JP6883847B2 - Method for measuring amyloid β protein and tau protein and / or phosphorylated tau protein - Google Patents

Description

The present invention relates to a method for simultaneously measuring the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein in an intranasal sample. The present invention also relates to a system for simultaneously measuring the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein in an intranasal sample.

Alzheimer's disease is a disease characterized by progressive dementia that occurs from presenile to old age, and it is said that the number of patients in Japan is currently more than 4.6 million. Furthermore, it is expected that the number will surely increase as the population ages. There has been no cure for Alzheimer's disease so far, but since the successful vaccine therapy in model mice in 1999, expectations for the development of a cure have increased. In order to effectively utilize these fundamental treatment methods, an early and simple method for diagnosing Alzheimer's disease is indispensable.

Clinical symptoms of Alzheimer's disease include memory impairment, higher brain dysfunction (aphasia, apraxia, agnosia, constructional apraxia) and the like. The symptom is often seen in other dementia diseases, and it is extremely difficult to make a definitive diagnosis of Alzheimer's disease based on clinical symptoms alone.

On the other hand, the characteristic histopathological findings of Alzheimer's disease are amyloid plaque and neurofibrillary tangles. The main constituent of the former is amyloid β protein with β-sheet structure, and that of the latter is hyperphosphorylated tau protein. It is known that in Alzheimer's disease, the above-mentioned pathological tissue changes such as accumulation of aggregated amyloid β protein have started long before the onset of clinical symptoms. In addition, the accumulation of hyperphosphorylated tau protein is thought to occur later than the accumulation of amyloid β protein, but neurofibrillary tangles are more severe than the accumulation of amyloid β protein. It is believed to be closely related. Therefore, by detecting both amyloid β protein and tau protein, it is possible to diagnose Alzheimer's disease at an early stage and its severity.

From this point of view, in recent years, positron emission tomography (PET), single photon tomography (SPECT), and magnetic resonance imaging (MRI) using a contrast medium that selectively binds to amyloid β protein in the brain. Research on the imaging of senile spots by Research on radioactive contrast media for PET and SPECT that bind to tau protein is also underway.

The present inventors have reported that the concentrations of amyloid β protein and tau protein can be measured by using an intranasal sample collected from the nasal cavity with a cotton swab or the like as a method for diagnosing Alzheimer's disease, which is less invasive (Patent Documents). 1, 2, Non-Patent Document 1). In the methods reported in Patent Documents 1 and 2 and Non-Patent Document 1, formic acid is added as a solubilizer in order to detect amyloid β protein. This is because the amyloid β protein is aggregated in the sample, and the amyloid β protein can be detected by disassembling the aggregation with formic acid to obtain a soluble amyloid β monomer.

However, formic acid decomposes proteins, making it impossible to measure tau protein or phosphorylated tau protein. Therefore, in order to measure both amyloid β protein and tau protein or phosphorylated tau protein, it is necessary to collect samples from two locations, which may be a burden on the patient.

International Publication No. 2011/092796 Japanese Patent No. 5850374

Nanjo T et al .: Development of a method to measure Aβ42 in nasal cavity and its application in normal human volunteers. Journal of Brain Science, 44: 5-23, 2014.

An object of the present invention is to provide a method for simultaneously measuring the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein using an intranasal sample obtained by one collection. Another object of the present invention is to provide a simultaneous measurement system for the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein using an intranasal sample obtained by one collection.

As a result of diligent research to achieve the above object, the present inventors have made an aggregate of amyloid β protein into a soluble amyloid β monomer by using guanidine hydrochloride as a solubilizer in an intranasal sample. It was found that tau protein and phosphorylated tau protein can be measured without being decomposed. As a result, it becomes possible to simultaneously measure the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein using the intranasal sample obtained by one collection.

The present invention has been completed by further studying based on these findings, and provides a method and a simultaneous measurement system for simultaneously measuring the amounts of the following amyloid β protein and tau protein and / or phosphorylated tau protein. It is a thing.

Item 1. A method for simultaneously measuring the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in an intranasal sample, which comprises the following steps (I) and (II):
(I) A step of eluting the intranasal sample collected from the nasal cavity into an extract containing a solubilizer and a surfactant, and
(II) A step of measuring the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in an intranasal sample from the extract obtained in step (I) by an immunological measurement method.
Item 2. Item 2. The method according to Item 1, wherein the solubilizer is guanidine or a salt thereof.
Item 3. Item 2. The method according to Item 1 or 2, wherein the surfactant is dodecyl maltoside.
Item 4. Item 4. The measuring method according to any one of Items 1 to 3, wherein the total protein amount is further measured in the step (II).
Item 5. In step (I), any one of Items 1 to 4, wherein the extract from which the intranasal sample is eluted is further subjected to at least one treatment selected from the group consisting of heat treatment, filter filtration and desalting. The method described in paragraph 1.
Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the intranasal sample is obtained by one collection.
Item 7. A means for eluting an intranasal sample collected from the nasal cavity into an extract containing a solubilizer and a surfactant,
In an intranasal sample comprising a means for measuring the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in the intranasal sample by an immunological measurement method from the extract obtained by the above means. Simultaneous measurement system for the amount of amyloid β protein and tau protein and / or phosphorylated tau protein.

According to the present invention, it is possible to simultaneously measure the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein using the intranasal sample obtained by one collection. Therefore, since the sample can be collected only once, the burden on the patient can be reduced in the diagnosis of a disease in which amyloid β protein and tau protein are accumulated, such as Alzheimer's disease.

3 is a graph showing the recovery rate of tau protein in Test Example 2 when a buffer containing no surfactant or various surfactants was used.

Hereinafter, the present invention will be described in detail.

It should be noted that, in the present specification, "comprise" also includes the meaning of "essentially consist of" and the meaning of "consist of".

The method for simultaneously measuring the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein in the intranasal sample of the present invention is characterized by including the following steps (I) and (II).
(I) Step of eluting the intranasal sample collected from the nasal cavity into an extract containing a solubilizer and a surfactant.
(II) A step of measuring the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in an intranasal sample from the extract obtained in step (I) by an immunological measurement method.

Unless otherwise specified, the "amyloid β protein" in the present invention refers to Aβ42 and Aβ40. Of particular importance among the amyloid β monomers are the amyloid β monomer (Aβ42) consisting of 42 amino acids and the amyloid β monomer (Aβ40) consisting of 40 amino acids. The formation of amyloid plaque, known as a characteristic phenomenon of Alzheimer's disease in brain tissue, is thought to be due to the cohesiveness and adsorptivity of Aβ42 and Aβ40. Currently, in actual clinical practice, Aβ42 and Aβ40 in cerebrospinal fluid are used as measurement indexes.

The intranasal sample is a nasal discharge or an intranasal mucosal scrape obtained by collecting nasal mucus by rubbing a collecting tool such as a cotton swab or a swab against the nasal mucosa of a test subject. The portion to be scraped is not particularly limited as long as nasal mucus can be collected, and examples thereof include olfactory fissure, superior nasal concha, middle nasal concha, inferior turbinate, and nasal septum.

The test subject in the present invention is a mammal (human, monkey, dog, cat, horse, cow, mouse, rat, etc.), and particularly a human.

The present invention is characterized in that the intranasal sample is obtained by one collection. Since only one collection is required in this way, the burden on the patient can be reduced.

The step (I) of the present invention is a step of eluting the intranasal sample collected from the nasal cavity into an extract containing a solubilizer and a surfactant.

In step (I), for example, a cotton swab or swab from which nasal mucus has been collected is immersed in the extract to elute the intranasal sample into the extract. Specifically, a cotton swab or swab itself from which nasal mucus has been collected is placed in a container such as a microtube containing an extract and stirred.

The solubilizer is used to disintegrate amyloid β protein aggregates eluted in the extract from the intranasal sample to form a soluble amyloid β monomer. By using a soluble amyloid β monomer, the amount of amyloid β protein can be accurately measured.

The solubilizer can be used without particular limitation as long as it can disintegrate amyloid β protein aggregates into a soluble amyloid β monomer. Of these, guanidine or a salt thereof is preferable, and guanidine hydrochloride is particularly preferable. By using guanidine or a salt thereof as a solubilizer, decomposition of tau protein and phosphorylated tau protein can be prevented. The solubilizer can be used alone or in combination of two or more. The concentration of the solubilizer in the extract is usually about 0.1 to 20 M, preferably about 1 to 10 M.

Surfactants are used to suppress the adsorption of proteins on the inner wall of the container and the adsorption of proteins on the nasal mucus. By using a surfactant, it becomes possible to measure the amounts of amyloid β protein, tau protein, and phosphorylated tau protein more accurately.

The surfactant can be used without particular limitation as long as it can suppress the adsorption of the protein on the inner wall of the container and the adsorption of the protein on the nasal mucus, and can be used as a cationic surfactant, an anionic surfactant, or both sexes. Both surfactants and nonionic surfactants can be used. Of these, nonionic surfactants are preferred, alkylglycosides, polyoxyethylene sorbitan fatty acid esters (such as Tween® 20), and octylphenol ethoxylates (such as Triton X-100) are more preferred, and dodecyl maltoside (n- Dodecyl-β-maltoside) is particularly preferred. The surfactant may be used alone or in combination of two or more. The concentration of the surfactant in the extract is usually about 0.001 to 10% by volume, preferably about 0.01 to 5% by volume.

The extract may contain additives other than solubilizers and surfactants as long as it does not inhibit the elution of amyloid β protein, tau protein and phosphorylated tau protein. Examples of such additives include buffers, metal salts and the like. Examples of the buffer include Tris-hydrochloric acid buffer, potassium phosphate buffer, acetate buffer, citric acid buffer, glycine-hydrochloric acid buffer, HEPES buffer, MES buffer and the like. Examples of the metal salt include potassium salt, sodium salt, magnesium salt and the like. The pH of the eluate is usually about 5-9. The solvent of the eluate is usually water.

In step (I), the extract from which the intranasal sample has been eluted can be further subjected to treatments such as heat treatment, filter filtration, desalting, and concentration.

The conditions of the heat treatment are not particularly limited as long as the proteins contained in the eluate can be solubilized, the heating temperature is usually 50 to 100 ° C., and the heating time is usually 1 to 60 minutes.

In filter filtration, impurities contained in the eluate are removed in order to prevent column clogging and the like. Examples of such a filter include those having a particle size of 0.2 μm or more or a molecular weight cut-off of 100,000 to 1,000,000.

Desalting involves removing the solubilizer and replacing the buffer. Desalting can be performed by known techniques, such as desalting columns, dialysis, ultrafiltration, gel filtration chromatography, precipitation / resuspension, etc., preferably desalting. It is a salt column.

At any point in step (I), it is desirable to divide the extract into two parts, one for measuring amyloid β protein and one for measuring tau protein and / or phosphorylated tau protein.

In step (II) of the present invention, the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in the intranasal sample is measured from the extract obtained in step (I) by an immunological measurement method. It is a process to do.

The immunological measurement method is a method using a binding substance such as an antibody that specifically binds to the peptide or protein to be measured, and can be carried out by using various known methods. Examples of immunological measurement methods include ELISA method, Western blotting method, radioimmunoassay, protein microarray and the like. When measuring by the ELISA method, a commercially available ELISA kit for measuring Aβ42, Aβ40, phosphorylated tau protein, tau protein and the like can be used.

In step (II), in addition to the above, the total protein amount can be further measured. The total protein amount can be measured by, for example, the BCA method, Bradford method, Lowry method, ultraviolet absorption method, pyrogallol red-molybdenum complex color development method, or the like.

The values of the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein obtained by the measuring method of the present invention can be used for diagnosing diseases in which amyloid β protein and tau protein are accumulated. For example, the risk of disease by comparing the values of the amounts of amyloid β protein, tau protein and phosphorylated tau protein, or the values obtained by performing various calculations using these values with a predetermined amount. Can be determined. Further, as described in Patent Document 2, the square root of the sum of the square of the amount of tau protein and the square of the amount of amyloid β protein is obtained, and this is compared with a predetermined threshold value. , Can determine the risk of disease.

Diseases in which amyloid β protein accumulates include Alzheimer's disease and Down's syndrome, and diseases in which tau protein accumulates include Alzheimer's disease, progressive supranuclear palsy (PSP), and cerebral cortex degeneration. Frontotemporal dementia, Pick disease, etc. can be mentioned.

The simultaneous measurement system for the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein in the intranasal sample of the present invention can be used.
A means for eluting an intranasal sample collected from the nasal cavity into an extract containing a solubilizer and a surfactant,
It is characterized by comprising a means for measuring the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in an intranasal sample by an immunoassay method from the extract obtained by the above means.

The system of the present invention is a system for realizing the above-mentioned method, and the intranasal sample, solubilizer, surfactant, extract, immunological measurement method and the like are the same as those described above.

As a means for eluting the above-mentioned intranasal sample into an extract containing a solubilizer and a surfactant, for example, a means for immersing a cotton swab or a swab from which nasal mucus has been collected in the extract can be mentioned. Is a means of putting a cotton swab or a swab itself from which nasal mucus is collected into a container such as a microtube containing an extract and stirring the nasal mucus.

As a means for measuring the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in the intranasal sample by the above immunoassay method, for example, an analyzer that carries out ELISA can be mentioned. ..

The system of the present invention may further include means for performing heat treatment, filter filtration, desalting, concentration, etc. on the extract in which the intranasal sample is eluted, means for measuring the total protein amount, and the like.

According to the method and system of the present invention, it is possible to simultaneously measure the amounts of amyloid β protein and tau protein and / or phosphorylated tau protein using an intranasal sample obtained from a single collection from a patient. .. Therefore, in the past, it was necessary to collect samples from two locations, but since it is only necessary to collect samples once, when diagnosing diseases such as Alzheimer's disease in which amyloid β protein and tau protein accumulate. , The burden on the patient can be reduced.

Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples and the like.

The concentration (%) of the surfactant in the following test examples indicates the volume%.

Test Example 1
With the approval of the Ethics Committee, 14 Alzheimer's (AD) cases and 17 healthy subjects were included. This was conducted for cases in which written consent was obtained by informed consent from among the examinees and patient caregivers of the Cranial Nerve Center, Shiga University of Medical Science Hospital. There was no significant difference in age and gender between the AD case and the normal elderly group. General blood tests showed no significant difference between the two groups.

<Method>
1) The nasal cavity (olfactory fissure, inferior turbinate) was scraped with a cotton swab.
2) In 100 μl of 50 mM Tris-hydrochloric acid buffer (pH 8.0) (abbreviated as guanidine solution in this test example) containing 200 mM NaCl, 8 M guanidine hydrochloride, 2 mM EDTA, and 0.05% dodecyl maltoside. I put a cotton swab and washed it well.
3) Heated at 70 ° C. for 10 minutes.
4) Filtered with a 0.2 μm PTFE filter.

About 100 μl of the filtrate was measured in the following two steps, A and B.

(Step A)
A-1. 45 μl of the filtrate was extrafiltered with a 300 K omega filter, and 90 μl of a buffer without guanidine (hereinafter abbreviated as substitution buffer) was added to wash the 300 K omega filter to obtain a total amount of 135 μl of the filtrate.
A-2. After filling the column with 1 ml of a G-10 column swollen with a substitution buffer, the swollen solution was centrifuged.
A-3. Guanidine hydrochloride in the sample was removed by adding 120 μl of the filtrate and 40 μl of the substitution buffer to the column in this order and collecting the gel filtrate by centrifugation (800 xg, 2 min).
A-4. Using this solution as a sample, Aβ42 and Aβ40 were measured and total protein was measured using a commercially available ELISA kit (Aβ42, Aβ40) and a total protein measurement kit. The reagents used are as follows.
・ Human / Rat βAmyloid (42) ELISA Kit, High Sensitive (Wako Pure Chemical Industries, Ltd., 292-64501)
・ Human / Rat βAmyloid (40) ELISA Kit II (Wako Pure Chemical Industries, Ltd., 294-64701)
・ Protein Assay Rapid Kit Wako (Wako Pure Chemical Industries, Ltd., 293-56101)

(B step)
B-1. After filling the column with 0.7 mL of G-10 column swollen with substitution buffer, the swollen solution was centrifuged.
B-2. Guanidine hydrochloride in the sample was removed by adding 40 μl of the filtrate and 10 μl of the substitution buffer to the column in this order and collecting the gel filtrate by centrifugation (800 xg, 2 min).
B-3. Using this solution as a sample, total tau / phosphorylated tau and total protein were measured using a commercially available ELISA kit (tTau / pTau) and a total protein measurement kit. The reagents used are as follows.
・ TAU (Total) Human ELISA Kit (Invitrogen, KHB0041)
・ TAU [pT181] Human ELISA Kit (Invitrogen, KHO0631)
・ Protein Assay Rapid Kit Wako (Wako Pure Chemical Industries, Ltd., 293-56101)

<Result>
The results are shown in Tables 1 and 2. In the olfactory cleft, the protein amount, Aβ42, Aβ40, tau protein, and phosphorylated tau protein could be measured simultaneously in 92.9% of AD cases and 94.1% of healthy cases. In the inferior turbinate, simultaneous measurement was possible in 100% of AD cases and 94.1% of healthy cases (Table 1). In the conventional method,
Simultaneous measurement of Aβ42 and tau protein (Test Example 3) and same measurement of tau protein and phosphorylated tau protein were not possible (Test Example 4), but according to the present invention, Aβ42, Aβ40, tau protein, and phosphorylated tau All simultaneous protein measurements are now possible.

The possibility of simultaneous measurement in this way has made it possible to compare biomarkers. For example, when the amount of phosphorylated tau protein was corrected by dividing it by the amount of tau protein, it showed a high tendency in the AD group in the olfactory fissure direction (p = 0.062, Table 2).

Test Example 2
Informed consent was given to cases with written consent.

<Method>
1) The nasal cavity was scraped with a cotton swab.
2) A cotton swab was placed in a buffer containing various surfactants and washed thoroughly. A certain amount of tau protein was added to the treatment, and the amount that could be recovered was examined.
The buffer is based on 50 mM Tris-hydrochloric acid buffer (pH 8.0) with 200 mM NaCl, 8 M guanidine hydrochloride, and 2 mM EDTA, with no surfactant added, or (A)-(E) below. ) Was added (in this test example, these solutions are referred to as guanidine solutions, and the solutions obtained by removing guanine hydrochloride from each solution are abbreviated as substitution buffer).
(A) 0.05% Tween-20
(B) 0.5% Tween-20
(C) 0.05% dodecyl maltoside
(D) 0.5% sodium deoxycholate
(E) 0.1% SDS
3) A tube containing a certain amount of tau protein in 100 μl of each solution was heated at 70 ° C. for 10 minutes.
4) Filtration was performed with a 0.2 μm PTFE filter.
5) 45 μl of the filtrate was extrafiltered with a 300 K omega filter, and 90 μl of a substitution buffer excluding guanidine was added to wash the 300 K omega filter to obtain a total amount of 135 μl of the filtrate.
6) After filling the column with 1 ml of a G-10 column swollen with a substitution buffer, the swollen solution was centrifuged.
7) 120 μl of the filtrate and 40 μl of the substitution buffer were added to the column in this order, and the gel filtrate was collected by centrifugation (800 xg, 2 min) to remove the guanidine hydrochloride in the sample.
8) 10 μl of the desalted solution was taken and the total protein amount was measured, and the total tau protein was measured with the remaining 150 μl. The TAU (Total) Human ELISA Kit was used to measure total tau protein, and the Protein Assay Rapid Kit Wako was used to measure total protein content.

<Result>
The results are shown in FIG. It was 0.05% dodecyl maltoside that was able to recover all of the added tau protein. With no additives, 0.5% Tween-20 was able to recover about 90%, 0.05% Tween-20 was able to recover about 80% tau protein, but 0.5% sodium deoxycholate and 0.1% SDS were not able to recover tau protein. It was.

Test Example 3
Simultaneous measurement of amyloid β protein and phosphorylated tau protein was attempted by the conventional method. It was conducted for 5 healthy subjects.

In this test example, the nasal mucosal tissue of the inferior turbinate was scraped with a cotton swab, and the cotton ball to which the nasal mucosal tissue was attached was put into a microtube to which 270 μl of ultrapure water was previously added. Then, it was cut at a position 3.0 cm from the tip of the cotton ball and stirred to elute the nasal mucosal tissue sample attached to the cotton swab. In this test example, a commercially available plastic swab (Cotton swab for medical assistance HUBY-COTIX EM3-50S (Yamayo Co., Ltd.)) was used.

Next, the cotton swab was pulled up to float the cotton ball from the ultrapure water, fixed to the microtube with a lid, and centrifuged at 14000 g to dehydrate the cotton ball. Then, the cotton swab was taken out from the microtube, and the solution in the microtube was repeatedly sucked and discharged 30 times with a syringe equipped with a 27G needle to suspend it. Of these, 10 μl was used to measure the total protein amount, and 60 μl was used to quantify tau protein. The TAU (Total) Human ELISA Kit was used to measure total tau protein, and the protein assay Rapid Kit Wako was used to measure total protein.

The remaining 200 μl of suspension was used to measure Aβ42 as follows.

200 μl of the suspended solution was transferred to a test tube, 500 μl of 100% formic acid was added as an extract, the mixture was stirred, and the mixture was allowed to stand in a constant temperature layer maintained at 70 ° C. for 1 hour. After 1 hour, the test tube was removed from the homeothermic layer, and the solution was fractionated and filtered through a filter (NANOSEP 100K OMEGA (PALL): a filter that excludes proteins of 100 kDa or more and allows only proteins having a molecular weight of 100 kDa or less to pass through). The filtrate was transferred to a test tube and concentrated under reduced pressure with a centrifugal evaporator until the solution volume reached 20 μl. After stirring the concentrated solution, 480 μl of 1 mol / l Tris (unadjusted pH) was added to prepare a 500 μl solution. The pH of each solution was measured and the success or failure of neutralization was recorded on a recording sheet, and it was confirmed that the pH was in the range of 7.0 to 8.6. These solutions were used as samples for measuring amyloid β protein.

Measurement of amyloid β protein was performed using an ELISA measurement kit for measuring amyloid β protein (Human / Rat βAmyloid (42) ELISA Kit, High-Sensitive). Prior to measurement, the solution for preparing the calibration curve was prepared to 0.05, 0.1, 0.2, 0.25, 1, 2, 2.5, 5, 10, or 20 pM using a standard solution of Aβ42. After preparation, 100 μl of each of the solution for preparing the calibration curve and 1 ml of the measurement sample was added to the microtiter plate of the measurement kit.

Table 3 shows the measurement results of Aβ42 and total tau protein. Aβ42 could be measured, but total tau protein could not be measured. The total protein amount was measurable.

Simultaneous measurement of total tau protein and phosphorylated tau protein was attempted by the conventional method.

With the approval of the Ethics Committee, the procedure was conducted in 6 patients with Alzheimer's disease (71-84 years, 3 males, 3 females) and 1 healthy subject (72 years, male).

From the nasal fissure, middle nasal meatus, inferior turbinate, and total nasal passage, scrape the nasal mucosal tissue with a cotton swab, and put the cotton ball with the nasal mucosal tissue into a microtube to which 270 μl of ultrapure water has been added in advance. I put it in. Then, it was cut at a position 3.0 cm from the tip of the cotton ball and stirred to elute the nasal mucosal tissue sample attached to the cotton swab.

Next, the cotton swab was pulled up to float the cotton ball from the ultrapure water, fixed to the microtube with a lid, and centrifuged at 14000 g to dehydrate the cotton ball. Then, the cotton swab was taken out from the microtube, and the solution in the microtube was repeatedly sucked and discharged 30 times with a syringe equipped with a 27G needle to suspend it. After this suspension was divided into halves, total tau protein and phosphorylated tau protein were measured. The TAU (Total) Human ELISA Kit was used to measure total tau protein, and the Tau [pT181] Human ELISA Kit was used to measure phosphorylated tau.

As a result, total tau protein could be measured, but phosphorylated tau protein could not be detected in all cases. That is, in the conventional method, total tau protein and phosphorylated tau protein could not be measured simultaneously using the same nasal sample.

Claims (5)

A method for simultaneously measuring the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in an intranasal sample, which comprises the following steps (I) and (II):
(I) A step of eluting the intranasal sample obtained by one collection from the nasal cavity into an extract containing a solubilizer and dodecyl maltoside, and
(II) A step of measuring the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in an intranasal sample from the extract obtained in step (I) by an immunological measurement method. The method according to claim 1, wherein the solubilizer is guanidine or a salt thereof. The measuring method according to claim 1 or 2 , further measuring the total protein amount in the step (II). Any of claims 1 to 3 , wherein in step (I), the extract from which the intranasal sample is eluted is further subjected to at least one treatment selected from the group consisting of heat treatment, filter filtration and desalting. The method described in item 1. A means for eluting the intranasal sample obtained by one collection from the nasal cavity into an extract containing a solubilizer and dodecyl maltoside, and
In an intranasal sample comprising a means for measuring the amount of amyloid β protein and tau protein and / or phosphorylated tau protein in the intranasal sample by an immunological measurement method from the extract obtained by the above means. Simultaneous measurement system for the amount of amyloid β protein and tau protein and / or phosphorylated tau protein.

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