WO1994018560A1

WO1994018560A1 - Method of assaying human tau protein, kit therefor, and diagnostic method

Info

Publication number: WO1994018560A1
Application number: PCT/JP1994/000196
Authority: WO
Inventors: Kenji Hosoda; Hiroshi Eguchi; Tadakatsu Nakamoto; Shinji Kobayashi; Takaharu Kubota; Hiroshi Mori
Original assignee: Teijin Limited
Priority date: 1993-02-12
Filing date: 1994-02-10
Publication date: 1994-08-18
Also published as: AU6010494A; JPH06239899A

Abstract

A method of immunoassaying a human tau protein present in a human cerebrospinal fluid by using an antibody immobilized on an insoluble support and a labeled antibody, each antibody having a recognition site in the amino acid sequence region ranging from the 251st to the 441st amino acid residues of a human tau protein; and an immunoassay kit therefor. These method and kit enable the concentration of a human tau protein of a normal man and that of a patient with central nerve cytopathy to be determined discriminatively at high sensitivity. Hence the assay results can be utilized for the test, study or diagnosis of patients with central nerve cytopathy.

Description

Specification

Measuring method of human tau protein, kit therefor and diagnostic method [Industrial application]

The present invention relates to a method for measuring tau protein in human cerebrospinal fluid, a kit therefor, and uses related thereto. More specifically, an immunological method that can accurately measure tau protein in human cerebrospinal fluid by distinguishing between persons with central nervous cell disorders and healthy persons who do not, and a kit therefor And related uses.

[Prior art]

Tau protein is a component of the paired helical filament (PHF), and its change in neurofibrils leads to the death of nerve cells, so it is known that it may be useful in diagnosing Alzheimer's disease. However, its measurement has mainly been examined immunohistologically in autopsy samples. However, as a diagnostic marker for Alzheimer's disease, it is effective to know its abundance in body fluids in vivo. An antibody may be used for the measurement.

As a method of using an antibody to measure tau protein, for example, cerebrospinal fluid is treated with ammonium sulfate, and the tau protein in the crude fraction is blotted using an antibody to confirm its presence. A method has been proposed [Peter Davis et al., Annals of Neurology Vol.22, (4), P.521 (1987)]. The antibody (A1z-50) used in this proposal is a crude brain homogenate from a patient with Alzheimer's disease that contains an abnormal phosphorylated tau protein (A-68). The antibody is obtained as an immunogen, and the detection method using this is a complicated method using Western plotting.

In addition, CR Harrington et al. Reported in J. Immunol. Methods, 134 (1990) p.261-271 that they obtained two antibodies against tau protein. Tells. Of the two antibodies (mAb423, 7/51) collected by them, the former does not recognize normal tau protein, and the latter is evening protein extracted with formic acid. Both recognize only natural tau protein, which is present in body fluids of living organisms, and cannot be used for the measurement.

U.S. Pat. No. 5,134,062 describes the removal of living fibroblasts from humans, culturing the cells in a medium, and indicating the metabolites of Alzheimer's disease from the cells. A method for screening Alzheimer's disease by immunological detection of a substance is described. This US patent shows skin cells as fibroblasts, and PHF (paired helical filament) and tau protein as indicators of metabolites of Alheimer's disease. This method requires removing and culturing the skin cells, and requires complicated means and a long time to obtain a predetermined amount of the indicator substance. In addition, the U.S. patent does not describe any specific examples showing the amount of PHF or protein involved.

Further, Canadian Patent No. 1,302,250 describes a method of measuring PHF or abnormal phosphorylated protein in cerebrospinal fluid to be useful for treating Alzheimer's disease. This method uses a monoclonal antibody that reacts with PHF.

[Problems to be solved by the invention]

A first object of the present invention is to provide a method for immunologically measuring soluble human tau protein present in a body fluid from a living body, particularly cerebrospinal fluid, and a kit therefor.

A second object of the present invention is to provide a method for early and easy measurement of soluble human tau protein in cerebrospinal fluid and a kit therefor.

A third object of the present invention is to provide a patient with central nervous An object of the present invention is to provide an immunological method and a kit for accurately measuring soluble human tau protein in cerebrospinal fluid by distinguishing it from that of a healthy human.

Another object of the present invention is to provide an immunological method capable of accurately measuring soluble human tau protein therein using a small amount of cerebrospinal fluid.

[Means for solving the problem]

According to the study of the present inventors, the object of the present invention is to provide a method for immunologically measuring human tau protein in human cerebrospinal fluid, which comprises an insoluble carrier-immobilized antibody and a labeled antibody. And that all antibodies have a recognition site in the amino acid sequence region of positions 251 to 441 of the human tau protein. Was found.

Further, according to the study of the present inventors, the object of the present invention is:

(a-1) an antibody against human tau protein immobilized on an insoluble carrier, (a-2) an antibody against human tau protein labeled with a labeling substance,

(b) Solubilizer

(c) cleaning agents and

(d) When the labeling substance is an enzyme, a substrate and a reaction terminator for measuring enzyme activity

A kit for immunologically measuring soluble human tau protein in human cerebrospinal fluid, wherein the antibodies (a-1) and (a-2) are all the second human tau protein. It has been found that this can be achieved by a kit characterized by having a recognition site in the amino acid sequence region from the 51st to the 44th amino acid.

Further, according to the study of the present inventors, the object of the present invention is as follows.

(i) Prepare human cerebrospinal fluid from human,

(ii) an insoluble carrier-immobilized antibody and a labeled carrier, and An antibody having a recognition site in the amino acid sequence region from the 25th to the 41st of the human tau protein is also prepared.

(iii) immunologically measuring soluble human tau protein in the human cerebrospinal fluid of (i) using the two antibodies of (ii);

(iv) determining the amount of soluble human tau protein in the human cerebrospinal fluid from the measured values; and

(V) diagnosing a disease of central nervous cell disorder in the human based on the amount thereof, which has been found to be achieved by a method for diagnosing disease of central nervous cell disorder in humans. .

The measurement method of the present invention and the kit therefor are characterized in that both the immobilized antibody and the labeled antibody have the amino acid sequence of human tau protein from the 25th position (Pro.) To the 25th position. It is characterized in that an antibody having a recognition site in the region up to the 1st (Leu.) Is used. By such a combination of antibodies, the soluble human tau protein contained in the cerebrospinal fluid can be measured with high sensitivity without any need for any treatment, and the measurement results indicate that the central nervous cell disorder There is a clear difference between people suffering from and healthy people. That is, according to the immunological measurement method of the present invention, the amount of the human protein in the cerebrospinal fluid from a person suffering from central nervous cell damage is detected at a clearly higher concentration than that of a healthy person. .

Therefore, the measurement results of the present invention can be used for examination, research, or diagnosis from a clinical or pathological viewpoint of a person having or suspected of having a disorder in central nervous cells.

A central nervous cell disorder is a disease or condition caused by any disorder of the central nervous cells, such as a disease or condition caused by cerebrovascular disorder, Alzheimer's disease and carbon monoxide poisoning. U. In particular, the present invention can be expected to be used for examination, research, or diagnosis of Alzheimer's disease or a person suspected of having Alzheimer's disease. All of the above-mentioned diseases based on disorders of central nervous cells are caused by soluble human tau in cerebrospinal fluid. The protein concentration shows a higher value than that of a healthy person.

In the present invention, the two antibodies, the immobilized antibody and the labeled antibody, are both intermediate regions that are the 251st to the 441st region in the amino acid sequence of 441 of the human tau protein. It has a recognition site in the amino acid sequence region at the C-terminus from the beginning, and it is not clear why highly sensitive and soluble human protein can be measured with it.

The present inventors have prepared several monoclonal and polyclonal antibodies specifically recognizing human tau protein and used them for immunoassay of soluble human tau protein in cerebrospinal fluid. Tried. For example, several monoclonal antibodies having a recognition site in the region N-terminal to the amino acid sequence of the amino acid sequence of the human tau protein were obtained. Immunological measurements were performed using a combination of several monoclonal and polyclonal antibodies. A calibration curve could be prepared with such a combination of antibodies, but when cerebrospinal fluid was actually measured as a test sample, the sensitivity was insufficient and stable measurement results could not be obtained. Alternatively, nothing could be used for diagnosis.

However, as in the present invention, when an antibody having a recognition site on the C-terminal side of the amino acid sequence of the human tau protein at the C-terminal side is used for both the immobilized antibody and the labeled antibody, Thus, soluble human tau protein could be measured stably and with high sensitivity.

The antibody used in the present invention may be a monoclonal antibody as long as it has a recognition site in the amino acid sequence of the amino acid sequence of the human tau protein in the 25th to 441st regions. Alternatively, it may be a polyclonal antibody. The combination may be any, but it is preferable to use a polyclonal antibody for both the immobilized antibody and the labeled antibody.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody as described above. In the case of a polyclonal antibody, Freund Complete Adjuvant or A1 (OH) 3 is used as an adjuvant. (Haraum) and purified human tau protein Can be obtained as an antiserum by immunizing animals by the method

On the other hand, in the case of monoclonal antibodies, hybridomas prepared by the cell fusion method of Keller and Milstein [G. Koeller and Milstein, Nature (Londa), 256, 495-497 (1975)] are cultured and It is prepared by separating the antibody reacting with the human tau protein from the culture solution. Cells used for cell fusion include P3U1 and the like.

In preparing the antibody, the human tau protein used as the immunogen is preferably one that is soluble in Trissalline having a pH of 6.5 to 8.5 (neutral) and purified from human brain. Among the antibodies obtained by immunizing the thus obtained human tau protein, those that recognize the protein [2 51-441] of the amino acid sequence of the human tau protein are selected and used. .

By using the antibody of the present invention, the immunoassay can specifically and sensitively measure the human protein in cerebrospinal fluid without modification. Examples of immunoassays include enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescence immunoassay. EIAs include, for example, “oxygen immunoassay” A publicly known method described in Eiji Ishikawa et al., 2nd edition, Medical Shoin 1982) can be used. EIA may be any of a method called a sandwich method and a competitive method.

In EIA by the sandwich method, quantification is performed using an antibody against the human tau protein of the present invention, for example, according to the following procedure. That is, one of the two antibodies (the first antibody) is immobilized on a suitable insoluble carrier (for example, a plastic container) (hereinafter, this is referred to as “immobilized antibody”). Next, the surface of the insoluble carrier is coated with a suitable substance (for example, bovine serum albumin) to avoid non-specific binding between the insoluble carrier and the reagent or sample to be measured. Like this The insoluble carrier on which the obtained first antibody is immobilized is brought into contact with the sample for a certain period of time and at a temperature for reaction. During this time, the solidified antibody (primary antibody) and the human protein in the sample bind. Next, the insoluble carrier is washed with an appropriate washing solution, and then contacted with a solution (eg, an aqueous solution) of the other antibody (second antibody) against the human tau protein labeled with an appropriate labeling substance (eg, an enzyme) for a certain period of time and at a temperature. The second antibody reacts with the human tau protein bound to the immobilized antibody. This is washed with an appropriate washing solution, and then the amount of the labeling substance labeled on the second antibody which is bound to the immobilized antibody on the insoluble carrier via the tau protein is measured.

The above sandwich method can also be carried out by simultaneously mixing an immobilized antibody, a labeled antibody and a sample containing a tau protein, and simultaneously bringing the three into contact with each other for a certain period of time and at a temperature to cause a reaction.

Thus, the amount of tau protein in the test 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 polyclonal antibodies, or one may be a monoclonal antibody and the other may be a polyclonal antibody. It can also be used as a monoclonal antibody. However, as described above, it is preferable in terms of sensitivity that both the first antibody and the second antibody are polyclonal antibodies.

As a competition method, for example, a fixed amount of a labeled antibody is allowed to react competitively with an antigen immobilized on a carrier and an antigen to be measured to form a carrier-immobilized antigen-labeled antibody complex, and a washing operation is performed. Then, a method of measuring the amount of the labeled substance of the labeled antibody bound to the carrier, or immobilizing the antibody on the carrier, and allowing the labeled antigen to react with the antigen to be measured in a competitive manner, the carrier-immobilized antibody After the washing operation, a labeled antigen complex is formed, and then the amount of the labeled substance of the labeled antigen bound to the carrier is measured. In each of the San Doi Tutsi method and the competitive method, the antibody may be a full antibody, also reducing the F (ab ') 2 F ( ab') 2 obtained by digesting the antibody with Bae trypsin The Fab 'or antibody Antibody fragments that can bind to an antigen, such as Fab obtained by digestion with papine, can be used as antibodies, and these can be labeled and used as labeled antibodies.

The insoluble carrier used in the method for measuring a protein according to the present invention may be any one which is usually used for immunological measurement, for example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, Examples thereof include polymers such as cross-linked dextran, agarose, and polysaccharide, as well as paper, glass, metal, and combinations thereof. The shape of the insoluble carrier can be various shapes such as tray shape, spherical shape, fibrous shape, rod shape, disk shape, disk shape, container shape, cell, test tube and the like.

Enzymes, fluorescent substances, luminescent substances, radioactive substances, and the like can be used as the labeling substance of the labeled antibody. Examples of enzymes include peroxidase, alkaline phosphatase, / 3-D-galactosidase, fluorescent substances such as fluorescein isothiosinate and phycopyriprotein, and luminescent substances such as isolucinol and luciferase. lucigenin and the like., and then as the radioactive material may be used, such as ^{^{1 2 5 I, 1 3 1}} K 1 4 C, 3 H, these are not limited to those illustrated, used to immunoassays Anything that can be done may be used.

When the labeling agent is an enzyme, a substrate and, if necessary, a color former are used to measure its activity. For example, when peroxidase is used as an enzyme, hydrogen peroxide is used as a substrate, and 2,2′-azinodi- (3-ethylbenzthiazoline sulfonic acid) ammonium salt is used as a coloring agent. (ABTS), 5-aminosalicylic acid, 0-phenylenediamine, 41-aminoantivirin or 3,3 ', 5,5'-tetramethylbenzidine, etc. When o-nitrophenyl phosphate is used as a substrate, and D-galact 'fern is used as an enzyme. When lyase is used, fluorescein di-(^-D-galacto viranoside) or 4-methylumbelliferyl-13-D-galact and pyranoside are used in combination as substrates.

In the assay kit of the present invention, the lysing agent (b) may be any of those usually used for immunological assays, and includes, for example, phosphate buffer, Tris-HCl buffer, acetate buffer, and the like. However, those having a pH in the range of 6.0 to 8.0 are shown as preferable examples. Further, (c) a detergent generally used for immunological measurement is also used as it is. Examples include physiological saline, phosphate buffer, Tris-HCl buffer, and mixtures thereof. Non-ionic surfactants such as Triton XI 00, Tween 20 or Brig 35, and ionic surfactants such as sodium dodecyl sulfate may be added to these detergents. Good. [Example]

Hereinafter, the present invention will be described in detail 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

The frozen human brain was dissolved in 4 volumes of TS Buffer (2 OmM Tris, 150 mM NaC; pH 7.6). The lysate was centrifuged (10 K rp.mX for 50 minutes), and the supernatant was fractionated using ammonium sulfate (33-50% ammonium sulfate). The resulting precipitate was dissolved in TBS Buffer (50 mM Tris, 500 mM NaCl; pH 7.6; 16 volumes of brain weight), homogenized uniformly, and boiled for 10 minutes. Then, the mixture was centrifuged (10 kr.p.mX10 minutes), and the supernatant was fractionated using Sephadex G-25 (equilibrium solution: 0.1 M MES: pH 6.5). Next, the salt was eluted by changing the salt concentration using phosphocellulose, and concentrated again using saturated ammonium sulfate. Shrunk. The obtained precipitate was suspended in 1 m of TS Buffer, dissolved, filtered through a Millipore filter HV (0.45 / zm), and then subjected to HPLC (column: J.T. Bakerbond C 4 4.6 × 2). 5 Omm), the second peak eluted was collected. This is the purified human tau protein.

(2) Preparation of antibody against human tau protein

The human tau protein purified in (1) above was mixed with Freund's complete adj uvant to prepare WZO / W emulsion. Rabbits were immunized three times, every two months. After three immunizations, whole blood was collected 7 days later. Next, the antiserum was applied to a protein A-Sepharose column equilibrated with 0.1 M phosphate buffer (pH 8.0), washed, and washed with 0.1 M quencher with pH 3.0. The acid Na was allowed to flow, and the anti-human tau protein antibody (IgG) was eluted from the column to obtain a purified antibody (IgG). Example 2 (Determination of recognition site of anti-human tau protein antibody)

It is known that human and human tau proteins have highly similar primary structures and also have cross-reactivity in quarantine. Thus, the recognition site of the anti-human tau protein antibody obtained in Example 1 was determined as follows using a large amount of easily prepared dicitau protein.

That is, ゥ sitau protein was cleaved by CNBr (Baker bond C4, column) in the presence of formic acid. The reaction was separated using reverse phase HPLC (acetonitrile 0 → 80% gradient in 40 min, flow rate lm / min) to give a fraction of 80. Figure 1 shows the reversed phase HPLC pattern.

A part of each of the obtained fractions (1 to 80) was added to a polystyrene microplate, and air-dried and fixed. After air-drying, the solution was after-coated with 1% BSA-PBS, and the purified antibody (IgG) obtained in Example 1 was diluted and added, followed by reaction for 1 hour. Then, F (ab ') ₂ — HRP label of anti-heron IgG antibody was added here, and after 1 hour reaction, A chromogenic substrate (ABTS) was added, and it was examined which fraction the purified antibody (IgG) obtained in Example 1 reacted with.

As a result, the fractions No.34 and No.44 showed high reactivity. That is, it was clarified that the peptides contained in these fractions specifically bound to the anti-human tau protein antibody obtained in Example 1. The N-terminus of the fraction N 0.34 peak is Pro-Asp-Leu-Lys, and the N-terminus of the No.44 peak may also be Pro-Asp-Leu-Lys. found.

より From the amino acid sequence of the sitau protein (Himmler et al .. MOLECULAR AND CELLULAR BIOLOGY, Apr. 1989, p. 1381-1388), it was confirmed that the C-terminal of methionine (M et) was cleaved by CNBr. Considering this, the No. 34 beak is estimated to be [258-C end], and the No. 44 peak is estimated to be [258-426]. The amino acid sequence of the corresponding human protein (see Sequence Listing, Goedert et al., 1989, Neuron, 3, 519-526) can be said to be [251-444]. Therefore, the epitope of the human tau protein recognized by this antibody can be called [251-441] of its amino acid sequence.

Example 3 (Measurement of human tau protein by sandwich method EIA using anti-human tau protein antibody)

(1) Preparation of antibody-immobilized beads

After thoroughly washing the polystyrene beads (diameter 6 mm), the antibody obtained in Example 1 was allowed to stand in a PBS solution having a concentration of 20 gZm at a temperature of 4 ° C for 24 hours, and then washed with PBS. In a 1% bovine serum albumin (BSA) solution in PBS at a temperature of 4 ° C. for one day and night, post-coating treatment was performed to obtain anti-human tau protein antibody-immobilized beads.

(2) Preparation of HRP-labeled antibody

2.0 mg / m PB of anti-human protein antibody collected in Example 1 To the lm S solution, 1 M acetate buffer (pH 4.2) 100 and 40 / g pepsin dissolved in 20 of the same buffer were added, and 37. C, and reacted for 4 hours. After completion of the reaction, separation was carried out using a Sephadex G25 column (2 cmX45 cm) equilibrated with PBS, and F (ab ') ₂ was collected. F (ab ') ₂ in lm gZm 0.01 M phosphoric acid 0.15 M NaC 1 (pH 7.4) solution 2 m in dimethylformamide solution MBS 10 mg / mi concentration 5 0 μ £ was added, and the mixture was reacted at a temperature of 25 ° C. for 30 minutes. Next, gel filtration was performed using 0.1 M phosphate buffer (O. IMPB) (pH 6.0) using force unevenness filled with Sephadex G-25, and The reaction MBS was separated.

On the other hand, to 2 mi of lO mg / m O.IMPB (pH 6.5) solution of HRP was added 60 mg / να-dimethylformamide solution 120 of S-acetylmercaptosuccinic anhydride, and 25 For 2 hours. Then add 0.1 M Tris-HCl buffer (pH 7.0)

O.IMEDTA160; a and 1.6 M £ of 1 M hydroxylamine were added and reacted at 37 ° C for 4 minutes. Thereafter, the reaction solution was placed in a collodion bag, and dialyzed at 4 ° C. for 3 days using a solution containing 0.1 M PB (pH 6.0) and 5 mM EDTA to obtain a thiolated HRP.

Next, 2 mg of maleimide 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 / mt. Leave at ~ 20 overnight. The solution was subjected to gel filtration through a column filled with Ultrogel AcA44 (LKB) to obtain an HRP-labeled anti-human tau protein antibody.

(3) Sandwich EIA measurement system

One anti-tau protein antibody-immobilized bead prepared in (1) and a purified human tau protein (standard substance) in a range of 0 to 20 ngZm containing 1% BSA in 0.05 MTBS (pH 8.0) 200 and the HRP-labeled human tau protein antibody prepared in (2) containing 1% BSA and 0.05 M Add 200 / TBS (pH 8.0) solution to each test tube,

Incubation was performed at 25 ° C for 2 hours. Next, aspirate the solution in the test tube, wash with 0.05M TBS (pH 8.0),

3, 3 ', 5,5' - Te tetramethyl benzidine hydrochloride 0.0 2% and 1 ₂ 0

2 Add 0.4 m of 0.1 M phosphate-noquenate buffer solution (pH 4.3) containing 2.5 mM to each test tube, react at 25 ° C for 30 minutes, and add 1 N The enzymatic reaction was stopped by adding 1 ml of sulfuric acid aqueous solution.

Next, the absorption intensity at a wavelength of 450 nm of this solution was measured using a spectrophotometer. This was plotted corresponding to a standard substance concentration of 0 to 20 ng / mi to obtain a calibration curve. This calibration curve is shown in FIG. From FIG. 2, it is understood that the measurement method of the present invention can accurately measure up to 0.05 ngZm. Example 4 (Measurement of tau protein in cerebrospinal fluid of patients)

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 patients with Alhaima's disease was measured. As a control, cerebrospinal fluid of 14 healthy subjects was measured. The results are shown in Figure 3. As shown in Fig. 3, a significantly high level of tau protein in cerebrospinal fluid of Alzheimer's disease patients is observed. (P <0.001)

According to FIG. 3, it is possible that Alzheimer's disease can be diagnosed in the case of a human protein concentration of 0.5 ngZm or more. Example 5 (Measurement of tau protein in patients with cerebrovascular disorder and carbon monoxide poisoning)

Using the method of Example 3 and the calibration curve obtained in Example 3, the concentration of ta protein in the cerebrospinal fluid of patients with cerebrovascular disease and carbon monoxide poisoning was measured. Fig. 4 shows the results. It is known that neuronal cells are destroyed in patients with carbon monoxide poisoning, and proteins in the patient's cerebrospinal fluid are An increase in white concentration is considered to be an indication of the destruction of neurons. Inferring from this, it is probable that cerebrovascular disorder patients also showed some destruction of their nerve cells. Reference Example 1 (Generation of monoclonal antibody against human tau protein and determination of recognition site)

Monoclonal antibodies against human tau protein were prepared according to the method of Keraichi and Milstein (Koeller & Milstein). That is, the mouse was immunized by mixing the human tau protein Songhead and Freund's complete adjvant. After four immunizations, fusion was performed three days later. After confirming the appearance of hybridomas, antibody-producing hybridomas were screened using solid-phase tau protein (fixed at 2 g nom).

As a result, eight clones having positive reactivity with the solid-phase tau protein were obtained. In order to determine the recognition sites of these eight clones thus established, differences in the reactivity between the human tau protein and the decitau protein were examined using the supernatants of these eight clones. As a result, these eight clones were all clones that produced monoclonal antibodies that specifically reacted with only the human tau protein. To examine this, the primary structures of human tau protein and ゥ sitau protein were compared. The human tau protein and the citrus protein have different amino acid sequence regions from the first to the 248th amino acid sequence region of the human tau protein, and the other regions have exactly the same primary structure. all right. From this, it is clear that the recognition sites of the antibodies produced by the eight clones are all present in the first to 248th regions. Reference Example 2 (EIA measurement system using monoclonal antibody and polyclonal antibody obtained in Example 1)

The 8 clones established in Reference Example 1 were cultured, and Lonal antibodies were purified. All of the eight monoclonal antibodies were each used as a 10 μg / mi solution, and each solution was immersed in a polystyrene ball to immobilize the monoclonal antibodies. A sandwich measurement system using each of the immobilized monoclonal antibodies and the HRP-labeled anti-human tau protein antibody of Example 3 was constructed. Only two of the eight monoclonal antibodies were able to obtain good calibration curves. The two San de I Tutsi measurement system the MCA with monoclonal antibodies and HRP-labeled anti-human Totau protein antibody (F - F n) - PCA and MCA (F - H ₅ H) abbreviated as one PCA. On the other hand, a sandwich assay system prepared by immobilizing the polyclonal antibody obtained in Example 1 in combination with an HRP-labeled anti-human tau protein antibody is abbreviated as “PCA-PCA”. FIG. 5 shows calibration curves obtained using the above three sandwich measurement systems. As apparent MCA from FIG _{5 (F - F ι α)} - PC A. MCA (F- H 5) - both of PCA and PCA- PCA was able to create a similar calibration curve. Example 6 (Measurement of human tau protein in cerebrospinal fluid)

Using the three measurement systems obtained in Reference Example 2, the concentration of human tau protein in the cerebrospinal fluid of healthy individuals and patients with Alheimer's disease was measured by the Sandwich method. The results are summarized in FIG.

A, B and C in FIG. 6 show the results of the following measurement systems, respectively.

A: PCA-PCA

B: M C A (F—F n) — P C A

C: M C A (F-H 5) One P C A

As is evident from Fig. 6, in the case of A (PCA-PCA), healthy subjects (Normal) and Alzheimer's disease patients (AD) were well distinguished in the amount of human tau protein, whereas monoclonal antibodies In the cases of B and C, the distinction was insufficient. That is, in the case of B, 5 cases in which the healthy human had a protein concentration of 0 (zero) There were 2 of them, and the concentration of Alzheimer's disease patients who could not be distinguished from healthy subjects was 1 (of 6). In the case of C, the concentration of human tau protein was 0 (zero) in four healthy subjects, indicating that there is a problem in the measurement method itself.

As is evident from FIG. 6, a measurement system (Λ) using two polyclonal antibodies having recognition sites in the 251st to 441st regions of the amino acid sequence of the human tau protein was clinically available. It is clear that the measurement system is meaningful and excellent. [Sequence List] (Amino acid sequence of human tau protein) Sequence length: 4 4 1

Sequence type: amino acid

Sequence type: protein

Array

Met Ala Glu Pro Arg Gin Glu Phe Glu Val Met Glu Asp His Ala Gly

1 5. 10 15

Thr Tyr Gl then eii Gly As Arg then ys Asp Gin Gly Gly Tr Thr Met His

20 25 30

Gin Asp Gin Glu Gly Asp Thr Asp Ala Gly then eu and ys Glu Ser Pro Leu

35 40 45

Gin Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser

50 55 60

Asp Ala ys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80

Asp Glu Gly Ala Pro Gly Lys Gin Ala Ala Ala Gin Pro His Thr Glu

85 90 95 lie Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly He Gly Asp Thr Pro

100 105 110

Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gin Ala Arg Met V'al

115 120 125

Ser and ys Ser Lys Asp Gly Thr Gly Ser Asp Asp and ys and ys Ala and ys Gly

130 135 140

Ala Asp Gly Lys Thr Lys lie A 1 a Thr Pro Arg G 1 y Ala Λ 1 a Pro Pro 145 150 155 160

Gly Gin Lys Gly Gin Ala Asn Ala Thr Arg lie Pro Ala Lys Thr Pro

165 170 175

Pro A 1 a Pro Lys Thr Pro Pro Ser Ser Gly G】 u Pro Pro Lys Ser G I y

180 185 190

Asp Arg Ser G 1 Tyr Ser Ser Pro G 1 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

Ys Val Ala Val Val Arg Thr Pro Pro ys Ser Pro Ser Ser la Lys 225 230 235 240

Ser Ar Leu G 1 n Thr A 1 a Pro Val Pro Met Pro Asp Leu Lys Asn Val

245 250 255

Lys Ser Lys 11 e Gly Ser Thr Glu Asn eu Lys His Gin Pro G 1 y Gly

260 265 270

Gly Lys Val G 1 n lie lie Asn then ys Lys then eu As then eu Ser Asn Va 1 Gin

275 280 285

Ser Lys Cys Gly Ser shi ys Asp Asn lie shi ys His Val Pro Gly Gly G 1 y 290 295 300 Ser Va 1 Gin 11 e Va I Tyr then ys Pro Va I Asp Leu Ser then ys Va 1 Tr Ser

305 310 315 320 ys Cys G 1 y Ser Leu G 1 Asn lie His His ys ys Pro G 1 y G I y G 1 y Gin

325 330 335

Va 1 G 1 u Va 1 then ys Ser G 1 u then ys Leu Asp Phe then ys Asp Arg Va 1 Gin Ser

340 345 350 shi ys 1] e G] y Ser Leu Asp Asn lie Thr His Va 1 Pro G] y G 1 y G] Asn

355 360 365

Lys Lys lie G】 u Thr His or ys Leu Thr Phe Arg G 1 u Asn Ala or ys A 1 a

370 375 380

Lys Thr Asp His G! y A 1 a G] u lie Va 1 Tyr Lys Ser Pro Va 1 Va 1 Ser 385 390 395 400

G 1 y Asp Thr Ser Pro rg His Leu Ser Asn Va 1 Ser Ser Thr G 1 y Ser

405 410 415

11 e Asp Met Va】 Asp Ser Pro Gin Leu Ala Thr Leu A 1 a Asp Glu Va 1

420 425 430

Ser A 1 a Ser Leu Ala Lys G 1 n G】 y Leu

435 440

[Brief description of drawings]

FIG. 1 shows the HPLC pattern of the CN Br degradation product of ゥ sitau protein.

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

FIG. 3 shows the results of measurement of tau protein concentration in cerebrospinal fluid of Alzheimer's disease patients and healthy subjects performed in Example 4. FIG. 4 shows the results of measurement of tau protein in cerebrospinal fluid of a patient suffering from cerebrovascular disorder and carbon monoxide poisoning in Example 5.

FIG. 5 shows respective calibration curves obtained in Reference Example 2 using three types of Sandwich measuring systems.

FIG. 6 shows the results of measurement of tau protein in cerebrospinal fluid of healthy individuals and patients with Alzheimer's disease obtained in Example 6 by using three types of sandwich measurement systems.

[The invention's effect]

According to the present invention, soluble human tau protein in cerebrospinal fluid can be measured simply and with high sensitivity. The measurement can be performed on the extracted human cerebrospinal fluid as it is without any processing, and it is measured as a distinct value between a person with central nervous cell disorder and a healthy person In addition, a small amount of cerebrospinal fluid as a test sample is sufficient. Thus, the present invention can be effectively used for examination, research, or diagnosis of patients having disorders of central nervous cells.

Claims

The scope of the claims

1. In a method for immunologically measuring human tau protein in human cerebrospinal fluid, the method uses an insoluble carrier-immobilized antibody and a labeled antibody, and both antibodies use the 25th protein of human tau protein. 1st to 44th A method characterized by having a recognition site in the first amino acid sequence region.

2. The method according to claim 1, wherein the human protein to be measured in human cerebrospinal fluid is a soluble protein.

3. The method according to claim 1, wherein the antibody immobilized on the insoluble carrier and the labeled antibody are both polyclonal antibodies.

4. The method according to claim 3, wherein the polyclonal antibody is an antibody obtained by immunizing a soluble human tau protein isolated from human brain as an immunogen.

5. (a-1) an antibody against human tau protein immobilized on an insoluble carrier, (a-2) an antibody against human tau protein labeled with a labeling substance,

(b) dissolving agent,-

(c) cleaning agents and

(d) When the labeling substance is an enzyme, a substrate and a reaction terminator for measuring the enzyme activity;

A kit for immunological measurement of soluble human tau protein in human cerebrospinal fluid, wherein the antibodies (a-1) and (a_2) are all the second of human tau protein. 5 Kits characterized by having a recognition site in the 1st to 44th amino acid sequence region.

6. The kit according to claim 5, wherein the antibodies (a-1) and (a-2) are both polyclonal antibodies.

7. The kit according to claim 6, wherein the polyclonal antibody is an antibody obtained by immunizing a soluble human protein isolated from human brain as an immunogen.

8. (i) Prepare human cerebrospinal fluid from human,

(ii) Antibodies that are immobilized on an insoluble carrier and labeled antibodies, and both antibodies have a recognition site in the amino acid sequence region from the 25th to the 41st amino acid sequence of the human tau protein. (Iii) immunologically measuring soluble human tau protein in the human cerebrospinal fluid of (i) using the two antibodies of (ii),

(iv) determining the amount of soluble human tau protein in the human cerebrospinal fluid from the measured value; and

(V) A method for diagnosing a disease of central nervous cell disorder in a human, wherein the disease is diagnosed based on the amount of the disease.

9. The diagnostic method according to claim 8, wherein the central nervous cell disorder is Alzheimer's disease.

0 The diagnostic method according to claim 9, wherein the diagnosis of Alzheimer's disease is distinguished from a healthy person by including soluble human tau protein in human cerebrospinal fluid at a concentration of at least 0.5 ng Zm.

1 The diagnostic method according to claim 8, wherein the antibody immobilized on the insoluble carrier and the labeled antibody are both polyclonal antibodies.

2. The diagnostic method according to claim 11, wherein the polyclonal antibody is an antibody obtained by immunizing a potential human tau protein isolated from human brain as an immunogen.