JPH06239899A - Antibody for human tau protein and determination of human tau protein in body fluid utilizing the same - Google Patents

Abstract

PURPOSE:To obtain an antibody recognizing the natural-type tau protein existing in the human body fluid, and to provide a method for easy and highly sensitive determination of such tau protein using this antibody. CONSTITUTION:This antibody recognizes the 251st to 419th amino acids of the amino acid sequence in the human tau protein. The objective method using this antibody is an immunoassay method for the tau protein in the human body fluid such as encephalofluid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an antibody which specifically recognizes animal tau protein and a method for measuring tau protein in body fluid using the antibody.

[0002]

2. Description of the Related Art Tau protein is a constituent of neurofibrils, and changes in the neurofibrils lead to death of nerve cells.
Although it is known to be useful for diagnosing Alzheimer's disease, its measurement has been examined mainly in autopsy samples by immunohistology. However, in order to make a diagnostic marker for Alzheimer's disease, it is effective to know the amount present in the body fluid in the living body. An antibody may be used for the measurement.

As a method of using an antibody for the measurement of tau protein, for example, there is a method of treating cerebrospinal fluid with ammonium sulfate and Western blotting the tau protein in the crude fraction to confirm its presence. Proposed [Pete
r Davis et al., Annals of Neurology Vol.22, (4), P.5
21 (1987)]. However, the antibody (Alz-50) used in this proposal is an antibody obtained by using a crude brain homogenate fraction of an Alzheimer's disease patient containing phosphorylated abnormal tau protein (A-68) as an immunogen. Yes, and the detection method using this is a complicated method using Western blotting.

CR Harrington et al., J. Immuno.
l. Methods, 134 (1990) p.261-271, it is reported that two kinds of antibodies against tau protein were obtained. However, the two antibodies they collected (mAb 423, 7
/ 51), the former does not recognize normal tau protein, the latter recognizes only tau protein extracted with formic acid, and neither recognizes natural tau protein present in body fluid in vivo And cannot be used for the measurement.

From these points, an antibody that recognizes a natural tau protein present in the body fluid of a living body is desired, and furthermore, the tau protein in the body fluid of an animal can be specifically and easily and sensitively measured. The method of doing was desired.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides an antibody which specifically recognizes a natural tau protein present in a body fluid of a living body and a simple and sensitive assay for tau protein in a body fluid using the antibody. The purpose is to provide a method.

[0007]

The present invention provides an antibody against natural tau protein of an animal and a method for measuring tau protein in a body fluid using the antibody.

In the present invention, examples of animals include humans and cattle. When humans, tau protein can be used as a diagnostic marker for Alzheimer's disease.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. In the case of a polyclonal antibody, Freund Compl as an adjuvant is used.
An antiserum can be obtained by immunizing an animal with a purified tau protein by a known method using eteAdjuvant or Al (OH) ₃ (alum).

On the other hand, in the case of a monoclonal antibody, the cell fusion method by Keller and Milstein [G. Koeller an
d Milstein, Nature (Londa), 256, 495-497 (1975)], and is prepared by culturing a hybridoma and separating an antibody that reacts with tau protein from the culture. Examples of cells used for cell fusion include P3U1.

In the production of antibodies, human tau protein is preferably used as the tau protein used as an immunogen. This human tau protein is Tris sali having a pH of 6.5 to 8.5 (neutral), which is purified from human brain.
Those soluble in ne are preferred. The antibody obtained by immunizing the tau protein thus obtained recognizes the protein [251-419] of the amino acid sequence of human tau protein, and this region is incorporated into the core of PHF in the brain of Alzheimer's disease patients. It is an important region in that it is a part of the tau protein that is processed.

[0012] Using the antibody of the present invention, the tau protein in the body fluid can be directly subjected to immunoassay without modification.
It can be measured specifically and sensitively. Examples of the immunological assay include enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescent immunoassay. Examples of EIA include "enzyme immunoassay" (second).
A method known per se, which is described in, for example, Ed. Ishikawa Eiji et al., Medical School 1982) can be used. EI
As A, methods called sandwich method and competitive method are known.

In EIA by the sandwich method,
Two antibodies against the tau protein of the present invention are used and quantified according to the following procedure, for example. That is, one of the two kinds of antibodies (first antibody) is immobilized on an appropriate insoluble carrier (for example, a plastic container) (hereinafter this is referred to as "immobilized antibody"). The surface of the insoluble carrier is then coated with a suitable substance (eg bovine serum albumin) to avoid non-specific binding between the insoluble carrier and the reagent or analyte sample to be measured. The thus obtained insoluble carrier on which the first antibody is immobilized is brought into contact with a sample for a certain period of time and temperature to react. During this period, the immobilized antibody (first antibody) is bound to the tau protein in the specimen sample. Then, the insoluble carrier is washed with an appropriate washing solution, and then the other antibody (the second antibody against the tau protein labeled with an appropriate labeling substance (eg enzyme) is used.
The antibody (for example, an aqueous solution) is contacted with the solution for a certain period of time and temperature to react the tau protein bound to the immobilized antibody with the second antibody. This is washed with an appropriate washing solution, and then the amount of the labeling substance labeled with the second antibody that is present by binding with the immobilized antibody on the insoluble carrier through the tau protein is measured.

The sandwich method described above uses an immobilized antibody,
It is also possible to simultaneously mix the specimen sample containing the labeled antibody and tau protein, and allow these three components to come into contact with each other at a certain time and temperature for reaction.

Thus, the amount of tau protein in the sample can be calculated from the value. Usually, in the sandwich method, both the first antibody and the second antibody may be monoclonal antibodies or may be polyclonal antibodies, or one may be used as a monoclonal antibody and the other may be used as a polyclonal antibody. .

As a competitive method, for example, a fixed amount of labeled antibody is competitively reacted with an antigen immobilized on a solid phase and an antigen to be measured to form a solid phase antigen / labeled antibody complex, and a washing operation is performed. After that, a method of measuring the amount of the labeled substance of the labeled antibody bound to the solid phase, or a method of immobilizing the antibody on the solid phase and causing the labeled antigen and the antigen to be measured to react competitively An example is a method of forming a complex and, after washing, measuring the amount of the labeling substance of the labeled antigen bound to the solid phase.

In each of the sandwich method and the competitive method, F (a) obtained by digesting IgG with pepsin was used.
Fab ′ obtained by reducing b ′) ₂ and F (ab ′) ₂
Alternatively, an antibody fragment that binds to an antigen, such as Fab obtained by digesting the antibody with papain, can be used as an antibody, or these can be labeled and used as a labeled antibody.

Examples of the insoluble carrier used in the method for measuring tau protein of the present invention include polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, agarose, and polysaccharides, as well as paper. , Glass,
Examples thereof include metals and combinations thereof. The shape of the insoluble carrier can be various shapes such as a tray shape, a spherical shape, a fibrous shape, a rod shape, a disk shape, a container shape, a cell, and a test tube.

As the labeling substance of the labeled antibody, an enzyme, a fluorescent substance, a luminescent substance, a radioactive substance or the like can be used. As the enzyme, peroxidase, alkaline phosphatase, β-D-galactosidase, etc., as the fluorescent substance, fluorescein isothiocyanate, phycobiliprotein, etc., as the luminescent substance, isorucinol, lucigenin, etc., and as the radioactive substance, ¹²⁵ I, ¹³¹ I, ¹⁴ C, ³ H and the like can be used, but these are not limited to the exemplified ones, and other substances may be used as long as they can be used in the immunological assay method.

When the labeling agent is an enzyme, a substrate and optionally a coloring agent are used to measure its activity. For example, when peroxidase is used as the enzyme, hydrogen peroxide is used as the substrate and 2,2′-azinodi- (3-ethylbenzthiazolinesulfonic acid) ammonium salt (ABT) is used as the color developing agent.
S), 5-aminosalicylic acid, o-phenylenediamine, 4-aminoantipyrine or 3,3 ', 5,5'
-When using tetramethylbenzidine or the like and alkaline phosphatase as the enzyme, o-nitrophenyl phosphate or the like as the substrate and β-D as the enzyme
When -galactosidase is used, it is used in combination with fluorescein-di- (β-D-galactopyranoside) or 4-methylumbelliferyl-β-D-galactopyranoside as a substrate.

As the body fluid containing tau protein measured by the method of the present invention, cerebrospinal fluid having a high content concentration is most preferable.

As described above, by using the antibody of the present invention, it can be measured as it is without modifying the tau protein in the body fluid. In addition, the method for measuring tau protein of the present invention can be used as a monitor for diagnosis and treatment of Alzheimer since it can easily and easily measure a diagnostic marker for Alzheimer's disease.

[0023]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Example 1 (Acquisition of anti-human tau protein antibody) (1) Purification of human tau protein Human tau protein was purified from human brain. Purification is performed using column chromatography, and the buffer is 0.1 M
Tris-HCl saline (pH 7.4) was used.

(2) Preparation of Antibodies to Human Tau Protein The human tau protein purified in (1) above was used in Freund's comp
A mixed W / O / W emulsion was prepared with lete adjuvant. Rabbits were immunized three times every two months. Whole blood was collected 7 days after the third immunization. Then, the antiserum was applied to a Protein A-Sepharose column equilibrated with 0.1 M phosphate buffer (pH 8.0), washed, and then washed with a pH of 3.0.
Flow 1 M Na citrate and remove anti-human tau protein I from the column.
GG was eluted to obtain purified IgG.

Example 2 (Determination of recognition site of anti-human tau protein antibody) It is known that human and bovine tau proteins have highly similar primary structures and also have cross-reactivity in immunoreactivity. There is. Therefore, the recognition site of the anti-human tau protein antibody obtained in Example 1 was determined as follows using bovine tau protein that can be easily prepared in a large amount.

That is, bovine tau protein was added in the presence of formic acid,
It was cleaved with CNBr (Baker bond C4, column).
The reactions were separated using reverse phase HPLC (acetonitrile 0 → 80% gradient in 40 min, flow 1 ml / min) to give 80 fractions. The reverse phase HPLC pattern is shown in FIG.

A part of each of the obtained fractions (1 to 80) was added to a polystyrene microplate, air-dried and fixed. After air-drying, after-coating with 1% BSA-PBS was performed, and the purified IgG obtained in Example 1 was diluted and added, and reacted for 1 hour. Then, here, anti-rabbit Ig
F (ab ′) ₂ -HRP labeling of G antibody was added, and after reacting for 1 hour, a chromogenic substrate (ABTS) was added to examine which fraction the purified IgG obtained in Example 1 reacts with.

As a result, the fraction No. 34 and N
o. 44 showed high reactivity. That is, it was revealed that the peptides contained in these fractions specifically bind to the anti-human tau protein antibody obtained in Example 1. Fraction No. The N-terminus of the 34 peak is Pro-Asp-Leu-Lys, N
o. N-terminal of 44 peak is also Pro-Asp-Leu-L
It turned out to be ys.

The amino acid sequence of bovine tau protein shown in SEQ ID NO: 1 in the sequence listing (Himmler et al., MOLECULAR AND CELLULA
R BIOLOGY, Apr.1989, p.1381-1388), considering that the C-terminal of methionine (Met) is cleaved by CNBr, No. 34 peaks are [258-C end], N
o. The 44 peaks are estimated to be [258-426]. Amino acid sequence of the corresponding human tau protein (SEQ ID NO: 2, Goedert et al., 1989, Neuron, 3, 519-526)
Can be said to be [251-419]. Therefore, the epitope of human tau protein recognized by this antibody can be referred to as [251-419] of its amino acid sequence.

This region is the P in the brain of Alzheimer's disease.
It is a part of tau protein incorporated in the core of HF (paired helical filament) (Kondo et a
l., 1988, Neuron 1, 827-834), and can be said to be an important region among tau proteins.

Example 3 (Measurement of human tau protein by side-itch method EIA using anti-human tau protein antibody) (1) Preparation of antibody-immobilized beads After polystyrene beads (diameter 6 mm) were thoroughly washed, Example 1 After allowing to stand overnight at 4 ° C in a PBS solution having a concentration of 20 µg / ml of the antibody obtained in step 1, PBS
The cells were washed with, and left in a PBS solution of 1% bovine serum albumin (BSA) at a temperature of 4 ° C. for one day and post-coated to obtain anti-human tau protein antibody-immobilized beads.

(2) Preparation of HRP-labeled antibody 2.0 mg / ml of the anti-human tau protein antibody collected in Example 1
1 ml of PBS solution in 1M acetate buffer (pH 4.2)
100 μl and 40 μg of pepsin were dissolved in 20 μl of the same buffer and added, and reacted at 37 ° C. for 4 hours. After the reaction was completed, separation was performed using a Sephadex G25 column (φ2 cm × 45 cm) equilibrated with PBS, and F (ab ′) ₂
Was collected. To 2 ml of a 1 mg / ml 0.01M phosphoric acid 0.15M NaCl (pH 7.4) solution of F (ab ') ₂ was added M
BS 10 mg / ml dimethylformamide solution 50
μl was added, and the mixture was reacted at a temperature of 25 ° C. for 30 minutes. Then, using a column packed with Ceradex G-25,
0.1M phosphate buffer (0.1M PB) (pH 6.
Gel filtration at 0) and unreacted MB with maleimidated antibody
Separated from S.

On the other hand, HRP 10 mg / ml 0.1 M P
To 2 ml of B (pH 6.5) solution, 60 mg / ml dimethylformamide solution of S-acetylmercaptosuccinic anhydride 12
0 μl was added, and the mixture was reacted at 25 ° C. for 2 hours. Then 0.1
800 μl of M Tris-HCl buffer (pH 7.0),
160 μl of 0.1 M EDTA and 1.6 ml of 1 M hydroxylamine were added, and the mixture was reacted at 37 ° C. for 4 minutes. After that, the reaction solution was placed in a collodion bag, and 0.1M PB was added.
(PH 6.0), using a solution containing 5 mM EDTA,
It dialyzed at 4 degreeC for 3 days, and thiolated HRP was obtained.

Next, 2 mg of the maleimidated antibody and 4 mg of thiolated HRP were mixed and concentrated using a collodion bag under ice cooling to a protein concentration of 4 to 10 mg / ml.
Left overnight at ~ 20 ° C. The solution is Ultrogel Ac
Gel filtration through a column packed with A44 (LKB)
An RP-labeled anti-human tau protein antibody was obtained.

(3) Sandwich EIA measurement system One anti-tau protein antibody-immobilized bead prepared in (1),
0.05M TBS (p) containing 1% BSA containing purified human tau protein (standard substance) in the range of 0 to 20 ng / ml.
H8.0) 200 μl and 0.05M TBS containing 1% BSA of the HRP-labeled human tau protein antibody prepared in (2)
200 μl of (pH 8.0) solution was added to each test tube and incubated at a temperature of 25 ° C. for 2 hours. Next, after removing the solution in the test tube by suction, 0.05M TBS
After washing with (pH 8.0), 3,3 ', 5,5'-
Tetramethylbenzidine hydrochloride 0.02% and H ₂ O ₂
Add 0.1M phosphate / citrate buffer (pH 4.3) containing 2.5mM to each test tube at 25 ° C.
After reacting at the temperature of 30 minutes, 1N as a reaction terminator
The enzymatic reaction was stopped by adding 1 ml of a sulfuric acid aqueous solution.

Then, using a spectrophotometer,
The absorption intensity at a wavelength of 50 nm was measured. This was plotted corresponding to the standard substance concentration of 0 to 20 ng / ml to obtain a calibration curve. This calibration curve is shown in FIG. It can be seen from FIG. 2 that the measurement method of the present invention can accurately measure up to 0.05 ng / ml.

Example 4 (Measurement of tau protein in patient cerebrospinal fluid) Using the method of Example 3 and the calibration curve obtained in Example 3,
The concentration of tau protein in the cerebrospinal fluid of 9 Alzheimer's disease patients was measured. As a control, the cerebrospinal fluid of 14 healthy persons was measured. The results are shown in Fig. 3. As shown in FIG. 3, a significantly high tau protein level in the cerebrospinal fluid of Alzheimer's disease patients is seen. (P <0.001)

[0038]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an antibody that specifically recognizes natural tau protein present in the body fluid of a living body. Further, the method for measuring tau protein of the present invention using this antibody can measure the amount of tau protein in body fluid easily and with high sensitivity, and can be effectively used for diagnosis and treatment of Alzheimer's disease. it can.

[0039]

[Sequence listing] SEQ ID NO: 1 Sequence length: 448 Sequence type: Type of amino acid sequence: Protein sequence Met Ala Glu Pro Arg Gln Glu Phe Asp Val Met Glu Asp His Ala Gln 1 5 10 15 Gly Asp Tyr Thr Leu Gln Asp Gln Glu Gly Asp Met Asp Pro Gly Leu 20 25 30 Lys Glu Ser Pro Leu Gln Thr Pro Ala Asp Asp Gly Ser Glu Glu Pro 35 40 45 Gly Ser Glu Thr Ser Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Ala 50 55 60 Thr Ala Pro Leu Val Asp Glu Gly Ala Pro Gly Glu Gln Ala Ala Ala 65 70 75 80 Gln Ala Pro Ala Glu Ile Pro Glu Gly Thr Ala Ala Glu Glu Ala Gly 85 90 95 Ile Gly Asp Thr Ser Asn Leu Glu Asp Gln Ala Ala Gly His Val Thr 100 105 110 Gln Ala Arg Met Val Ser Lys Gly Lys Asp Gly Thr Gly Pro Asp Asp 115 120 125 Lys Lys Thr Lys Gly Ala Asp Gly Lys Pro Gly Thr Lys Ile Ala Thr 130 135 140 Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr 145 150 155 160 Arg Ile Pro Ala Lys Thr Thr Pro Thr Pro Lys Thr Ser Pro Ala Thr 165 170 175 Met Gln Val Gln Lys Lys Pro Pro Pro Ala Gly Ala Lys Ser Glu Arg 180 185 190 Gly Glu Ser Gly Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly 195 200 205 Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr 210 215 220 Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro 225 230 235 240 Lys Ser Pro Ser Ala Ala Lys Ser Arg Leu Gln Ala Ala Pro Gly Pro 245 250 255 Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn 260 265 270 Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys 275 280 285 Leu Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile 290 295 300 Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val 305 310 315 320 Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His 325 330 335 His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp 340 345 350 Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr 355 360 365 His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr 370 375 380 Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val 385 390 395 400 Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser 405 410 415 Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu 420 425 430 Ala Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 435 440 445 448

SEQ ID NO: 2 Array length: 441 Array type: Type of amino acid sequence: Protein Sequence Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val 115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly 130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175 Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190 Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205 Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly 260 265 270 Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln 275 280 285 Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly 290 295 300 Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln 325 330 335 Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340 345 350 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn 355 360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala 370 375 380 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser 405 410 415 Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val 420 425 430 Ser Ala Ser Leu Ala Lys Gln Gly Leu 435 440

[Brief description of drawings]

FIG. 1 is an HPLC pattern of a CNBr degradation product of bovine tau protein.

FIG. 2 is a calibration curve obtained in the sandwich EIA measurement system of Example 3 (3).

FIG. 3 shows the results of measurement of tau protein concentration in cerebrospinal fluid of Alzheimer's disease patients and healthy subjects, which were carried out in Example 4.

[Procedure amendment]

[Submission date] July 16, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Title of invention

[Correction method] Change

[Correction content]

Title: Antibody to human tau protein and method for measuring human tau protein in body fluid using the antibody

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical indication location G01N 33/53 D 8310-2J (72) Inventor Tadakatsu Nakamoto 4-3 Asahigaoka, Hino-shi, Tokyo 2 Teijin Limited Tokyo Research Center (72) Inventor Shinji Kobayashi 4-3-2 Asahigaoka, Hino City, Tokyo Teijin Limited Tokyo Research Center (72) Inventor Kei Mori 3-3-Hikarigaoka, Nerima-ku, Tokyo 4-1123

Claims (14)

[Claims] 1. An antibody against animal tau protein. 2. The antibody according to claim 1, wherein the animal is human. 3. The antibody according to claim 1, wherein the animal is bovine. 4. A 251-amino acid sequence of human tau protein.
The antibody according to claim 2, which recognizes 419. 5. A bovine tau protein amino acid sequence 258-
The antibody according to claim 3, which recognizes 426. 6. The antibody according to claim 1, which is directed against a core of PHF. 7. The antibody according to claim 1, which is obtained by using human tau protein as an immunogen. 8. The human tau protein is neutral Tris sali.
The antibody according to claim 8, which is dissolved by ne. 9. The antibody according to claim 1, wherein the antibody is a polyclonal antibody. 10. The antibody according to claim 1, wherein the antibody is a monoclonal antibody. 11. A method for measuring tau protein in a body fluid of an animal, which comprises using the antibody according to claim 1 as an antibody in the immunological measurement of the tau protein in a body fluid of an animal. 12. The method for measuring tau protein in body fluid according to claim 11, wherein the animal is a human. 13. The method for measuring tau protein in body fluid according to claim 11, wherein the animal is bovine. 14. The measuring method according to claim 12, which is used for diagnosis of Alzheimer's disease.