WO2005047897A1

WO2005047897A1 - Method of diagnosing alzheimer’s disease

Info

Publication number: WO2005047897A1
Application number: PCT/JP2004/017218
Authority: WO
Inventors: Shinobu Kitazume; Yuriko Tachida; Ritsuko Oka; Yasuhiro Hashimoto; Takaomi Saidoh
Original assignee: Riken
Priority date: 2003-11-12
Filing date: 2004-11-12
Publication date: 2005-05-26
Also published as: JP2005145837A

Abstract

A method of diagnosing isolated Alzheimer’s disease through detection of any increase of β-secretase activity. There is provided a method of diagnosing Alzheimer’s disease, comprising detecting or measuring any secretory sialic acid transferase broken by β-secretase contained in a sample from human.

Description

Specification

Technical field of diagnosis of Alzheimer's disease

The present invention relates to a method for diagnosing Alzheimer's disease, an antibody capable of specifically recognizing secreted sialyltransferase of human origin cleaved by i3-secretase, and a kit for diagnosing Alzheimer's disease containing the above antibody It is. Background art

Panorezheimer's disease is a disease with widespread neurodegenerative dementia. The production of amyloid peptide (A / 3) is one of the causes of Alzheimer's disease (Selkoe, DJ (2001) Physiol. Rev. 81, 741-766; Iwata, N., et al. , (2001) Science 292, 1550-1552)). Upon formation of A, amyloid precursor protein (APP) is - is more cut-secretase soluble Nyuita ₂ terminal fragments (APP s] 3) and 1 2 kD a of CO OH-terminal fragment (C99) is generated, The latter remains attached to the membrane. C99 is further cleaved by y-secretase to form pathogenic Α (De Strooper, B., et al., (1998) Nature (London) 391, 387-390; and Wolfe, MS, et al. (1990) Nature (London) 398, 513-517). In an alternative pathway, APP is cleaved within the AjS sequence by α-secretase to form a soluble NH ₂ -terminal fragment (AP PSQ!) And a 10 kDa membrane-bound CO OH-terminal fragment (C 83). (Buxbaum, JD, et al., (1998) J. Biol. Chera. 273, 27765-27767; and La 脑 ich, S., et al., (1999) Proc. Natl. Acad. Sci. USA 96, 3922- 3927).

Many markers have been reported to correlate with Alzheimer's disease, but many of them have not been identified for their relationship to pathological changes and their diagnostic value is not necessarily established. Cerebrospinal fluid markers directly associated with pathological changes include a decrease in the amyloid peptide (A) 31-42) consisting of 42 amino acids and an increase in (phosphorylated) tau. However, although A 1-42 correlates well with late-stage Alzheimer's disease, No difference from normal was observed in the last stage (mild cognitive impairment: MCI) or in the early stage of the disease. In this regard, phosphorylated tau also increased in MCI and is considered to be the best biomarker. However, neuronal cell death has already progressed when phosphorylated tau is rising, and complete recovery of neurological function cannot be expected even if treatment is started at this time. The ideal marker should be one that can be diagnosed before the death of nerve cells, but such a marker has not been reported so far.

Previous studies by the present inventors have demonstrated that human secretase cleaves rat α2,6 sialyltransferase and detected soluble sialyltransferase, the product of this cleavage. Are antibodies that specifically recognize the newly generated Ν-terminal (cleaved end) ^]. Using this antibody, it was possible to evaluate J3 secretase activity in vitro or at the level of cultured cell experiments (S. Kitazume, et al., (2001) Proc. Natl. Acad. Sci. USA, Vol. 98). , No. 24, 13554-13559) ₀ It is also possible to screen for 3 secretase inhibitors using this evaluation system. Disclosure of the invention

As noted above, sporadic Alzheimer's disease, which accounts for more than 95% of Alzheimer's disease, is an end product due to the increased activity of 3) secretase and increased cleavage of amyloid precursor protein by this enzyme. Amyloid β-peptide (A j3) production increases and deposits in the brain. These deposits are called senile plaques and are the initial lesions of Alzheimer's disease. In other words, increased activity of] 3 secretase has triggered sporadic Alheimer's disease. If the increase in j3 secretase activity can be detected, Alzheimer's disease can be diagnosed before the onset of disease, and effective prevention can be achieved. However, conventionally, there was no method for diagnosing this increase in activity. The present inventors have found that the / 3 secretase cleaves not only amyloid precursor protein but also α; 2,6-sialyltransferase as a physiological substrate. For the first time in the world. The present invention provides a method for diagnosing sporadic Alzheimer's disease by detecting an increase in / 3 secretase activity using an antibody that specifically recognizes sialyltransferase cleaved by secretase. Was an issue to be solved. Furthermore, an object of the present invention is to provide an antibody that specifically recognizes a human-derived secretory sialyltransferase cleaved by a / 3 secretase and a diagnostic kit using the antibody.

In Japanese Patent Application No. 2002-141414, which is an earlier patent application of the present inventors, after contacting β-secretase with sialyltransferase, the enzyme is cleaved by the enzymatic activity of secretase. A method for measuring the J3-secretase sialyltransferase cleaving activity by detecting or measuring the secreted sialyltransferase derived from a rat using an antibody that specifically recognizes the enzyme is described. I have. On the other hand, it has been shown that the cut end of human << 2,6 sialyltransferase by secretase has a different amino acid sequence from the rat enzyme. Thus, in the present invention, when a new antibody recognizing the cleavage end of human α2,6 sialyltransferase was prepared, the antibody reacts strongly with a human soluble enzyme (secreted sialyltransferase). It became clear that it showed the property. When diagnosing human samples, it is essential to use an antibody that recognizes the cut end of human 2,6-sialyltransferase. The use of this antibody has made it possible for the first time to detect or measure soluble sialyltransferase in human serum and cerebrospinal fluid. The amount of soluble enzyme reflects the / 3 secretase activity, which makes it possible to diagnose Alzheimer's disease before the onset of disease or at an early stage of onset.

That is, according to the present invention, there is provided a method for diagnosing Alzheimer's disease, comprising detecting or measuring secretory sialyltransferase cleaved by 3-secretase contained in a human-derived sample. .

Preferably, the secretory sialyltransferase is detected or measured using an antibody that specifically recognizes the secretory sialyltransferase.

Preferably, as an antibody that specifically recognizes secreted sialyltransferase, an animal is immunized with a peptide having the amino acid sequence Glu-Phe-Gin-Val-Leu-Lys-Cys as an antigen. An antibody obtained by the above method is used.

Preferably, the sample from human is a sample from brain.

Preferably, the risk of developing Alzheimer's disease is predicted by detecting or measuring secretory sialyltransferase.

According to another aspect of the present invention, there is provided an antibody capable of specifically recognizing secreted sialyltransferase of human origin, which is cleaved by one secretase. Preferably, the antibody of the present invention is an antibody obtained by immunizing an animal with a peptide having the amino acid sequence Glu-Phe-Gin-Val-Leu-Lys-Cys as an antigen. According to still another aspect of the present invention, there is provided a diagnostic kit for Alzheimer's disease, comprising the above-described antibody of the present invention. Brief Description of Drawings

FIG. 1 shows the results of detection of soluble << 2,6 sialyltransferase in human cerebrospinal fluid by Western blotting using an anti-human E44 antibody.

FIG. 2 shows the results of detecting soluble <2,6 sialyltransferase in human serum by Western blotting using an anti-human E44 antibody.

FIG. 3 shows the results of quantifying soluble sialyltransferase using an anti-human E44 antibody (left figure) or a C-terminal antibody end (right figure). BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

The method for diagnosing Alzheimer's disease of the present invention is a method characterized by detecting or measuring secretory sialyltransferase cleaved by 3-secretase, which is contained in a sample derived from human. By detecting or measuring secretory sialyltransferase according to the method of the present invention, the risk of developing Alzheimer's disease can be predicted. The expression of a2,6 sialyltransferase in the brain was low among experts in glycosyltransferases at the time of this application, and the amount of soluble enzyme was considered to be below the detection sensitivity. Therefore, soluble sialyltransferase is detected in cerebrospinal fluid even if antibodies that specifically recognize secreted sialyltransferase cleaved by secretase can be obtained from human samples. It was not known whether it was present in a possible amount or not. The present invention demonstrated for the first time that a secretory sialyltransferase can be detected or measured by the present invention.

Also, ? It has been reported that / 3 secretase activity is increased in patients with urticaria-induced Alzheimer's disease. However, not all cases are elevated, and in some patients the activity remains normal. In this patient, accumulation may be due to increased production of amyloid J3 peptide due to factors other than increased J3 secretase activity, or reduced degradation without a change in amyloid 3 peptide production There is a possibility. In this case, in the former case, the secretase inhibitor is a radical treatment method in which the cause is fundamentally normalized. On the other hand, the latter may require the use of another therapeutic agent. As described above, it is common that amyloid] 3-peptide is deposited and causes dementia, but it is possible to diagnose and treat Alzheimer's disease with a different underlying cause. Conventionally, the measurement of / 3 secretase activity in Alzheimer's disease patients was performed using brain tissue obtained by necropsy (dissection after death). A method for measuring [3] secretase activity in the brain of a living patient has not yet been reported, and the method of the present invention is the first measurement method.

In one example of the diagnostic method of the present invention, the amount of secreted sialyltransferase present in a sample derived from a subject and cleaved by 3-secretase is reduced by j3-secretase present in a sample of a control subject. By comparing with the amount of secreted sialyltransferase to be diagnosed, Alzheimer's disease can be diagnosed.

Detection or measurement of secretory sialyltransferase can be performed by immunoassay using an antibody that specifically recognizes secretory sialyltransferase. That is, the analysis of the binding between the secretory sialyltransferase and the antibody is performed by enzymatic labeling, coloring labeling, radiolabeling, or luminescence of the antibody or the secondary antibody binding to the antibody or the secretory sialyltransferase. By binding a label such as a label, and detecting or measuring this label, It can be carried out. Examples of the immunoassays that can be performed in the present invention include ELISA, Western blot, immunoprecipitation, slot or dot blot assay, immunohistochemistry, radioimmunoassay (RIA), fluorescent immunoassay, avidin-biotin or streptavidin. Examples include, but are not limited to, Imnoassy using a biotin system.

In the diagnostic method of the present invention, a human-derived sample can be used. The human-derived sample is not particularly limited as long as it can detect or measure secretory sialyltransferase cleaved by 3-secretase. Use body fluids such as serum, urine, and saliva. Possible force Preferably, a sample derived from the brain can be used. Samples derived from the brain include, but are not limited to, lumbar cerebrospinal fluid, ventricular cerebrospinal fluid, homogenates of brain tissue, or thin sections of brain tissue. The use of lumbar cerebrospinal fluid is particularly useful because the diagnostic methods of the present invention can be performed using samples obtained from living subjects.

The present invention further relates to an antibody capable of specifically recognizing secreted sialyltransferase of human origin cleaved by secretase. The antibody of the present invention is obtained, for example, by immunizing an animal with a complex obtained by covalently binding a peptide having the amino acid sequence Glu-Phe-Gln-Val-Leu-Lys-Cys (SEQ ID NO: 1) to a KLH protein as an antigen. Can be obtained by The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody. The production of the antibody of the present invention can be performed by a conventional method.

For example, a polyclonal antibody capable of specifically recognizing secretory sialyltransferase is a mammalian antibody using a peptide having the above-described amino acid sequence Glu-Phe-Gin-Val-Leu-Lys-Cys as an antigen. By immunizing the mammal, collecting blood from the mammal, and separating and purifying the antibody from the collected blood. For example, mammals such as mice, /, musters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep, and birds can be immunized. The immunization may be performed by using an ordinary immunization method known to those skilled in the art, for example, by administering the antigen one or more times. It can be carried out.

The antigen can be administered, for example, two or three times at intervals of 7 to 30 days, particularly 12 to 16 days. The dose can be set at a time, for example, about 0.05 to 2 mg of the antigen. The route of administration is not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc. can be appropriately selected.Intravenous, intraperitoneal or subcutaneous injection It is preferred to administer Also, the antigen may be in a suitable buffer, such as complete Freund's adjuvant, RAS

[MPL (Monophosphoryl Lipid A) + TDM (Synthetic Trehalose Dicorynomycolate) + CWS (Cell Wall Skeleton) adjuvant system], can be used by dissolving in an appropriate buffer containing a commonly used adjuvant such as aluminum hydroxide. However, the above adjuvant may not be used depending on the administration route and conditions. Here, an adjuvant means a substance that, when administered together with an antigen, nonspecifically enhances an immune response to the antigen.

After rearing the immunized mammal for 0.5 to 4 months, a small amount of serum from the mammal is sampled from the ear vein or the like, and the antibody titer can be measured. When the antibody titer rises, administer the antigen appropriately as many times as necessary. For example it is possible to perform additional immunized with 1 0 antigen _§ ~ 1 0 0 0 mu g. One to two months after the last administration, blood is collected from the immunized mammal by a conventional method, and treated at 56 ° C for 30 minutes to inactivate the complement system. Purify the specific antibody at Matdara Fil. As the affinity carrier, for example, an antigen peptide immobilized on Affigel or the like can be used. The blood is separated by a common method such as centrifugation, precipitation using ammonium sulfate or polyethylene dalicol, chromatography such as genole filtration chromatography, ion exchange chromatography, affinity chromatography, etc. By purifying, the polyclonal antibody of the present invention can be obtained as a polyclonal antiserum. The serum may be inactivated by, for example, treating it at 56 ° C. for 30 minutes. When the antibody of the present invention is a monoclonal antibody, the globulin type of the monoclonal antibody is not particularly limited, and examples thereof include IgG, IgM, IgA, IgE, and IgD. Further, the monoclonal antibody of the present invention may be a humanized antibody or a human antibody.

The cell line that produces the monoclonal antibody of the present invention is not particularly limited. For example, it can be obtained as a hybridoma by cell fusion of an antibody-producing cell and a Myeoma cell line. The hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.

As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used. As the antigen, the same peptide as in the case of polyclonal antibody

(Ie, a peptide having the amino acid sequence Glu-Phe-Gin-Val-Leu-Lys-Cys) can be used. Mice, rats, etc. are used as animals to be immunized, and administration of the antigen to these animals is performed according to a conventional method. For example, prepare a suspension or emulsion of an adjuvant, such as complete Freund's adjuvant or incomplete Freund's adjuvant, and an antigen peptide, and administer it several times to the vein, subcutaneous, intradermal, intraperitoneal, etc. of animals. To immunize the animals. For example, spleen cells are obtained as antibody-producing cells from the immunized animal, and the cells are fused with myeloma cells by a method known per se (G. Kohler et al., Ature, 256 495 (1975)) to obtain a hybrid. Domas can be made.

Examples of myeloma cell lines used for cell fusion include P3X63Ag8, P3U1, and Sp2Z0 strains in mice. Cell fusion is performed using a fusion promoter such as polyethylene glycol or Sendai virus, and hypoxanthine-aminopterin-thymidine (HAT) medium is used for selection of hybridomas after cell fusion according to standard methods. can do. Hybridomas obtained by cell fusion can be cloned by a limiting dilution method or the like. In addition, screening by enzyme immunoassay, etc., can specifically recognize secreted sialyltransferase derived from human cleaved by] -secretase. A cell line producing the possible monoclonal antibody can be obtained.

To produce the desired monoclonal antibody from the thus obtained hybridoma, the hybridoma is cultured by a conventional cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. Just fine. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, and the like can be used in appropriate combination.

In addition, fragments of various antibodies as described above are also within the scope of the present invention. Examples of antibody fragments include F (ab ') 2 fragment, Fab' fragment and the like. The antibodies of the present invention can also be used as labeled antibodies. By producing a labeled antibody, detection and measurement of human-derived secretory sialyltransferase cleaved by β-secretase can be easily performed. Alternatively, the antibody of the present invention or a secondary antibody that binds to a secretory sialyltransferase that is an antigen thereof can be used after being labeled. The type and labeling method of the label of the antibody of the present invention or its secondary antibody can be appropriately selected from those known to those skilled in the art.

When an enzyme is used as a label, for example, horseradish peroxidase, anolyte phosphatase, glucose oxidase, β-galata tosidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase And gnorecose-6-phosphate dehydrogenase can be used as a label. As a method of labeling the enzyme of the present invention or the secondary antibody thereof or a fragment thereof (F (ab ') 2 fragment, Fab' fragment, etc.), the sugar chain of the enzyme is oxidized with periodic acid, A method of binding an amino acid such as the antibody to the generated aldehyde group, a method of introducing a maleimide group or a pyridyl sulfide group or the like into the enzyme, and binding the thiol group present in the Fab or fragment of the antibody. Can be mentioned.

When using an enzyme as a label, after incubating the test sample with the labeled antibody, the released labeled antibody is removed by washing, and then the substrate of the above labeled enzyme is allowed to act. Then, the labeled antibody can be detected by measuring the reaction by color development or the like. For example, when labeled with peroxidase, it produces a brown or yellow color in combination with hydrogen peroxide as a substrate and diaminobenzidine or o-phenylenediamine as a chromogenic reagent. In the case of labeling with glucose oxidase, for example, 2,2,2-acid-di (3-ethylbenzothiazoline-16-sulphonic acid (ABTS)) can be used as a substrate.

When a fluorescent dye is used as a label, for example, it is possible to label the antibody of the present invention or its secondary antibody with a fluorescent dye such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocyanate). it can. The binding between the antibody of the present invention or its secondary antibody and a fluorescent dye can be performed by a conventional method.

When a colored labeling substance is used as a label, for example, colloidal metal and colored latex can be used as the label. Representative examples of the colloidal metal include metal colloid particles which are dispersed particles of gold sol, silver sol, selenium sol, tellurium sol, platinum sol and the like. The size of colloidal metal particles is usually about 3 to 6 O nm in diameter. As a typical example of the colored latex, a synthetic latex such as polystyrene latex colored with respective pigments such as red and blue can be given. Natural latex such as natural rubber lattas can be used as the latex. The size of the colored latex can be selected from several tens nm to several hundred nm in diameter. Commercially available products can be used for these coloring labeling substances as they are, but they may be further processed in some cases or produced by a method known per se.

The binding between the antibody of the present invention or its secondary antibody and the color labeling substance can be performed by a conventional method. For example, when the color labeling substance is colloidal gold particles that are dispersed particles of gold sol, it is usually possible to physically bind the antibody and gold sol by mixing them at room temperature. .

In addition, as a label, in addition to the above, an affinity label (for example, biotin or the like), Or isotopic label (e.g., ^{1 2 5} I, etc.) can also be used like. Detection or measurement of secreted sialyltransferase cleaved by secretase using the antibody of the present invention can be performed by ELISA, Western blot, immunoprecipitation, slot or dot plot assay, immunostaining, radioimmunoassay. (RIA), fluorescent imnoassay, iminoassay using the avidin-biotin or streptavidin-biotin system, and such analysis is a method known to those skilled in the art.

For example, the detection of soluble sialyltransferase using an anti-human E44 antibody by Western blotting can be performed according to the method described in Example 2 of the present specification. In addition, it is considered that quantification can be performed by the sandwich ELISA method using the same sample as in Example 2. Coat the ELISA plate in advance with C-terminal antibody, and trap the soluble sialyltransferase in the sample on the plate. After thorough washing, the trapped sialyltransferase is reacted with an anti-human E44 antibody. The secondary antibody is reacted with horseradish peroxidase labeled anti-Peacock IgG (Cappel). Color is developed by adding a chromogenic substrate, and the concentration is measured using an absorptiometer. A calibration curve is prepared in advance using a standard sample containing a known concentration of soluble sialyltransferase, and a quantitative value can be calculated.

Furthermore, the present invention also relates to a diagnostic kit for Alzheimer's disease, which comprises an antibody capable of specifically recognizing the above-mentioned secretory sialyltransferase derived from human cleaved by J3-secretase. By using the diagnostic kit of the present invention, it is possible to detect or measure secretory sialyltransferase cleaved by 3-secretase contained in a sample derived from human, thereby diagnosing Alzheimer's disease. it can.

The diagnostic kit of the present invention includes, for example, an antibody (primary antibody) of the present invention capable of specifically recognizing a secretory sialyltransferase derived from human cleaved by (1) / 3-secretase;ぴ (2) A labeled substance capable of generating a signal is bound A secondary antibody can be included. As the secondary antibody used herein, an antibody capable of binding to the primary antibody or an antibody capable of recognizing and binding to the secretory sialyltransferase (however, recognizing and binding to a site different from the binding site of the primary antibody) Which is an antibody) can be used. When an antibody capable of recognizing and binding to the secretory sialyltransferase is used as the secondary antibody, sandwich immunoassay (eg, sandwich ELISA) can be performed.

In the diagnostic kit of the present invention, the primary antibody of the present invention may be immobilized in advance, or the primary antibody of the present invention may be labeled in advance. The solid phase that can be used in the diagnostic kit of the present invention is not particularly limited, and examples thereof include polymers such as polystyrene, insoluble carriers such as glass beads, magnetic particles, microplates, filter paper for immunochromatography, and glass filters. Can be mentioned.

The diagnostic kit of the present invention may further contain other optional components. Other optional components include, for example, an enzyme used for labeling, a substrate thereof, a radioisotope, a luminescent substance, a fluorescent substance, a coloring substance, a buffer, a plate, and the like, but are not limited thereto. Absent.

Further, the form of the diagnostic kit of the present invention is not particularly limited, but for the purpose of performing a diagnosis quickly and easily, an integrated diagnostic kit in which the components of the diagnostic kit of the present invention are integrated can be provided. . The form of the integrated diagnostic kit is also not particularly limited, and examples thereof include a cassette type using an immunochromatography method and a cartridge type for performing a competitive immunoassay.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example

Example 1: Preparation of antibody

(1) antibodies against human _alpha 2, 6 sialyltransferase Ν- terminus of a soluble Peptide plus cysteine residue corresponding to the newly generated amino terminal sequence (EFQVL...) When human a 2,6 sialyltransferase is cleaved by j3 secretase

(Glu-Phe-Gln-Val Leu-Lys-Cys) and KLH was converted to MBS

Activated with m-maleimidobenzoyl-N-hydroxysuccinimide ester) to bind to ralia protein. This is immunized together with Freund adjuvand to the egret to obtain antiserum. The obtained antiserum is applied to an antigen peptide 'afficity' column to elute only the bound antibody. AffigenlO or 15 is used as an affinity carrier. Dissolve 1 mg of hapten peptide in 100 mM HEPES buffer (pH 7.5). Next, filter the 2nd Aff igwl on a glass filter with suction filtration, wash with distilled water, add the peptide solution, rotate and stir at 4 ° C all day and night, and then glass again to remove free peptide. Filter with suction on the filter. Wash thoroughly with 30% acetic acid or 20% ethanol, finally equilibrate with PBS and store at 4 ° C. Pack the affinity carrier into the column and wash with PBS. Dilute 10 ml of the immobilized antiserum with the same volume of PBS, pass through a filter (0.45 μl), and add to the column. Then, wash with 50 ml of PBS. The antibody is eluted from the affinity gel with 5 ml of 50 mM citrate buffer (pH 3.0), and immediately neutralized with 2 M Tris buffer (pH 9.5). The eluate is dialyzed against 20% glycerol-PBS and finally the antibody concentration is determined by measuring the absorbance at 280 nm. The obtained antibody was named anti-human E44 antibody.

(2) antibodies against human _alpha 2, 6 C-terminal side of sialyltransferase

The catalytic domain of sialyltransferase is located at the C-terminal side. Among these, 18 amino acid sequences that are well preserved among animal species

(Cys-Asp-Gln-Val-Asp-lie-Tyr-Glu-Phe-Leu-Pro-Ser-Lys-Arg ~ Lys-Tnr-Asp-Va1) (SEQ ID NO: 2) was selected as an antigen peptide. The preparation method is the same as that for the anti-human E44 antibody. This was named C-terminal antibody. Example 2: Detection of soluble sialyltransferase using anti-human E44 antibody (Western plot method) Since human serum samples (equivalent to the amount of protein for 200 μg) contain a large amount of albumin, remove albumin by preliminarily defatting and concentrating by acetone precipitation using an albumin removal kit. For human cerebrospinal fluid, use a sample obtained by ordinary lumbar puncture. Confirm that the cerebrospinal fluid used as the sample is free from blood contamination. After dissolving these samples (equivalent to the protein amount of 50 to 100 g) in PBST solution (50 mM phosphate buffer (ρ （7.2), 0.15 M NaCl, 0.1% Tween20), Mix with Lae li sample buffer, heat and use for SDS / PAGE. The separated proteins are electrically transferred to a nitrocellulose membrane. After blocking the membrane with 5% milk in TTBS buffer (50 mM Tris-HCl (pH 7.2), 0.15 M NaCl, 0.1% Tween20), anti-human E44 antibody (l / g / ml) or C-terminal React with antibody (5 / ig / ml). A secondary antibody is reacted with horseradish peroxidase-labeled anti-Peacock IgG (Cappel). Visualize the sialyltransferase band using a chemiluminescent substrate (Pierce, Super Signal). The results are shown in FIGS. 1 and 2.

Reactivity with a standard sample containing a known concentration of soluble sialyltransferase was detected and quantified using a lumino image analyzer (LAS-IOOOplus: Fuji Film). Figure 3 shows the results. Industrial applicability

According to the present invention, a novel method for diagnosing Alheimer's disease is provided. The method for diagnosing Alzheimer's disease according to the present invention is based on the detection or measurement of β-secretase activity, and can diagnose Alzheimer's disease before onset or in the early stage of onset. That is, according to the method of the present invention, in particular, Alzheimer's disease can be diagnosed before the onset of the disease, and effective prevention can be achieved. At the same time, personality changes, difficulty in uttering words, loss of memory, loss of physical It is also possible to diagnose Alheimer's disease early in the onset of patients with early symptoms of Alzheimer's disease, such as misplacement or forgetting of a person's name. Further, according to the diagnostic method of the present invention, It will be possible to distinguish Alzheimer's disease from other diseases that have symptoms similar to those of Alzheimer's disease. Further, in the diagnostic method of the present invention, lumbar cerebrospinal fluid can be used as a human-derived sample, so that a living subject can be easily diagnosed with a low-risk attestation.

Claims

The scope of the claims

1. A method for diagnosing Alzheimer's disease, comprising detecting or measuring secretory sialyltransferase cleaved by 3-secretase contained in a sample derived from human.

2. The method according to claim 1, wherein the secretory sialyltransferase is detected or measured using an antibody that specifically recognizes the secretory sialyltransferase.

3. As an antibody that specifically recognizes secreted sialyltransferase, an antibody obtained by immunizing an animal with a peptide having the amino acid sequence Glu-Phe-Gln-Val-Leu-Lys-Cys as an antigen is used. 3. The method of claim 2 for use.

4. The method according to any one of claims 1 to 3, wherein the human-derived sample is a brain-derived sample.

5. The method according to any one of claims 1 to 4, wherein the risk of developing Alzheimer's disease is predicted by detecting or measuring secretory sialyltransferase.

6. An antibody capable of specifically recognizing human-derived secretory sialyltransferase cleaved by -secretase.

7. The antibody according to claim 6, wherein the antibody is obtained by immunizing an animal with a peptide having the amino acid sequence Glu-Phe-Gin-Val-Leu-Lys-Cys as an antigen.

8. A diagnostic kit for Alzheimer's disease, comprising the antibody according to claim 6 or 7.