimer's disease or a sign thereof can be diagnosed. .

[IV] Diagnostic kit

The kit used for the diagnosis of Alzheimer's disease of [III-1] is composed of at least one antibody that recognizes the protein of [I] or a fragment of the protein as a component, and the antibody and the brain or other than brain. It is characterized by including means for detecting a reaction with a sample collected from a living body.

Examples of the antibody used include a polyclonal antibody [I I] and a monoclonal antibody, and a monoclonal antibody is preferable.

Methods using one type of antibody include, for example, immunohistochemical techniques (eg, EIA), and methods using two types of antibodies include, for example, ELISA using the sandwich method. .

Means for detecting the reaction include those used in the detection method described in [III-11]. For example, in the case of immunohistochemical techniques (eg, EIA), enzyme-labeled anti-mouse IgG, substrate, Examples include a buffer solution, a blocky antibody (eg, goat serum), and in the case of ELISA, an enzyme-labeled anti-mouse IgG, a substrate, a carrier (eg, beads, plates, etc.), a buffer solution, a reaction stop solution, and the like. Can be BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a photograph showing a silver-stained secondary electrophoresis gel of a sample of an Alzheimer's disease patient (A1 zheimer's Disease) (left) and a sample of a normal person (Contro1) (right). . The arrow indicates the protein of the present invention detected in Alzheimer's disease patients. It shows that it is not detected by ordinary people.

The numbers displayed in the center vertically indicate the molecular weight (kD) of the molecular weight marker, and the numbers displayed in the lower side indicate the isoelectric point (pi) of the isoelectric point marker.

FIG. 2 is a photograph of immunostaining of an Alzheimer's disease patient using the antibody of the present invention.

FIG. 3 is a photograph of immunostaining of a normal person using the antibody of the present invention. The arrow in FIG. 2 indicates the protein of the present invention detected in the Alzheimer's disease patient recognized by the antibody of the present invention, which is recognized at the same position as in the case of the silver staining shown in FIG. 1, and the normal person in FIG. Indicates that it has not been detected. Next, the present invention will be described in detail with reference to examples.

Example 1 Extraction of protein expressed in Alzheimer's disease patient and preparation of electrophoresis sample

The brains after the death of patients with Alzheimer's disease (8 cases, 72 to 78 years old) and normal persons (8 cases, 69 to 85 years old) were taken out and cryopreserved at minus 90 ° C.

7 to 10 g of the cerebral cortex was excised and homogenized in a double volume of HEPS buffer (containing 25 mM HEPES, 0.25 mM EDTA, 50 mM magnesium chloride: PH6,8).

The obtained homogenate was centrifuged at 20 ° C., 20 ° C. and 60 ° C. for 60 minutes, and the supernatant was freeze-dried by an ordinary method. This lyophilized sample was mixed with 2 volumes of sample buffer [9.9 M urea, 4% (v / v) Noridet P—40, 2.2% (vZv) Am pholyte (Nippon Millipore: PH 3 ~ 10), 100 mM MD ithiothreitol (DTT)] and incubated at 37 ° C for 1 hour to prepare a sample for electrophoresis. Finally, the protein concentration was about 5 mgZm1. Example 2 Separation of proteins using high-resolution two-dimensional electrophoresis

Primary electrophoresis is performed by isoelectric focusing using Investigator 2—DE electrophoresis system (manufactured by Nippon Millipore) as the electrophoresis device, and sodium dodecyl sulfate—polyacrylamide is used for secondary electrophoresis. Electrophoresis (SDS-PAGE) was performed.

(1) Primary electrophoresis

The primary electrophoresis gel was prepared as follows.

First, 9.5 M urea, 2.0% (V / V) Nonidet P—40, 4.1% (vZv) acrylamide monobis solution (30.8% T, 2.6% C) Was stored as a stock in 5.65 ml portions and stored at minus 30 ° C.

Next, this stock was dissolved at the time of use, and Ampho 1 yte 3501 used in Example 1 and 10% ammonium persulfate (APS) 401 were added. mm diameter).

The first electrophoresis was performed using 85% phosphoric acid as the anolyte and 10 N-sodium hydroxide as the catholyte.

Overlay buffer [0.5 M urea, 0.2% (V / V) N 0 nidet P—40, 0.1% (V / V) Am pho 1 ite, 5.0 mM DTT, 0. [M M—Mercaptoethanol] was layered in 101 layers, and pre-electrophoresis was performed at room temperature for 2 hours at a maximum voltage of 150 V and a maximum current of 110 A / ge 1.

The protein sample from the cerebral cortex prepared in Example 1 was layered between the overlay buffer and the gel in a layer of 101, and the maximum voltage was set to 200 V and the maximum current was set to 110 μA / ge1. Electrophoresis was performed at room temperature until Volt — Ours reached 180 000 V. As an isoelectric point marker, IEF manufactured by Biorad Standards (PI 4.6 to 9.6) were used.

(2) Secondary electrophoresis

After the first electrophoresis, the one-dimensional gel is taken out in 10% glycerin, and the one-dimensional gel equilibration buffer (0.3 MT ris Base, 0.075 MT ris HC 1, 3%) is taken for 2 minutes. After equilibration in sodium dodecyl sulfate (SDS), 50 mM DTT, 0.1% bromphenol blue), it was placed on a secondary electrophoresis gel. The secondary electrophoresis gel was Duracry 1 (manufactured by Nippon Millipore) 29.6 lml, Tris buffer (130.8 g / 1 Tris Base, 66.3 g / l Tris) HC 1) 2 20.5 ml, M i 1 1i- Q water 3 65.4 ml, 10% SDS 9.0 ml, tetramethylethylenediamine (TEMED) 0.447 ml, 2.24 ml of 10% APS was mixed and prepared to a thickness of 1 mm between 26 × 26 cm glass plates.

The two-dimensional electrophoresis buffer was adjusted with the composition of 25 mM Tris Base, 19 2 mM glycine, 0.1% SDS, and the electrophoresis was performed at 20 ° C at a maximum voltage of 500 V and maximum power. The procedure was performed at 1,600 mW / ge 1 until the stained surface became 1 cm from the bottom of the gel.

As a molecular weight marker, an SDS-PAGE standard (Low, MW 1440000 to 9-740) manufactured by Piorad was used.

Example 3 Characterization of proteins detected in Alzheimer's disease patients

(1) In order to detect proteins expressed in Alzheimer's disease patients, the secondary electrophoretic gel obtained in Example 2 (2) was silver-stained by a conventional method.

Fig. 1 shows secondary electrophoresis gels of each sample of Alzheimer's disease patients and normal subjects stained with silver.

In all samples (8 cases) of Alzheimer's disease patients, One protein spot was detected, which was clearly different from the sample (8 cases).

No spots of this protein were detected in all normal samples.

The isoelectric point and molecular weight of this protein specific to Alzheimer's disease patients were determined by comparison with the respective markers.

The isoelectric point was P 1 = 6.9, and the molecular weight was 30 kD.

(2) The protein specifically detected in this Alzheimer's disease patient was subjected to plotting in order to determine the N-terminal amino acid sequence. For plotting, use Imm obilon (manufactured by Nippon Millipore) for the membrane, and use Mi 1 iB10t (manufactured by Nippon Millipore) for the blotter. 50 A / sq-cm) and a maximum power of 100 W for 30 minutes.

The blotting membrane was stained with PhastGelBlueR (Pharmacia) to cut out the target spots. For the determination of the N-terminal amino acid sequence, the protein in this spot was analyzed by an ordinary method using Applied Biosystems' 477 APRoitenSeeqenccer and 120 AAnalyzer.

The amino acid sequence up to the 15th residue from the N-terminus of the protein specific to this Alzheimer's disease patient is, as shown in the sequence listing below,

(1) (5)

X a a -X a a -V a 1-P ro -X a a -G l y-A 1 a-X a a-

(10) (15) T hr — Gly—I1e-Tyr-G1u—Xaa—Leu. (Where X aa represents an unidentified amino acid) In this amino acid sequence, Xaa is an unidentified amino acid, but Xaa at the first residue is Ala, Xaa at the second residue is A1a, Xaa at the fifth residue is Ser, It is highly probable that Xaa at residue 8 is Ser and that Xaa at residue 14 is Trp or A1a.

Example 4 Preparation of polyclonal antibody

The amino acid sequence (Ala-A1a-Val-Pro-Ser-Gly-A1a—Ser-Thr-) obtained by the sequence analysis of the protein spot of Example 3 (2) Based on G 1 y-I 1 e-T yr-G lu-A 1 a-Leu), Proc. Natl. Acad. Sci USA (Vo186, 90084) The peptide was synthesized by the Fmoc solid-phase method using Advanced ChemTec 350 according to the method described in 99088). The peptide was synthesized from the C-terminal side to the N-terminal side together with a polystyrene resin with a MAP core. N, N-Dimethylformamide (DMF) was used as the system solution, and 20% piperidine was used to deprotect 1-hydroxybenzotriazole (HO bt) and 1,3-diisopropylcarbodiimide (DIC). Was used as a coupling agent. The synthesized peptide was washed with methanol and dried. After separation from the resin, the protecting groups on the side chains were removed using a solution of 90% trifluoroacetic acid, 5% trianisole, 3% ethanedithiol, and 2% anisol. The resulting peptide was washed five times with ether, dissolved in purified water, freeze-dried and stored until use. The first immunization was carried out by subcutaneously injecting 0.5 mg of the peptide synthesized in this way into 20 egrets together with Freund's vaccine leteadjvant. The second immunization was performed in the same manner by subcutaneously injecting 20 mg of 0.5 mg in a dose together with Freundsinco mp leteadjvant, blood was subsequently collected from the ear, and centrifuged to collect serum. Obtained. Using the polyclonal antibody contained in this serum as the primary antibody, the following test was performed.

Example 5 Stamp stamp using polyclonal antibody

(1) Preparation of water-soluble protein samples from the cerebral cortex of Alzheimer's disease patients (8 cases) and normal persons (8 cases) was performed in the same manner as in Example 1.

(2) Separation of cerebral cortical water-soluble proteins using high-resolution two-dimensional electrophoresis was performed by primary electrophoresis in the same manner as in Example 2 (1), and then in the same manner as in Example 2 (2). Secondary electrophoresis was performed to obtain a two-dimensional electrophoresis gel. (3) In order to immunologically detect the protein of the present invention which seems to be specifically expressed in Alzheimer's disease patients, the above two-dimensional electrophoresis gel was subjected to easter blotting. For blotting, use Immo bilon-P (MI LLIPO RE) for the membrane and Mi 1 i Blot (MILLI PO RE) for the plotter, and use a maximum voltage of 100 V (125 / A / sq-cm). ) The test was performed at a maximum power of 100 W for 30 minutes. The blotting membrane was stored frozen at 180 ° C and then returned to room temperature in 20 mM Tris saline (TBBS) containing 0.1% Tween-20. After washing three times with TBBS, the blotting membrane was incubated for 24 hours at room temperature with the above-mentioned antibody (primary antibody) diluted 1: 1000. After washing three times with TBBS, the cells were incubated for 1 hour and 30 minutes with a goat antibody (secondary antibody) of an alkaline phosphatase-labeled anti-magpie antibody. After washing three times with TB BS, an alkaline phosphatase reaction was carried out using Western Blue AP—substrate solution (Promega, ffb3841) as a substrate until protein spots appeared. Washed twice. This procedure stained many spots. It is considered that the nature of the primary antibody (polyclonal antibody) has cross-reactivity with many proteins. Alzheimer's as shown in Figure 2 In the affected patients, clearly different spots were detected in all the samples as compared with the normal subjects in the places identified by the silver staining of Example 3 (1) described above. Conversely, as shown in FIG. 3, these spots were not detected in normal subjects or were very small in quantity.

The immunostaining using the antibody of the present invention is relatively easy to distinguish between Alzheimer's disease patients and normal subjects by two-dimensional electrophoresis because of its higher sensitivity and less staining of other proteins than silver staining. I can do it. In addition, this antibody can be used, for example, as one antibody when performing the ELASA (sandwich) method to examine the presence or amount of a protein specifically expressed in this Alzheimer's disease patient in a subject sample. it can. Industrial applicability

The protein of the present invention can be obtained from at least the brain of Alzheimer's disease patients, and is detected in Alzheimer's disease patients but not in normal persons. Therefore, an antibody useful for diagnosing Alzheimer's disease using this protein Can be adjusted. In addition, this protein is expected to be of great use in studies such as investigating the causes of Alzheimer's disease.

The antibody of the present invention recognizes the protein of the present invention or a fragment thereof which is expressed in the brain of a patient with Alzheimer's disease or a living body other than the brain, and reacts with the protein or a protein having a fragment of the protein to diagnose Alzheimer's disease. Useful for

According to the method for diagnosing Alzheimer's disease of the present invention, the degree or presence or absence of a reaction with a sample is detected using the above-described antibody of the present invention, so that a definitive diagnosis of Alzheimer's disease in the brain or the like can be easily performed. . In addition, it is highly likely that early diagnosis of Alzheimer's disease can be easily performed using peripheral samples. You.

According to another method for diagnosing Alzheimer's disease of the present invention, a sample collected from a subject is analyzed to check for the presence or amount of the protein of the present invention, so that diagnosis of Alzheimer's disease can be performed only reliably. And an early diagnosis can be made.

According to the kit for diagnosing Alzheimer's disease of the present invention, Alzheimer's disease can be easily diagnosed. Sequence listing

SEQ ID NO: 1

Array length: 1 5

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Fragment type: N-terminal fragment

Array

X a a X a a V a 1 P ro X a a G 1 y A l a X a a

1 5

T h r G 1 y l i e T y r G 1 u X a a L e u

1 0 1 5

Claims

The scope of the claims

1. A protein with at least the following properties that can be obtained from the brain of at least Alzheimer's disease patients.

(1) Molecular weight: sodium dodecyl sulfate-polyacrylamide gel "30 kD as measured by electrophoresis

(2) Isoelectric point: p I = 6.9

(3) Amino acid sequence from the N-terminal to the 15th residue:

(1) (5)

X aa -X aa -V a 1 -Pro -X aa -G ly-A 1 a- ¹⁰ (1 0) (1 5)

Xaa-Thr-Gly-Ile-Tyr-Glu-Xaa-Leu (where Xaa represents an unidentified amino acid)

2. An antibody that recognizes the protein according to claim 1 or a fragment of the protein.

¹⁵ 3. Reacting a sample collected from a subject with at least one antibody that recognizes the protein of claim 1 or a fragment of the protein, and detecting the degree or presence or absence of the reaction. Method of diagnosing Alzheimer's disease.

4. As a component, at least one kind of antibody that recognizes the protein according to claim 1 or a ²⁰ fragment of the protein, and means for detecting a reaction between the antibody and a sample collected from a living body A diagnostic kit for Alzheimer's disease, comprising:

5. A method for diagnosing Alzheimer's disease, comprising analyzing a sample collected from a subject to check for the presence or amount of the protein according to claim 1.

256. If the analysis method is electrophoresis, chromatography, or bioassay. 6. The method for diagnosing Alzheimer's disease according to claim 5, which is a method.