JP5991666B2 - Method and kit for detecting Alzheimer's disease - Google Patents

Description

  The present invention relates to a detection method and kit for Alzheimer's disease. More specifically, the present invention relates to a method and kit for detecting Alzheimer's disease characterized by quantifying amyloid β protein-binding albumin in a blood sample of a subject.

Conventionally, for diagnosis of Alzheimer's disease, image diagnosis or neuropsychological diagnosis using CT, MRI, PET, or the like has been used. In addition, the amount of biomarkers (eg, amyloid β protein, tau protein) in cerebrospinal fluid is used as auxiliary data for diagnosis of Alzheimer's disease.
However, image diagnosis and psychological diagnosis require a high degree of specialized knowledge, require time for diagnosis, and are expensive.
Moreover, the collection of cerebrospinal fluid is highly invasive, and the patient's psychological and physical burden is large.

Therefore, there is a demand for a simple method that is less invasive when collecting a test sample.
Recently, the “withdrawal” hypothesis that brain Aβ is drawn into the blood as the amount of Aβ in the blood decreases is proposed. Based on this hypothesis, it is considered that the blood Aβ amount reflects the brain Aβ amount.
However, no difference in the amount of blood amyloid β protein has been observed between Alzheimer's disease patients and healthy individuals.

In addition, a method for measuring amyloid β protein in plasma has been developed (Non-Patent Document 1), which predicts the degree of future cognitive decline, and diagnoses Alzheimer's disease at the present time. It wasn't something to do.
On the other hand, it is reported that about 90% of amyloid β protein in blood is bound to albumin (Non-patent Document 2).
However, there is no report that measures amyloid β protein in blood in association with albumin.

Yaffe, K. et al., JAMA, 2011, 305 (3): 261-266 Biere, A. L. et al., J. Biol. Chem., 1996, 271 (51): 32916-32922

  Therefore, it has been desired to develop a method and kit that can collect a test sample by a less invasive method and can easily detect Alzheimer's disease.

The present invention provides a method for detecting Alzheimer's disease characterized by quantifying amyloid β protein-binding albumin in a blood sample of a subject.
The present invention also provides a kit for detecting Alzheimer's disease comprising a substance capable of binding to amyloid β protein-binding albumin.

  As a sample, a blood sample that is less invasive at the time of collection is used, so that the collection of the sample is easy and the burden on the subject can be reduced.

It is a figure explaining the measurement principle of the amount of amyloid protein binding albumin in one form of the present invention. It is a scatter diagram which shows the measured value in an Example. It is a statistical graph (box-and-whisker figure) explaining the measurement data in an Example.

<Method for detecting Alzheimer's disease>
The method for detecting Alzheimer's disease of the present invention is characterized in that amyloid β protein-binding albumin is quantified in a blood sample of a subject.
The present invention is based on the new finding that the amount of “amyloid β protein binding” albumin in the blood sample of Alzheimer's disease patients is significantly less than that in healthy individuals.

A “subject” includes humans and non-human mammals, but is preferably a human, particularly a human suspected of suffering from Alzheimer's disease.
A “blood sample” is a whole blood sample, a serum sample or a plasma sample.
“Amyloid β protein” (hereinafter sometimes referred to as “Aβ”) is a peptide consisting of 37 to 43 amino acids generated from amyloid precursor protein (APP), and is typically Aβ _1-40 or Aβ _{1. -42} and Aβ _1-43 , in particular Aβ _1-42 . Representative amino acid sequences of human Aβ _1-40 , Aβ _1-42 and Aβ _1-43 are shown in the sequence listing as SEQ ID NOs: 1, 2 and 3, respectively.
“Albumin” in the context of the present invention refers to serum albumin. A representative amino acid sequence of human (serum) albumin is shown in the sequence listing as SEQ ID NO: 4.

Aβ-binding albumin in a blood sample can be quantified by various methods such as a dye-binding method, immunoassay, electrophoresis, chromatography, mass spectrometry, and combinations thereof.
Preferably, Aβ-binding albumin is quantified as albumin bound to Aβ (Aβ-binding albumin), in particular as Aβ-binding albumin immobilized on a support.

Quantification of immobilized Aβ-bound albumin can be performed by an albumin quantification method known in the art.
For example, in the dye-binding method, a reagent that reacts with albumin to develop color or change its color tone is used. Examples of such a reagent include bromcresol green (BCG) and bromcresol purple (BCP).

In the immunoassay, an antibody against albumin (anti-albumin antibody) is used.
In the present invention, “antibody” refers to a complete antibody and a fragment having an antigen-binding site thereof (including single-chain antibody (scFv), Fab antibody, F (ab ′) ₂ antibody, Fab ′ antibody). The antibody may be a known antibody or a newly prepared antibody. The antibody may be monoclonal or polyclonal, but is preferably monoclonal.

  Methods for producing antibodies that bind to specific proteins or peptides are known in the art. Briefly, such antibodies can be conjugated to the protein or peptide, either alone or in a suitable carrier (e.g., keyhole limpet hemocyanin, diphtheria toxin, tetanus toxin) and optionally an adjuvant (e.g., Animals (e.g., mice, rats, hamsters, guinea pigs) administered once or more (subcutaneously, intravenously, intraperitoneally, etc.) with complete or incomplete Freund's adjuvant, aluminum hydroxide or aluminum phosphate (alum)) By purifying from serum of rabbits, goats, sheep, pigs, horses, camels, chickens, ostriches, etc. using a purification method such as affinity chromatography (e.g., immunoglobulin fractions). Or as a purified antibody). The whole serum (antiserum) may be used as it is without purification.

  Methods for producing monoclonal antibodies are also known in the art (eg, Kohler and Milstein (Nature, 256: 495-497, 1975; Antibodies: A Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor Laboratory, 1988)). Briefly, spleen cells (B cells) of an animal immunized with the protein or peptide are fused with appropriate myeloma cells by a cell fusion method to produce immortalized cells (hybridomas), and have the desired binding ability. A hybridoma producing the antibody is selected, and the hybridoma may be grown in vitro or in vivo (eg, intraperitoneally in the mouse) to obtain the produced antibody.

  It is preferred that the anti-albumin antibody recognizes albumin of the subject mammal while not recognizing albumin of other mammals (which may be included in other reagents such as blocking agents). More preferably, the anti-albumin antibody is an antibody that recognizes human albumin but does not recognize bovine, rabbit, goat and / or sheep albumin. Such anti-human albumin antibodies are available from domestic and foreign suppliers as labeled or unlabeled anti-human serum albumin-mouse monoclonal antibody or anti-human serum albumin-rabbit (goat, sheep, etc.) polyclonal antibody or serum. Alternatively, it can be easily produced by the above method.

The antibody is preferably labeled with a detectable label. The label of the antibody is known in the art and can be appropriately selected depending on the detection means used. For example, an enzyme, a radioisotope, a fluorescent substance, a coloring substance, a luminescent substance, or the like can be used for the label. Examples of the enzyme include peroxidase (particularly HRP), β-galactosidase, phosphatase (particularly alkaline phosphatase), luciferase, glucose oxidase, β-glucosidase, β-glucuronidase and the like. Examples of the radioisotope include ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²⁵ I, and ¹³¹ I. Examples of the fluorescent dye include fluorescein isothiocyanate (FITC), rhodamine, Cy2 and Cy3. Examples of the coloring substance include 3,3′-diaminobenzidine (DAB), 3,3 ′, 5,5′-tetramethylbenzidine (TMB), and the like. Examples of the luminescent substance include luminol and luciferin.

Immobilized Aβ-binding albumin can also be quantified using aptamers or peptides that can specifically bind to albumin.
Aptamers are single-stranded nucleic acids (DNA, RNA or nucleic acid analogs (eg LNA)) that recognize specific targets. Methods for making aptamers that can specifically bind to specific targets are known in the art. Briefly, a nucleic acid group having a random base sequence (for example, 30 to 150 bases) is prepared, and a nucleic acid group that specifically recognizes the target of interest is selected by a screening method such as the SELEX method.

Peptides that can specifically bind to albumin include, for example, bacteria-derived albumin binding properties described in WO96 / 14416 [Japanese Patent Publication No. 10-509311] and WO2009 / 016043 [Japanese Patent Publication 2010-534486] A domain-containing one or a variable domain derived from an anti-albumin antibody as described in WO2008 / 096158 [Japanese Patent Publication No. 2010-518062].
The aptamer or peptide that can specifically bind to albumin is preferably provided with a detectable label. For example, the radioisotope, the fluorescent substance, the coloring substance, the chemiluminescent substance, and the like as described above are used for the label. Nucleic acid labeling methods are known in the art.

  For measurement of the signal from the label, a spectrophotometer, a fluorometer, a CCD camera, a CMOS camera, a photodiode, a photomultiplier tube, etc., which are appropriately selected, are used together with a light source and / or a filter as necessary. It can be carried out.

Immobilization of Aβ-binding albumin can be performed, for example, using a support on which a substance capable of binding to Aβ is fixed. In this case, Aβ-binding albumin is immobilized on the support via Aβ in the sample. The immobilization can also be performed using a support on which Aβ is fixed.
Examples of substances that can bind to Aβ include an antibody against Aβ (anti-Aβ antibody) and an aptamer that can specifically bind to Aβ.

The anti-Aβ antibody is an N-terminal region of Aβ (for example, amino acids 1-5, 1-6, 1-7, 3-7, 1-8, 3-8, 1-10, 3-9, 4-10, 5-9, 9-10, 1-11, 5-11, 1-12, 1-16, 5-16, 10-16, Antibodies recognizing positions 12-16, 1-17, 11-17), central region (e.g. amino acids 11-26, 11-28, 12-28, 13-28, 15-24) , 15-30, 16-24, 17-22, 18-21, 18-22, 22-35, 25-34, 25-35), Aβ ₄₀ , C-terminal region of Aβ ₄₂ and / or Aβ ₄₃ (for example, amino acids 31 to 40, 32 to 40, 33 to 40, 33 to 42, 34 to 40, 35 to 40, 34 to 42) , 35-42, 35-43, 36-42, 37-42, 38-43) are known (eg Johnson-Wood, K et al., Proc. Natl. Acad Sci. USA, 94: 1550-1555 (1997); Asami-Odaka A et al., Bioc hemistry, 34 (32): 10272-10278, 1995). These antibodies can be obtained from domestic and foreign suppliers, and can be easily obtained by the above-described methods (for example, using Aβ or a fragment having an epitope as described above). Preferably, the anti-Aβ antibody is an antibody (more preferably a monoclonal antibody) that recognizes the N-terminal region or C-terminal region of Aβ.

  Aptamers that can specifically bind to Aβ are described, for example, in Takahashi et al. (Bol. Biosyst., 5 (9): 986-991, 2009), and can be easily obtained as described above.

Quantification of immobilized Aβ-bound albumin can also be performed by Aβ quantification methods known in the art.
For example, immobilized Aβ-bound albumin can be quantified in an assay using an anti-Aβ antibody or an aptamer that can specifically bind to Aβ as described above. The aptamer capable of specifically binding to anti-Aβ antibody or Aβ is preferably labeled as described above.
For such an assay, Aβ-bound albumin can be immobilized using a support on which a substance capable of binding to albumin is immobilized. Examples of the substance capable of binding to albumin include an anti-albumin antibody as described above or an aptamer or peptide that can specifically bind to albumin.

  When quantifying Aβ-binding albumin is performed by the dye-binding method, immunoassay, or method using an aptamer as described above, particularly when using a kit (described later) for carrying out the method, Inexpensive and relatively quick detection is possible. For this reason, the method of the present invention can be carried out relatively easily not only in hospitals and laboratories equipped with clinical examination facilities and equipment but also in clinics and clinics.

  As will be apparent from the examples described later, the amount of Aβ-binding albumin in the blood sample from the Alzheimer's disease patient tends to be lower than the amount in the blood sample of a healthy person. Therefore, the amount of Aβ-binding albumin in the blood sample is useful as an indicator of Alzheimer's disease.

  Preferably, the ratio (Aβ-binding albumin) in the blood sample is determined from the measured Aβ-binding albumin amount and the total albumin (Aβ-binding albumin + Aβ non-binding albumin) amount measured in another sample from the same subject. Amount) / (total albumin amount) is obtained. When this ratio is lower compared to a predetermined threshold level (cutoff value), the subject who provided the blood sample is considered likely to have Alzheimer's disease. Alternatively, when the ratio is higher compared to a threshold level (cutoff value), the subject is considered less likely to have Alzheimer's disease. The threshold level (cut-off value) is based on statistically significant numbers of measured data on patients with a definite diagnosis of Alzheimer's disease and healthy individuals (which are clearly not affected by Alzheimer's disease). Can be determined.

In order to measure the amount of Aβ-binding albumin relative to the total amount of albumin, the blood sample may be previously diluted with a diluent (for example, a buffer solution) so that the total amount of albumin becomes a predetermined value.
The buffer is appropriately selected from those known in the art. For example, phosphate buffer, Tris-HCl buffer, citrate buffer, carbonate buffer, borate buffer, succinate buffer, acetate buffer Etc. The buffer may contain NaCl, a surfactant (eg, Tween 20, Triton X-100) and / or a preservative (eg, sodium azide) as necessary. Specific examples of the buffer are PBS, PBS-T, TBS or TBS-T.

Specifically, the method of the present invention is performed, for example, as follows.
In this embodiment, in order to immobilize Aβ-binding albumin in a blood sample, a support on which a substance capable of binding to Aβ is immobilized is used.
Conditions suitable for binding a blood sample to a support and a substance capable of binding Aβ-binding albumin in the sample to Aβ on the support via Aβ (for example, temperature: 20 ° C. to 40 ° C., more Preferably 25-38 ° C .; time: 5-120 minutes, more preferably 10-90 minutes, more preferably 30-60 minutes) under contact (incubation; FIG. 1A).

After incubation, the support is washed one or more times with a wash solution (eg, buffer) to remove material (including Aβ non-binding albumin) that has not been immobilized on the support.
At this point, Aβ non-binding albumin in the sample is removed and only Aβ binding albumin is immobilized on the support. Thus, the amount of albumin immobilized on the support reflects the amount of Aβ binding albumin in the blood sample.

  Albumin immobilized on the support can be quantified using a dye binding method such as BCG method, BCP method, or BCP improvement method.

Alternatively, albumin immobilized on the support can be quantified by immunoassay, for example, as follows (FIG. 1B).
A labeled anti-albumin antibody under conditions suitable for the binding of the support (with Aβ-bound albumin immobilized) and the anti-albumin antibody and albumin (eg, temperature: 20 ° C. to 40 ° C., more preferably 25 to 38 ° C .; Time: 5 to 120 minutes, more preferably 10 to 60 minutes).
Unsupported anti-albumin antibody is removed by washing the support once or more with a washing solution.
The signal from the label is quantified by a method corresponding to the label attached to the anti-albumin antibody. For example, photometry is performed using a microplate reader together with a light source and / or a filter as necessary.

  Standard curve using blank and standard substance (standard solution) before, during or after quantifying the immobilized albumin on the support, depending on the quantification method used (substance capable of binding to Aβ-binding albumin) A (calibration curve) may be created.

  In the above-mentioned embodiment, the method for quantifying the Aβ-binding albumin immobilized on the support by a dye-binding method or an immunoassay has been specifically described. However, Aβ-binding albumin in a sample can be obtained by other methods (for example, Western blotting). Needless to say, it can be quantified by the above method.

The method of the present invention can be used as a method for evaluating the risk of suffering from Alzheimer's disease by performing it on a blood sample of a subject who does not show symptoms or signs of Alzheimer's disease. When the measured value is below the threshold value, the subject can be estimated to be at high risk of suffering from Alzheimer's disease, and when the measured value is not below the threshold value but is lower than the average value of healthy individuals and close to the threshold value, It can be estimated that the subject has a relatively high risk of suffering from Alzheimer's disease. On the other hand, when the measured value exceeds the threshold value, it can be estimated that the subject has a low risk of suffering from Alzheimer's disease.
Since the method of the present invention can be carried out with a small invasiveness, the risk of future morbidity can be estimated, for example, by carrying out it during regular medical examinations. As a result, it is possible to promptly promote prevention efforts.

  The method of the present invention is also performed on a blood sample at two or more time points from a subject (patient) undergoing treatment for Alzheimer's disease to quantify Aβ protein-binding albumin in the blood sample over time. By comparison, it can also be used as a method for monitoring the progress of Alzheimer's disease. When the amount of Aβ-binding albumin in the sample at a later time point is increased compared to that in the sample at an earlier time point, it can be estimated that the Aβ-binding albumin is in a recovery trend from Alzheimer's disease and conversely decreases. It can be assumed that Alzheimer's disease is ongoing.

Quantification over time is, for example, every 5, 10, 15 or 20 days, every 1, 2 or 3 weeks, every 1, 2, 3, 4, 5 or 6 months, 1, 2, 3, 4 or 5 years Do it every time.
If the patient is being treated for Alzheimer's disease, this method can be used to assess the effectiveness of the treatment. Treatment is not limited to drug therapy and includes, for example, diet therapy, non-drug therapy such as exercise therapy, and a combination of drug therapy and non-drug therapy.

<Alzheimer's disease detection kit>
The kit for detecting Alzheimer's disease of the present invention comprises a substance capable of binding to amyloid β protein-binding albumin.
This kit is suitable for carrying out the method of the present invention described above.

Substances that can bind to Aβ-binding albumin include, for example, BCG, BCP, anti-albumin antibodies as described above, aptamers that can specifically bind to albumin as described above, and specifically bind to albumin as described above. The obtained peptide, the anti-Aβ antibody as described above, and the aptamer capable of specifically binding to Aβ as described above, preferably anti-albumin antibody or anti-Aβ antibody.
The substance capable of binding to Aβ-binding albumin may be provided in a solution form, or in the case of a protein or peptide, may be provided in a lyophilized form.
If necessary, the substance capable of binding to Aβ-binding albumin may be labeled as described above. Alternatively, the substance capable of binding to Aβ-binding albumin has one partner (for example, biotin) attached to a binding pair such as (strept) avidin-biotin and the other partner (for example, (strept) avidin). May be capable of binding to a label to which is attached.

The kit of the present invention preferably further comprises a support on which a substance capable of binding to the Aβ protein or a substance capable of binding to albumin is immobilized. The substance capable of binding to Aβ is preferably an anti-Aβ antibody as described above. The substance capable of binding to albumin is preferably an anti-albumin antibody as described above.
The form of the support can be appropriately selected from those known in the art, such as microtiter plates, tubes, capillary tubes, beads, particles, filter paper, filters, membranes, disks, sticks, strips, slide glasses, chips, etc. possible.

Any material known in the art can be used as the support material. For example, glass, resin (for example, polystyrene, polyester, polyacrylamide, polyacrylate, polymethacrylate, polypropylene, polyvinyl butyrate, polyvinyl chloride, Tetrafluoroethylene, polyvinylidene fluoride, nylon, rayon), cellulose and its derivatives (for example, nitrocellulose), and the like.
The substance capable of binding to Aβ or albumin may be immobilized on the support by physical adsorption, covalent bond, or non-covalent bond (ionic bond, electrostatic bond, hydrophobic interaction, etc.).

The support is preferably subjected to a blocking treatment to prevent nonspecific adsorption. Any blocking agent known in the art can be used. For example, serum albumin of a mammal other than the subject mammal (for example, bovine serum albumin (BSA) when the subject is a human), skim milk powder, casein And so on.
The surface of the support on which a substance capable of binding to Aβ or albumin is immobilized and subjected to blocking treatment is a water-soluble polymer (for example, polyvinyl pyrrolidone, polyvinyl alcohol, or cellulose esters such as hydroxypropylmethylcellulose and hydroxyethylcellulose) or non-reducing It may be coded with a polysaccharide (for example, sucrose, trehalose, raffinose) and the like. By such coding, the solid phase containing a substance capable of binding to Aβ or albumin on the support is very stabilized and suitable for long-term storage.

The kit of the present invention may further contain a label signal generating agent (for example, a color former) and / or a reaction stopping solution as necessary.
The label signal generating agent can be appropriately selected from known ones according to the label. For example, if the label is peroxidase, TMB, DAB, o-phenylenediamine (OPD), 2,2-azino-di- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 10-acetyl -3,7-dihydroxyphenoxazine (ADHP), and in the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (4-MUP), etc. obtain.
The reaction stopper may be, for example, sulfuric acid or sodium hydroxide.

The kit of the present invention may also contain a standard solution for preparing a standard curve (calibration curve), and optionally a standard solution diluent.
The kit of the present invention may also contain a washing solution (e.g., a buffer as described above) or a sealant (e.g., a plate seal if the support is a plate) that seals the support during incubation. Good.
The kit of the present invention is used for evaluating the risk of suffering from Alzheimer's disease and / or for monitoring the progress of Alzheimer's disease and / or for evaluating the effectiveness of Alzheimer's disease treatment as described above. can do.

In each case, serum was collected from 15 Alzheimer's disease (AD) patients (60-87 years old) and 17 healthy persons (65-87 years old) who obtained informed consent. The definitive diagnosis of AD patients was based on neuropsychological diagnosis, imaging diagnosis, and exclusion diagnosis of other diseases causing dementia.
The collected samples were subdivided into three, one for total albumin concentration and apolipoprotein E (ApoE) phenotype and two for Aβ-binding albumin quantification.
Total albumin concentration and ApoE phenotype were measured at SRL by immunoblotting with BCP modification and isoelectric focusing, respectively.

Based on the total amount of albumin, the corresponding serum sample for quantifying Aβ-binding albumin was diluted with TBS so that the total albumin concentration was 150 μg / ml.
100 μl of the diluted sample was put into each well of a 96-well microplate to which a monoclonal antibody (BAN50; Takeda Pharmaceutical Co., Ltd.) that recognizes the N-terminal region (amino acids 1 to 16) of Aβ was fixed, and then at 37 ° C. for 1 hour. After incubation, the plate was washed 4 times with TBS-Tween 20. Next, an HRP-labeled anti-human albumin polyclonal antibody diluted 1: 30000 (Goat anti-Human Albumin Antibody HRP Conjugated Catalog No. A80-129P; Bethyl) was added to each well and incubated at 37 ° C. for 1 hour. Thereafter, it was again washed 4 times with TBS-T.

The TMB / H ₂ O ₂ solution was added to each well to start the color reaction, and after 15 minutes, the color reaction was stopped by adding with a stop solution. Subsequently, photometry was performed for 0.1 second using a 450 nm filter with a microplate reader.
The results are shown in FIGS. 2 is a scatter diagram, and FIG. 3 is a box plot. A result is shown as an average of two measurement data independently performed about the same sample.
According to two independent t-tests, the difference was significant in the 95% confidence interval (p = 0.021), and was also significant in the multinomial logistic analysis taking into account age and ApoE4 (p = 0.021).
It was found that albumin bound to amyloid β protein in the AD patient group was significantly less than that in the healthy group.

  The above embodiments and examples are described as examples for facilitating the understanding of the present invention, and the present invention is limited only to the specific forms and examples described in this specification or the accompanying drawings. Is not to be done. The specific arrangements, means and methods described herein can be replaced with many others known in the art without departing from the spirit of the invention.

Claims (9)

A method for obtaining data for detecting Alzheimer's disease, comprising quantifying amyloid β protein-binding albumin in a blood sample of a subject. The method according to claim 1, wherein the quantification of the amyloid β protein-binding albumin is performed by quantifying albumin bound to amyloid β protein (amyloid β protein-binding albumin). Quantification of the amyloid β protein-binding albumin is performed by quantifying amyloid β protein-bound albumin immobilized on the support via a substance capable of binding to amyloid β protein or a substance capable of binding to albumin, The method according to claim 1 or 2. The method according to any one of claims 1 to 3, wherein the quantification of the amyloid β protein-binding albumin is performed using an anti-albumin antibody. The method according to any one of claims 1 to 3, wherein the quantification of the amyloid β protein-binding albumin is performed using an anti-amyloid β protein antibody. A kit for detecting Alzheimer's disease, comprising a substance capable of binding to amyloid β protein-binding albumin. The kit according to claim 6, further comprising a support on which a substance capable of binding to amyloid β protein or a substance capable of binding to albumin is immobilized. The kit according to claim 6 or 7, wherein the substance capable of binding to amyloid β protein-binding albumin is an anti-albumin antibody. The kit according to claim 6 or 7, wherein the substance capable of binding to amyloid β protein-binding albumin is an anti-amyloid β protein antibody.