JP2002040023A - Methods for detecting Alzheimer's disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sure and simple detection method of Alzheimer's disease having excellent specificity and using blood as a sample. SOLUTION: A blood sample obtained from an individual having the possibility of having Alzheimer's disease is denatured by heating or the like in the presence of a protein solubilizing agent, to remove impurity proteins, and then existence of τ protein in the protein-removed sample solution is analyzed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting Alzheimer's disease, and more particularly, to a method for detecting Alzheimer's disease by measuring the presence of tau protein in blood.

[0002]

2. Description of the Related Art Alzheimer's disease is in old age (45-65).
Years of age, progressive dementia that develops in cerebral cortex, and pathological changes include degeneration of nerve cells and atrophy of the cerebral cortex due to a decrease in the number of nerve cells. Plaques and neurofibrillary tangles are noted. Senile dementia due to so-called spontaneous aging that occurs in the senile period of 65 years or older is also referred to as Alzheimer's senile dementia because no essential difference is recognized pathologically.

[0003] The number of patients with this disease has increased with the increase in the elderly population, and has become a socially important disease. However, although there are various theories about the cause of this disease, the result is still unknown, and early elucidation is desired.

It has been elucidated that a major component of senile plaque, one of the pathological changes of Alzheimer's disease, is amyloid β protein (Annu. Rev. Neurosci., 12 , 46).
3-490 (1989)). Another pathological change, neurofibrillary tangles, is a phenomenon in which a paired helical filament (hereinafter referred to as “PH”) is introduced into a nerve cell.
F ") is accumulated.
Tau protein has been identified as one of its components (J. Biochem., 99 , 1807-1810 (1986); Proc. Natl. A
cad. Sci. USA, 83 , 4913-4917 (1986)).

[0005] Tau proteins are a group of closely related proteins that usually form several bands at a molecular weight of 48 to 65 kD by SDS polyacrylamide gel electrophoresis, and are known to promote the formation of microtubules.

[0006] Tau protein incorporated into PHF in the brain of Alzheimer's disease patients is more abnormally phosphorylated than tau protein in the brain of healthy subjects, indicating that polyclonal antibodies against PHF (anti-p-tau; J. Biochem., 99 , 18
07-1810 (1986)) and monoclonal antibodies against tau protein (tau-1;... .. Proc Natl Acad Sci USA, 83 491
3-4917 (1986)). Also, PHF
The phosphorylation site of the tau protein in Alzheimer's disease has been elucidated, for example, the phosphorylation site of the phosphorylated tau protein incorporated therein has also been determined (JP-A-6-239893).

As for the tau protein, a kit for measuring the concentration in cerebrospinal fluid (Japanese name: Finoscalar hTAU; European and American names: INNOTEST)
hTAU Ag (manufactured by Innogenetics) is commercially available, and although it has a problem in specificity, it is considered to be clinically useful. On the other hand, a method for detecting Alzheimer's disease by focusing on the phosphorylation site of phosphorylated tau protein in PHF has also been developed (Neurosci. Lett., 270 , 91-94).
(1999)). This method may have better specificity than the method for measuring tau protein.

All of these methods use cerebrospinal fluid as a sample, and there has been a demand for a method for detecting Alzheimer's disease using a blood sample or the like that is less invasive to a patient.

Regarding tau protein in blood as a marker for Alzheimer's disease, a report denying its usefulness (Dement. Geriat. Cong. Disord., 10 , 442-445 (199)
9)) and increased in about 1/3 of patients with Down's syndrome but not in Alzheimer's disease (Neurosci. Lett.,
275 , 159-162 (1999)), and there is no method for detecting Alzheimer's disease by measuring the amount of tau protein in blood.

[0010] In each report, a method of directly measuring blood is used, and the value of tau protein is not constant, and there is no significant difference between Alzheimer's disease patients and healthy subjects. The conclusion is that it is not useful for detection. The reason why the value of tau protein is not constant in these methods is that the amount of protein present in blood is 100 times or more as compared with the case where cerebrospinal fluid is used as a sample.

[0011]

SUMMARY OF THE INVENTION The present invention has been made to provide a reliable, highly sensitive, and simple method for detecting Alzheimer's disease with excellent specificity.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, if plasma was heated in the presence of a protein solubilizing agent to remove contaminating proteins, It was found that tau protein in plasma could be reliably measured, and the concentration of tau protein in plasma thus measured was significantly changed in Alzheimer's disease patients. The present invention has been achieved based on these findings.

That is, the present invention provides (1) a step of denaturing a blood sample obtained from an individual suspected of having Alzheimer's disease in the presence of a protein solubilizing agent to remove contaminating proteins, and removing contaminating proteins. A method for detecting Alzheimer's disease, comprising a step of analyzing the presence of tau protein in a sample solution obtained.

According to a preferred embodiment of the present invention, (2)
The method of the above (1) including a step of further concentrating the sample solution from which the contaminating protein has been removed, and (3) the analysis of the presence of the tau protein is carried out by examining the reactivity between the sample and the anti-tau protein antibody. A method according to (1) or (2) is provided.

Further, according to another preferred embodiment of the present invention, (4) the method according to any one of the above (1) to (3), wherein the protein solubilizing agent is a guanidine salt and a sulfhydryl group-containing agent. Is provided.

According to another aspect of the present invention, there is provided (5) Alzheimer's disease according to any one of the above (1) to (4), which comprises at least an anti-tau protein antibody and a protein solubilizing agent. A reagent kit for performing the detection method is provided.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail.

The method for detecting Alzheimer's disease of the present invention comprises:
A blood sample obtained from an individual suspected of having Alzheimer's disease is denatured in the presence of a protein solubilizing agent to remove contaminating proteins (hereinafter, this may be referred to as "deproteinization").
And then analyzing the presence of tau protein in the deproteinized sample solution. The sample solution from which the protein has been removed can be further concentrated, if desired, and then analyzed for the presence of tau protein. Analysis of tau protein in the sample solution
It is preferably carried out by examining the reactivity between the sample and the anti-tau protein antibody.

As used herein, the term “tau protein”
Unless otherwise specified, it means non-phosphorylated tau protein, phosphorylated tau protein, or a partial fragment thereof. The "blood sample" to be analyzed for tau protein is obtained by collecting blood from an individual suspected of having Alzheimer's disease, for example, from the cubital vein using a blood collection tube, and separating plasma or serum.

In the present invention, in order to prevent dephosphorylation of phosphorylated tau protein in a blood sample, a phosphatase inhibitor is preferably added during or after blood collection. Examples of the phosphatase inhibitor to be used include EDTA, EGTA, okadaic acid, pyrophosphate, phosphate, sodium fluoride, β-glycerophosphate, cyclosporin A, and the like. Among them, EDT
A, EGTA is preferred. The concentration of the phosphatase inhibitor is not particularly limited as long as it has an inhibitory effect, and is, for example, generally 1 to 1000 mM, preferably 1 to 1 mM.
00 mM is appropriate. Further, it is preferable to determine and use an optimal combination of concentrations depending on the type of the phosphatase inhibitor.

Therefore, the blood sample of the present invention is EDT
Plasma is preferably obtained by collecting blood using a blood collection tube containing A, adding the above kinase inhibitor if necessary, and centrifuging the blood. The obtained blood sample is preferably stored frozen at, for example, −20 ° C. or lower until subjected to analysis of tau protein.

The blood sample thus obtained is first subjected to a denaturation treatment in the presence of a protein solubilizing agent to solubilize the tau protein, and then to remove contaminating proteins from the sample.

The protein solubilizing agent used in the present invention is not particularly limited as long as it has a function of dissolving a protein which is hardly soluble in water, and specifically, for example, guanidine or a salt thereof, a sulfhydryl group-containing agent ( Less than,
), Urea, and a surfactant, and these substances are used alone or in combination. Above all, it is preferable to use a guanidine salt and an SH group-containing agent in combination.

Examples of the guanidine salt used include various acid salts such as isothiocyanate, hydrochloride, and perchlorate. Of these, guanidine isothiocyanate and guanidine perchlorate are exemplified. preferable. SH
Examples of the group-containing agent include β-mercaptoethanol, dithiothreitol, and N-acetylcysteine, among which β-mercaptoethanol is preferable. As a surfactant, Tween 20 (ICI
Americas Inc. Made), Triton X
-100 (Rohm & Haas) and Nonide
t P40 (Shell International)
Petroleum Company Limited
And nonionic surfactants such as

Among these, guanidine perchlorate and β
-It is particularly preferred to use a combination of mercaptoethanol, guanidine isothiocyanate and β-mercaptoethanol.

The concentration of the protein solubilizer used in the present invention is generally 0.01 to 10 M, preferably 0.1 to 6 M.
M is used, but it is preferable to determine and use the optimum concentration depending on the type of protein solubilizing agent and the combination thereof. For example, the concentration of the guanidine salt is usually 1 mM to 6 mM.
M, preferably 0.5 to 1M. The concentration of the SH group-containing agent is generally 1 mM to 1M, preferably 50 to 50M.
0 mM. The concentration of the surfactant is usually 0.0
01 to 0.5% (v / v), preferably 0.01 to 0.
2% (v / v). The concentration of urea is usually 2 to 10
M, preferably 5-7M.

The tau protein can be solubilized by adding the above protein solubilizing agent to the sample, diluting the sample by 2 times or more, preferably 2 to 10 times, and mixing and stirring.

The protein contaminants in the sample solution thus obtained are denatured and removed from the sample solution. As a method for denaturing contaminating proteins, for example, a method using a protein denaturing agent such as trichloroacetic acid, trifluoroacetic acid, ammonium sulfate, urea, an organic solvent, a method using an interface, and a method using heating are used alone or in combination.

In the present invention, a method of heating is preferably used, and particularly preferably a method of boiling, that is, a method of heating to 100 ° C. while boiling is used. The boiling (heating) time is usually 1 to 15 minutes, preferably 3 to 10 minutes.

The denatured contaminating protein can be removed by a known solid-liquid separation method such as membrane separation or centrifugation, but is preferably separated by centrifugation.

Under the above conditions, the tau protein is recovered in the supernatant of the centrifugation without being denatured by heating.

The tau protein in the sample from which contaminant proteins have been removed can be concentrated by a method known per se, such as ultrafiltration. Alternatively, after the salt concentration is reduced by dialysis, gel filtration, or the like, specific concentration can be performed by immunoprecipitation using magnetic beads or the like on which an anti-tau protein antibody is immobilized. Alternatively, the sample from which contaminating proteins have been removed can be concentrated by solid phase extraction. Specifically, for example,
A sample from which contaminating proteins have been removed is added to a general solid phase for extraction in which low-molecular-weight hydrocarbons are immobilized on silica gel, etc., and the tau protein is retained on the solid phase. I do. Next, an organic solvent or a mixed solvent of water and an organic solvent is flowed to collect the tau protein, and the protein can be concentrated by removing the solvent. In addition, a step of removing immunoglobulin-G by column chromatography or the like can be performed as necessary. It is preferable to use a Protein G-Sepharose column or the like for column chromatography.

The sample solution concentrated by ultrafiltration or the like
If desired, the above-described denaturation and removal operations of contaminating proteins may be repeated.

The sample solution thus obtained is analyzed for the presence of tau protein. The method for analyzing tau protein is not particularly limited, but preferably, the reactivity between the sample and the anti-tau protein antibody, that is, the immunoreactivity between the sample and the anti-tau protein antibody is compared with the reactivity between the positive control and the anti-tau protein antibody. It is done by checking.

The anti-tau protein antibody used for the immune reaction may be any antibody that can specifically bind to tau protein and / or phosphorylated tau protein. Examples include anti-tau protein monoclonal antibody HT7 (binding to amino acids 159-163 of tau protein) and BT2 (binding to amino acids 193-198 of tau protein) marketed by Innogenetics. Further, WO 97/34145,
No. 00-34300, anti-phosphorylated tau protein antibodies, or anti-tau protein antibodies prepared according to the descriptions of these publications, such as anti-PS199, anti-PS202, anti-PT205, anti-PT231, Anti-PS23
5, anti-PS262, anti-PS396, anti-PS404, anti-P
Antibodies such as S413, anti-PS422, anti-PT231PS235, anti-tau154-168 and the like can be mentioned. Among them, it is particularly preferable to use antibodies such as anti-PS199 and anti-tau154-168.

Anti-PS 199 is a phosphorylated peptide of S (serine) 199 (Lys-Ser-Gly-) in which K (lysine) is added to the amino terminus of amino acids 195 to 205 of tau protein.
An antibody obtained using Tyr-Ser-Ser (p) -Pro-Gly-Ser-Pro-Gly-Thr; SEQ ID NO: 1) as an antigen, and specifically binds to phosphorylated tau protein. In addition, anti-tau 154-168
Is a peptide (Cys-Pro-Ar) in which C (cysteine) is added to the amino terminus of amino acids 154 to 168 of tau protein.
g-Gly-Ala-Ala-Pro-Pro-Gly-Gln-Lys-Gly-Gln-Ala-Asn-
Ala; SEQ ID NO: 2: This is hereinafter referred to as "Tau154-168".
WO97 / 3414 as an antigen
An antibody obtained according to the method described in Japanese Patent Publication No. 5 and which binds to tau protein regardless of phosphorylation.

In this specification, the amino acid number of the tau protein is the number of the longest human tau protein described in Neuron, 3 , 519-526 (1989).

The positive control used for measuring the amount of tau protein of the present invention may be any positive control as long as it immunoreacts with the anti-tau protein antibody used in the present method. Specifically, it may be a tau protein purified from humans and rats, or a synthesized peptide. As a method for purifying tau protein, Journal of Neuron
Science Research, 25 , 412-419 (1990). As the chemically synthesized peptide, for example, the peptide shown in SEQ ID NO: 3 can be used. If necessary, a commercially available kit (name in Japan: Finoscalar hTAU; name in Europe and America: IN
NOTEST hTAU Ag; Innogeneti
cs) can also be used. Tau protein or peptide used as a positive control is, for example, 1 mg of lyophilized powder in PBS (0.15
M NaCl, 10 mM phosphate buffer pH 7.4) 10
It is preferable to dissolve in 0 μL, aliquot 10 to 20 μL, freeze-preserve, and use.

The analysis of tau protein by an immune reaction can be performed by a commonly used method known per se. Specifically, for example, an immunoblot, a sandwich method, a competition method and the like can be mentioned, and latex or the like may be used for them.

Among the above, the analysis of the amount of tau protein in a sample and the molecular weight thereof by immunoblotting can be performed, for example, as follows.

To the deproteinized sample obtained above, an appropriate treatment solution, for example, a Laemmli sample treatment solution (0.125 M Tris-HCl (pH 6.8),
% SDS, 20% glycerol, 20 μg / mL BP
B, 0.7 M β-mercaptoethanol), and heat-treated. The mixture was electrophoresed on a polyacrylamide gel according to the method of Laemmli (Nature 227 , 680-685 (1970)), and a commonly used protein transfer device, for example, a semi-dry blotter For example, a membrane usually used for transferring proteins, for example, PVDF (Polyvinylidene d)
(fluoride) film or the like.

After blocking the membrane, an anti-tau protein antibody to which a first substance having a specific binding property generally used is bound is reacted. After washing the membrane, the labeled substance used for signal detection was bound, the second substance that specifically binds to the first substance was reacted, and the membrane was washed and then bound to the second substance. A signal emitted from the labeling substance is detected, and the molecular weight and abundance of the tau protein can be known from the position and intensity of the signal. It is preferable to use a reaction other than an antigen-antibody reaction, which is a reaction with a substance having specific binding ability, for detecting the signal, in order to eliminate the influence of an antibody brought in from the other. That is, as the first substance and the second substance, for example, a combination of biotin and streptavidin, a combination of digoxigenin and an anti-digoxigenin antibody, and the like are used, and among these, a combination of biotin and streptavidin is particularly preferable. Examples of the labeling substance include an enzyme, a fluorescent substance, a radioactive substance and the like. Among them, alkaline phosphatase and ho
rsadish peroxidase (hereinafter sometimes referred to as “HRP”) is particularly preferably used.

As another analysis method using an immune reaction,
A method for measuring tau protein by an immunoassay can be used. For example, the sample solution obtained above is added to a 96-well EIA plate on which an anti-human tau protein antibody is immobilized, and the tau protein is bound to the solid phase. Further, an anti-human tau protein antibody for detecting the tau protein, that is, an antibody that recognizes an epitope different from the immobilized antibody used as the detection antibody is added and incubated. After washing with a washing solution, the detection antibody is reacted with a label, for example, an enzyme-labeled anti-rabbit IgG antibody or an enzyme-labeled streptavidin. After washing with a washing solution, the activity of the label bound to the solid phase is measured, and the reactivity with the above-mentioned positive control having a known concentration is measured, whereby the amount of tau protein in the sample can be measured.

In this manner, the amount of tau protein present in a blood sample of an individual suspected of having Alzheimer's disease is analyzed, and the amount of tau protein present in the sample is compared with that of an individual without Alzheimer's disease. If it has changed significantly, the individual is identified as having Alzheimer's disease. If the control amount of an individual without Alzheimer's disease is similar to that of the control, the individual is confirmed as not having Alzheimer's disease. Thus, the detection of Atzheimer's disease can be performed according to the present invention.

The reagent kit for detecting Alzheimer's disease, which comprises a step of denaturing in the presence of the protein solubilizing agent to remove contaminating proteins and a step of analyzing tau protein by an immunological reaction, comprises a normal immunological reaction. It is provided by the configuration according to the kit used. That is, the reagent kit of the present invention contains at least a protein solubilizer and an anti-tau protein antibody, and further, as an optional element, a washing solution, a labeled antibody,
Includes positive controls, etc.

More specifically, for example, in the case of the sandwich method, it includes a pretreatment solution containing at least a protein solubilizer, an immobilized anti-tau protein antibody, and an anti-tau protein antibody for labeling.
In the case of the competitive method, a pretreatment solution containing at least a protein solubilizing agent, a labeled tau protein, and an anti-tau protein antibody are included. In the case of the sandwich method using latex, a pretreatment solution containing at least a protein solubilizing agent, an anti-tau protein antibody-immobilized magnetic latex, and an anti-tau protein antibody for labeling are included. In the case of the competition method using latex, a pretreatment solution containing at least a protein solubilizing agent, an anti-tau protein antibody-immobilized magnetic latex, and a labeled tau protein antibody are included.

[0047]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

In the following embodiment, "TBS"
Is a 20 mM Tri containing 150 mM NaCl.
s-HCl buffer (pH 7.5), "TBST" is 0.
TBS containing 05% Tween20. “BPB” is Bromophenol Blue. "HRP" is horseradish perox
Indicates the idase.

Example 1 Immunoblotting of Tau Protein in Plasma
Detection by Tsu preparative (1) (bonded to amino acid number 159-163 of tau protein) antibodies anti-tau protein monoclonal antibody HT7 and BT2 (binds to amino acid numbers 194-198 of the tau protein), In
Nogenetics purchased and used.

Anti-PS 199 and anti-tau 154-168
Was prepared as follows according to the description in WO97 / 34145.

The chemically synthesized phosphorylated tau peptide (S
(Serine) 199 phosphorylated peptide: SEQ ID NO: 1) was bound to hemocyanin of keyhole limpet, and rabbits were repeatedly immunized to obtain antiserum. The antiserum is passed through a non-phosphorylated peptide column to collect unadsorbed fractions, thereby removing components that react with non-phosphorylated peptide. This fraction is passed through a phosphorylated peptide column, and the adsorbed fraction is collected by Pierce. Gentle elution
Elution with buffer ^™ gave an affinity-purified phosphorylation site-specific antibody (anti-PS199).

The chemically synthesized non-phosphorylated peptide, tau 1
A peptide in which cysteine was added to the amino terminus of 54-168 (SEQ ID NO: 2) was bound to hemocyanin of keyhole limpet, and rabbits were repeatedly immunized to obtain an antiserum. This antiserum was treated with non-phosphorylated peptide tau 15
The adsorbed fraction was passed through a 4-168 column and eluted with a Genel elution buffer ^™ of Pierce to obtain an affinity-purified specific antibody (anti-tau 154-168). The specificity of this anti-tau 154-168 was confirmed to bind to the recombinant tau protein by enzyme immunoassay (EIA) as follows.

First, recombinant tau protein 2-249 was added to a plate on which anti-tau protein 194-198 monoclonal antibody BT2 (Innogenetics) was immobilized, incubated, and then washed. Next, anti-tau 1
After incubation with the addition of 54-168, the plate was washed. Further, after an HRP-labeled anti-rabbit IgG antibody (goat) was reacted as a secondary antibody, the antibody was washed. HRP in solid phase
The activity was measured, and the enzyme activity (HRP activity) was increased in accordance with the concentration of the recombinant tau protein 2-249, whereby the antibody against the synthetic tau protein 154-168 peptide was bound to the recombinant tau protein 2-249. confirmed.

The above antibody was biotinylated for use in immunoblotting. The biotinylated antibody was prepared in 0.16 M sodium borate buffer (pH 8.5) using sulfo-NHS-
LC-biotin was added at 80 equivalents of IgG, and the reaction was allowed to proceed overnight at 4 ° C., followed by NAP-5 ^™ (Amersham Pharm).
(Asia Biotech) to remove small molecular weight substances and add BSA to 1%.

(2) Removal of contaminating proteins from human plasma E obtained from three healthy subjects and three Alzheimer's disease patients
A final concentration of 1 mM EDTA, 1 m in 7 mL of DTA plasma
M EGTA, 1M guanidine perchlorate, 0.5M
NaCl, 0.1 M β-mercaptoethanol, 10
The mixture was diluted 2-fold with 7 mL of a 2-fold concentration buffer so as to have a 0 mM phosphate buffer (pH 5.0), and heated in boiling water for 5 minutes. The heat-denatured protein was removed by centrifugation at 10,000 rpm for 15 minutes, and the tau protein was recovered in 12.6 mL of supernatant. Ultrafiltration (Amicon minicon; ma
The solution was concentrated to 0.2 mL with a chromosome concentrate and heated again in boiling water for 5 minutes. The protein denatured by heat was removed by centrifugation at 10,000 rpm for 15 minutes, and tau protein was recovered in 0.2 mL of supernatant.

The supernatant was added to a 10 mM sodium acetate buffer (p
H7.0). Transfer the solution to ProteinG-S
The suspension was added to 0.16 mL of epharose gel, stirred overnight by a tag rotor, and flash centrifuged at 12,000 rpm to obtain Protein G-Sepha.
Human immunoglobulin bound to rose was removed. Tween 20 was added to the supernatant at a final concentration of 0.05%, NaC
1 was added to a final concentration of 0.5M. The antibody beads were added thereto, and the tau protein was bound to the antibody beads while stirring with a tag rotor overnight, to perform immunoprecipitation of the tau protein.

The antibody beads were prepared by using the anti-tau protein monoclonal antibody HT7 as an anti-mouse IgG-Dynabeads ^™.
(Dynal) to dimethyl methylim
It is chemically bonded using idate.2HCl (DMP).

The beads were mixed with 100 μL of TBST for 3 hours.
The antibody beads that have been washed once and precipitated are suspended in 5 μL of TBS, and a double-concentration sample treatment solution of Laemmli (0.125 M Tris-HCl (pH 6.8),
4% SDS, 20% glycerol, 20 μg / mL B
5 μL of PB, 0.7M β-mercaptoethanol)
In addition, the mixture was heated at 95 ° C. for 5 minutes to solubilize the tau protein.

(3) Immunoblot The above tau protein was purified by Laem using 9% polyacrylamide.
Electrophoresis was performed by the method of MLI (Nature 227 , 680-685 (1970)), and was transferred to a PVDF membrane by a semi-dry blotter. The PVDF membrane was subjected to immunoblotting with biotinylated anti-tau 154-168 as follows.

Blocking and antibody treatment TBS containing 5% skim milk and 1.5% normal goat serum
The membrane was blocked for 1 hour with 20 mL. 1.8 ng
/ ML of biotinylated anti-tau 154-168 / mL
Was placed on a membrane and reacted overnight at 4 ° C. in a wet box. TB film
Unreacted antibodies were removed by washing three times with 20 mL of ST for 10 minutes.

HRP-streptavidin treatment and detection of chemiluminescence HRP-streptavidin (Amersham Ph)
armacia Biotech) was immersed in a 1,200-fold diluted solution and allowed to react for 1 hour.
Unreacted HRP-streptavidin was removed by washing 6 times with 0 mL for 5 minutes. Amersham Pharm
The membrane is moistened with a mixture of 2 mL of solution A and 50 μL of solution B of ECLplus ^{™ from} Acia Biotech, sandwiched between transparent sheets, and adhered to Hyperfilm ECL ^™ for several tens of seconds to several tens of minutes to determine the presence of antigen on the membrane. Detected by luminescence.

Further, after a luminescent substrate of ECL plus ^™ was added to the PVDF membrane, a luminescent image was detected with a lumino image analyzer LAS-1000plus manufactured by Fuji Photo Film Co., Ltd.

From the results of the above immunoblotting, human blood had an amino acid sequence of tau 154-168 and molecular weights of 1,200,000, 54,000, 44,000,
38,000, 32,000, 27,000 and 25,
It was confirmed that 2,000 or less tau proteins and fragments thereof were present.

Analysis of Detection Results Further, the luminescence image was analyzed by Image Gauge, image analysis software manufactured by Fuji Photo Film Co., Ltd., and compared with the luminescence amount of a known amount of a positive control, the molecular weight to which anti-tau 154-168 bound was about 32. 2,000 bands were quantified. As a result, the plasma tau protein concentration was 5.0 pM, 7.8 pM, and 6.9 pM in three healthy subjects, respectively. On the other hand, in three Alzheimer's disease patients, the values were 8.3 pM, 9.8 pM, and 8.1 pM, respectively, and the tau protein concentration in plasma showed a significantly higher value in the Alzheimer's disease patient.

Example 2 EIA of tau protein in plasma
According Detection (1) removing healthy subjects three contaminating proteins from human plasma, obtained from Alzheimer's disease patients three E
A final concentration of 1 mM EDTA, 1 m in 7 mL of DTA plasma
M EGTA, 1M guanidine perchlorate, 0.5M
NaCl, 0.1 M β-mercaptoethanol, 10
The mixture was diluted 2-fold with 7 mL of a 2-fold concentration buffer so as to have a 0 mM phosphate buffer (pH 5.0), and heated in boiling water for 5 minutes. The heat-denatured protein was removed by centrifugation at 10,000 rpm for 15 minutes, and the tau protein was recovered in 12.6 mL of supernatant.

Ultrafiltration (Amicon minicon; m
acrosolute concentrators)
And heated again in boiling water for 5 minutes. The protein denatured by heat was removed by centrifugation at 10,000 rpm for 15 minutes, and tau protein was recovered in 0.2 mL of supernatant.

This supernatant was added to the assay buffer (0.1% BS).
A, 1 mM EDTA, 1 mM EGTA, 20 mM Tris, 0.15 M NaCl, 0.05% Tween2
(0, pH 7.4). An assay buffer solution was added to the internal dialysis solution to make 0.35 mL, and a 20-fold concentrated solution was used as a sample for enzyme immunoassay (EIA) shown below.

(2) Immobilization of EIA anti-human tau protein antibody on a 96-well EIA plate 0.1 M sodium carbonate buffer solution of anti-human tau protein monoclonal antibody HT7 (pH
9.0) 0.1 mL was added, and the mixture was incubated at 4 ° C. for 3 hours in a wet box to be immobilized. After removing the supernatant, 0.1 M
Washed with sodium carbonate buffer, PBS containing 1% BSA, 1% skim milk, 0.5% gelatin (10 mM sodium phosphate, 150 mM NaCl; pH 7.4)
0.2 mL of the solution was added and blocking was performed at 4 ° C. for 2 hours. Wash solution (20 mM Tris, 0.05% Tween2
0; pH 7.4) and used immediately after washing. When not used immediately, they were further washed with pure water, dried in vacuum, placed in a laminate bag, and stored at 4 ° C.

Measurement of phosphorylated tau protein by EIA According to the method described in Example 3 of JP-A-2000-34300, phosphorylated tau protein was measured as follows.

A known amount of a positive control (synthetic peptide: SEQ ID NO: 3) quantified using a tau protein measurement kit manufactured by Innogenetics was assayed using an assay buffer (0.1% BSA, 1 mM EDTA, 1 mM EGT).
A, 20 mM Tris, 0.15 M NaCl, 0.05%
Tween 20; pH 7.4)
50 μL was added to the wells of the EIA plate prepared above. Further, the human plasma sample prepared in (1) above was
Similarly, 50 μL was added to the wells of the EIA plate prepared above.

To each well containing a positive control having a known concentration or the human plasma sample prepared in the above (1), 25 ng / mL of an affinity-purified anti-phosphorylated tau protein rabbit antibody (anti-PS199) (1% normal goat serum, 1%
50 μL of normal mouse serum-containing assay buffer) was added,
The plate was sealed with a plate sealer and incubated at 4 ° C. overnight with shaking.

After washing with a washing solution, a peroxidase-labeled anti-rabbit IgG antibody (ENVISION + / HRP dextran polymer reagent (DAKO)) was added to an assay buffer solution containing 1% normal goat serum, 5% skim milk and 0.5% normal mouse serum. 100-fold diluted solution)
L, sealed with a plate sealer, and incubated at 4 ° C. for 2 hours with shaking.

After washing with a washing solution, TMB (3, 3 ', 5,
5'-Tetramethyl Benzidine)
2.64 mg was weighed and added to DMSO (Dimethyl
After dissolving in 0.1 mL of Sulfoxide, 10 mL of 0.1 M citrate buffer (pH 4.4) was added, and 3.3 μL of 30% aqueous hydrogen peroxide was added to prepare a substrate solution, and 0.1 mL of this was added. With HRP bound to the solid phase at room temperature
The reaction was performed for 0 to 40 minutes. The reaction was stopped by adding 0.1 mL of 1 M sulfuric acid, and the absorbance at 450 nm was measured with a plate reader to determine the concentration of phosphorylated tau protein.

Measurement of Tau Protein by EIA Using an EIA kit for measuring tau protein purchased from Innogenetics, 25 μL of each of the human plasma samples prepared in the above (1) was used, and the tau protein was measured according to the method described in the kit. The concentration was determined.

Measurement Results The concentration of phosphorylated tau protein in the blood obtained above and below was lower than the detection limit (<0.04p) in three healthy subjects.
M), 0.15 each in 3 Alzheimer's disease patients
pM, 0.69 pM and 0.26 pM. The tau protein concentration in the blood was 0.2 for each of three healthy subjects.
2 pM, 0.28 pM, 0.28 pM, 1.25 pM, 1.03 pM, 3 patients with Alzheimer's disease, respectively
1.04 pM.

As described above, both the phosphorylated tau protein and the tau protein are higher in Alzheimer's disease patients than in healthy subjects, whereby Alzheimer's disease can be detected.

[0077]

According to the present invention, tau protein and / or phosphorylated tau protein in a blood sample can be measured. This makes it possible to more easily detect Alzheimer's disease without using cerebrospinal fluid, which is an auxiliary means for determining a treatment policy, determining a therapeutic effect, and determining a care standard.

[0078]

[Sequence List] <110> Method for detecting Alzheimer's disease <130> J05530 <160> 3 <210> 1 <211> 12 <212> PRT <213> Homo sapiens < 220> <221> SITE <222> 6 <223> Xaa = phosphoserine <400> 1 Lys Ser Gly Tyr Ser Xaa Pro Gly Ser Pro Gly Thr 1 5 10 <210> 2 <211> 16 <212> PRT <213> Homo sapiens <400> 2 Cys Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala 1 5 10 15 <210> 3 <211> 48 <212> PRT <213> Homo sapiens <220> <221> SITE <222> 42 <223> Xaa = phosphoserine <400> 3 Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala 1 5 10 15 Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Glu Pro Pro 20 25 30 Lys Ser Gly Asp Arg Ser Gly Tyr Ser Xaa Pro Gly Ser Pro Gly Thr 35 40 45

Claims (5)

[Claims] 1. A step of denaturing a blood sample obtained from an individual suspected of having Alzheimer's disease in the presence of a protein solubilizing agent to remove contaminating proteins, and removing tau in the sample solution from which contaminating proteins have been removed. A method for detecting Alzheimer's disease, comprising a step of analyzing the presence of a protein. 2. The method according to claim 1, further comprising the step of further concentrating the sample solution from which the contaminating proteins have been removed. 3. The method according to claim 1, wherein the analysis of the presence of tau protein is performed by examining the reactivity of the sample with an anti-tau protein antibody. 4. The method according to claim 1, wherein the protein solubilizing agent is a guanidine salt and a sulfhydryl group-containing agent.
The method described in the section. 5. The reagent kit for performing the method for detecting Alzheimer's disease according to any one of claims 1 to 4, comprising at least an anti-tau protein antibody and a protein solubilizing agent.