Mild cognitive impairment test

Abstract

Provided is a means for detecting, or for providing assistance for detecting, a subject suffering from mild cognitive impairment. A biomarker for use in a test for mild cognitive impairment, the biomarker comprising claudin-5.

Description

[Technical Field]

[0001] Techniques relating to the testing or diagnosis of mild cognitive impairment are disclosed. [Background technology]

[0002] Japan is currently experiencing rapid population decline and an aging population with a declining birthrate. The aging rate is expected to continue rising even after the number of elderly people reaches its peak in 2042, and it is estimated that by 2065, one in 2.6 people will be 65 or older. Meanwhile, while the average life expectancy is 81 years for men and 87 years for women, the healthy life expectancy, which means people can live independently without needing nursing care, is 72 years for men and 75 years for women, resulting in a 10-year gap between average life expectancy and healthy life expectancy. Therefore, in order to maintain a sustainable society in the coming super-aging society, the most important issue is to extend healthy life expectancy so that people can live healthy lives without needing nursing care.

[0003] Dementia is one of the triggers for the need for caregiving. It is estimated that one in seven elderly people suffers from dementia, with the number exceeding seven million by 2025. Furthermore, the number of dementia patients worldwide is estimated to exceed 50 million and reach 150 million by 2050. Dementia countermeasures were a major topic of discussion at the 2013 G8 Summit in London, and dementia countermeasures have been promoted on a global scale. While drugs that slow cognitive decline are already on the market, and early intervention could potentially slow the progression of dementia and extend healthy lifespan, the development of highly reproducible, objective biomarkers that facilitate early intervention remains a major obstacle to treatment development. As a result, the contribution of drugs and treatment satisfaction for dementia remain among the lowest of all diseases, and the number of patients continues to increase.

[0004] Dementia develops over a period of more than 30 years, progressing from the preclinical stage to mild cognitive impairment (MCI). Current dementia treatments target dementia patients and involve drugs that activate neurons in the cerebral cortex and hippocampus, which control cognitive function. However, neurodegeneration progresses during dementia, and insufficient neurons remain. Once neurodegeneration has progressed, dementia medications are no longer effective. On the other hand, neurodegeneration is not yet advanced in the pre-symptomatic stage. It is believed that administering therapeutic drugs to pre-symptomatic individuals can slow cognitive decline and delay or prevent the onset of dementia. Therefore, the most important strategy for dementia prevention is to identify pre-symptomatic dementia patients at the pre-symptomatic stage and intervene early for prevention and treatment. However, objective biomarkers for detecting pre-symptomatic dementia patients have yet to be developed. Furthermore, because cognitive decline is assessed by questionnaires, patients diagnosed with dementia already have advanced neurodegeneration, making effective prevention and treatment less likely. Therefore, a method for identifying pre-symptomatic individuals (pre-clinical stage / MCI) is needed for dementia prevention and treatment.

[0005] Alzheimer's disease (AD), which accounts for approximately 70% of dementia cases, is known to involve the frequent occurrence of senile plaques, which are aggregates of amyloid and tau proteins, in the brain, and the breakdown of the blood-brain barrier (BBB), allowing albumin to enter the brain (Non-Patent Document 1). AD progresses in the following order: accumulation of amyloid and tau proteins in the brain, brain atrophy, and cognitive decline. Because the deposition of amyloid and tau proteins in the brain precedes cognitive decline, efforts have been made to develop dementia biomarkers using senile plaques as indicators. However, although analytical techniques for amyloid and tau proteins in cerebrospinal fluid (CSF) have been established, collection of CSF is highly invasive and generally requires hospitalization (2-3 hours of rest after collection). Furthermore, side effects such as headache, nausea, and vomiting are common, as well as the risk of blood contamination in the sample and the difficulty of lumbar puncture in elderly patients due to lumbar degenerative disease. These issues pose challenges in terms of convenience and versatility. Therefore, it is difficult to put this technology into practical use as a screening technique for people with MCI. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2018 / 207638 [Patent Document 2] WO2018 / 105560 [Non-patent literature]

[0007] [Non-Patent Document 1] Zenaro, E. et al., The blood-brain barrier in Alzheimer's disease. Neurobiol Dis 2017, 107, 41-56, doi:10.1016 / j.nbd.2016.07.007. [Non-patent document 2] Hashimoto, et al., Claudin-5-Binders Enhance Permeation of Solutes across the Blood-Brain Barrier in a Mammalian Model. J Pharmacol Exp Ther 2017, 363, 275-283 [Non-patent document 3] Hashimoto, et al., Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5. Sci Rep 2018, 8, 8383 Summary of the Invention [Problem to be solved by the invention]

[0008] Under these circumstances, one challenge is to provide a means for detecting or assisting in the detection of subjects suffering from mild cognitive impairment. [Means for solving the problem]

[0009] Section 1. A biomarker for testing mild cognitive impairment consisting of claudin 5. Section 2. Item 1. The biomarker according to Item 1, wherein the mild cognitive impairment test comprises distinguishing mild cognitive impairment from a group of diseases consisting of Parkinson's disease, multiple sclerosis, Alzheimer's disease, obsessive-compulsive disorder, and bipolar disorder. Section 3. A method for testing a subject for mild cognitive impairment using claudin 5 in a body fluid collected from the subject as an indicator. Section 4. Item 4. The method according to item 3, wherein the test comprises distinguishing mild cognitive impairment from a group of diseases consisting of Parkinson's disease, multiple sclerosis, Alzheimer's disease, obsessive-compulsive disorder, and bipolar disorder. Section 5. Item 5. The method according to Item 3 or 4, comprising measuring the amount of claudin 5 in a body fluid collected from the subject. Section 6. Item 6. The method according to item 5, wherein the amount of claudin 5 in the body fluid is measured using an anti-claudin 5 monoclonal antibody. Section 7. Item 7. The method according to any one of Items 3 to 6, comprising comparing the amount of claudin 5 in the body fluid of the subject with the amount of claudin 5 in the body fluid of a healthy subject. Section 8. Item 8. The method according to any one of Items 3 to 7, wherein the subject is a subject suspected of having cognitive impairment. Section 9. Item 9. The method according to any one of Items 3 to 8 (excluding medical procedures), further characterized by combining the method with one or more diagnoses selected from the group consisting of a medical interview, brain image analysis, a phosphorylated tau test, an amyloid-β test, an electrophysiological test, a cerebrospinal fluid test, a urine test, a blood test, a chest X-ray, a cerebral blood flow SPECT test, a cerebral glucose metabolism PET test, and a genetic test. Section 10. A reagent containing an anti-claudin-5 antibody for carrying out the method according to any one of Items 3 to 9. Section 11. A step of mixing an anti-claudin 5 antibody with a body fluid collected from a subject to prepare a sample, and a step of detecting a substance bound to the anti-claudin 5 antibody in the sample. A method for obtaining information about cognitive impairment of a subject using a substance in the sample as an index, comprising: Section 12. A reagent comprising an anti-claudin-5 antibody for carrying out the method according to item 11. [Effects of the Invention]

[0010] It makes it possible to detect (or assist in detecting) subjects suffering from mild cognitive impairment. [Brief explanation of the drawings]

[0011] [Figure 1] 1 shows the results of detecting claudin-5 micelles using a monoclonal antibody that recognizes the extracellular domain of claudin-5. [Figure 2] The results of investigating the specificity of claudin-5 detection using an enzyme immunoassay using a monoclonal antibody that recognizes the extracellular domain of claudin-5 are shown. [Figure 3] The results show that claudin-5 in human biological samples can be quantitatively measured by enzyme immunoassay using a monoclonal antibody that recognizes the extracellular domain of claudin-5. DETAILED DESCRIPTION OF THE INVENTION

[0012] Claudin 5 is an expression product of the claudin 5 (also called claudin-5, CLDN-5, Cldn-5, CLDN5, Cldn5, etc.) gene and is a protein expressed in living organisms. The amino acid sequences of CLDN-5 proteins derived from various biological species are known.

[0013] As shown in the Examples below, the amount of claudin 5 in the blood of subjects suffering from mild cognitive impairment is significantly higher than that in healthy individuals or subjects suffering from other neurodegenerative diseases (e.g., Alzheimer's disease, multiple sclerosis, Parkinson's disease) or psychiatric disorders (obsessive-compulsive disorder, bipolar disorder), and is specifically detected in subjects with mild cognitive impairment. Therefore, claudin 5 in body fluids can be used as a biomarker for detecting subjects suffering from mild cognitive impairment. Furthermore, by using this marker, it is possible to identify (or diagnose or determine) that a subject is suffering from mild cognitive impairment rather than the above-mentioned neurodegenerative disease or psychiatric disorder.

[0014] The type of body fluid is not limited, but is preferably blood or cerebrospinal fluid. The type of blood is not limited, but is preferably peripheral blood. The site from which peripheral blood is collected is not limited, and examples include the arm, leg, and neck. In one embodiment, the blood is preferably blood collected from the forearm. The blood may be whole blood, plasma, or serum, preferably plasma or serum, and more preferably serum.

[0015] The type of subject is not particularly limited, and examples include animals including humans and non-human mammals (e.g., dogs, cats, monkeys, chimpanzees, gorillas, horses, cows, sheep, etc.), with humans being the preferred subject.

[0016] In one embodiment, the subject is preferably a subject suspected of having cognitive impairment. A subject suspected of having cognitive impairment may be, for example, a subject who is subjectively or objectively suspected of having a decline from a previous level of performance in one or more cognitive domains (complex attention, executive function, learning and memory, language, perceptual-motor, social cognition). In one embodiment, the subject is preferably of a certain age or older (e.g., 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, or 75 years or older).

[0017] Mild cognitive impairment is a condition that is neither dementia nor normal cognitive function. For example, mild cognitive impairment is characterized by the following: (1) subjective or objective complaints of cognitive decline; (2) cognitive decline but not meeting the diagnostic criteria for dementia; and (3) normal basic daily function and independent living. According to the diagnostic criteria of the National Institute of Aging-Alzheimer's Association workgroup (NIA-AA), mild cognitive impairment is characterized by the following (A) to (D): (A) cognitive decline compared to previous years, which can be noted by the individual, informants, or clinicians; (B) impairment in two or more cognitive domains: memory, executive function, attention, language, and visuospatial cognition; (C) independent daily activities, even if they take longer, are less efficient, or result in more errors than before; and (D) not dementia. Mild cognitive impairment is classified into amnestic MCI and non-amnestic MCI depending on whether or not memory impairment is present, and amnestic MCI is known to be more likely to progress to Alzheimer's disease (AD) dementia.

[0018] As shown in the Examples below, claudin-5 is not detected in the blood of subjects suffering from Parkinson's disease, multiple sclerosis, Alzheimer's disease, obsessive-compulsive disorder, or bipolar disorder, but is detected in the blood of subjects with mild cognitive impairment. Therefore, using claudin-5 in the blood as an indicator, it is possible to identify (or diagnose or determine) that a subject has mild cognitive impairment, rather than Parkinson's disease, multiple sclerosis, Alzheimer's disease, obsessive-compulsive disorder, or bipolar disorder (or to assist in the identification or diagnosis).

[0019] In one embodiment, the mild cognitive impairment test refers to an aid in identifying (or diagnosing, determining) whether a subject has mild cognitive impairment. In one embodiment, the mild cognitive impairment test refers to an aid in identifying (or diagnosing, determining) whether a subject has mild dementia based on blood claudin-5 measured for the subject.

[0020] In one embodiment, a method for testing a subject for mild cognitive impairment is provided, using claudin-5 in the subject's blood as an indicator. This method can be used to assist in identifying (or diagnosing or determining) whether a subject has mild cognitive impairment. The testing method can include collecting a body fluid from the subject and / or measuring the amount of claudin-5 in the body fluid collected from the subject. The method for collecting the body fluid from the subject and the method for measuring the amount of claudin-5 in the body fluid can be any method.

[0021] The method for measuring blood claudin-5 is not particularly limited, and can employ, for example, a measurement method using an immunological technique, high-performance liquid chromatography, or liquid chromatography-tandem mass spectrometry. In one embodiment, blood claudin-5 is preferably measured by an immunological measurement method using an anti-claudin-5 antibody. The anti-claudin-5 antibody used to measure blood claudin-5 is preferably a monoclonal antibody, and preferably a monoclonal antibody that recognizes the extracellular domain of claudin-5. Examples of such monoclonal antibodies include the monoclonal antibodies disclosed in Patent Documents 1 and 2, and Non-Patent Documents 2 and 3. Any immunological measurement method can be used.

[0022] The immunological assay is not particularly limited, and examples thereof include enzyme immunoassay, chemiluminescence immunoassay, fluorescence immunoassay, radioimmunoassay, immunoturbidimetry, immunoprecipitation, Western blotting, affinity chromatography, etc. Among these, the enzyme immunoassay ELISA, particularly the sandwich ELISA, is preferred.

[0023] A method for testing for mild cognitive impairment can include comparing the amount of claudin-5 in the blood of a subject with the amount of claudin-5 in the blood of a healthy subject. A healthy subject refers to an animal (preferably a human) of the same species as the subject that has no medical history of cognitive dysfunction and no objective or subjective suspicion of cognitive dysfunction. A method for testing for mild cognitive impairment can suggest that the subject is likely to have mild cognitive impairment when the amount of claudin-5 measured in the blood of the subject is significantly higher than the amount of claudin-5 in the blood of a healthy subject.

[0024] The method for testing for mild cognitive impairment involves presetting a cutoff value for the amount of claudin-5 in blood, and using this as a criterion to suggest whether a subject is likely to have mild cognitive impairment, or to assist in identifying (or diagnosing or determining) whether a subject has mild cognitive impairment. The cutoff value can be determined, for example, based on the amount of claudin-5 in blood measured in subjects with mild cognitive impairment and the amount of claudin-5 in blood measured in control subjects (which may include dementia patients) without mild cognitive impairment. The cutoff value can be set by creating an ROC curve.

[0025] In one embodiment, the method for testing for mild cognitive impairment may be a method for determining whether a subject has a high probability of having mild cognitive impairment or for assisting in identifying (or diagnosing or determining) that the subject has mild cognitive impairment when the subject's blood claudin-5 concentration is 0.001 ng / ml or higher. The blood claudin-5 concentration may be 0.005 ng / ml or higher, 0.01 ng / ml or higher, 0.05 ng / ml or higher, or 0.1 ng / ml or higher.

[0026] The method for testing for mild cognitive impairment can be combined with other tests for mild cognitive impairment. This can improve the accuracy of identification (or diagnosis, judgment). Examples of other tests include interviews, brain image analysis, phosphorylated tau tests, amyloid beta tests, electrophysiological tests, cerebrospinal fluid tests, urine tests, blood tests, chest X-rays, cerebral blood flow SPECT tests, cerebral glucose metabolism PET tests, and genetic tests. One or more of these tests can be combined with the method for testing for mild cognitive impairment described above.

[0027] The interview included cognitive function tests such as the Hasegawa's Dementia Scale-Revised (HDS-R), Mini-Cog, Montreal Cognitive Assessment (MoCA), Dementia Assessment Sheet for Community-based Integrated Care System-21 items (DASC-21), Mini-Mental State Examination (MMSE), and ABC-DS (ABC dementia scale).

[0028] In one embodiment, a reagent is provided for assisting in identifying (or diagnosing or determining) whether a subject has mild cognitive impairment using the above-described method. The reagent may include a reagent for measuring claudin-5 in the blood (e.g., an anti-claudin-5 antibody). In addition, the reagent may include a buffer solution or any other component (e.g., a biotin-conjugated antibody, a streptavidin-conjugated particle, an enzyme-labeled antibody, an enzyme substrate, a chemiluminescent substrate, etc.).

[0029] In one embodiment, a method for obtaining information about a cognitive impairment of a subject using a substance in the sample as an indicator is provided, the method comprising the steps of mixing an anti-claudin-5 antibody with a body fluid collected from the subject to prepare a sample, and detecting a substance in the sample to which the anti-claudin-5 antibody binds. In this method, the subject, body fluid, and anti-claudin-5 antibody described above can be used. The substance to which the anti-claudin-5 antibody binds can be a complex of the anti-claudin-5 antibody and the substance to which it binds, and examples of substances to which the anti-claudin antibody binds include claudin-5, fragments thereof, and exosomes containing them.

[0030] The information obtained by the above method is not particularly limited, and may include, for example, information on whether claudin 5 is detected in a body fluid or sample, information on the amount of a complex between an anti-claudin 5 antibody and a substance to which it binds, information on the amount of claudin 5 (including its fragments) contained in the complex, etc. Based on such information, it is possible to identify (or diagnose or determine) (or assist in) the cognitive impairment of a subject.

[0031] The anti-claudin-5 antibody used in the above method may be used alone, or, if necessary, may be in the form of a reagent combined with a buffer solution or any other component (e.g., biotin-conjugated antibody, streptavidin-conjugated particles, enzyme-labeled antibody, enzyme substrate, chemiluminescent substrate, etc.). [Example]

[0032] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0033] 1. Development of immunoassay using anti-claudin-5 antibody To detect claudin-5 in the blood, we decided to construct a quantitative system using the sandwich ELISA method of enzyme immunoassay, taking into consideration versatility and quantitative performance. As a model for claudin-5 in the blood, we decided to use claudin-5 micelles, in which claudin-5 is expressed on the membrane of liposomes using an improved wheat germ cell-free synthesis system that we developed ourselves (Non-Patent Document 3). Furthermore, as an antibody for detecting claudin-5, we used an antibody that we developed ourselves that recognizes the extracellular domain of claudin-5 (Patent Documents 1 and 2). A 96-well ELISA plate (Nunc Maxisorp) was used. TMTo each well of a flat-bottom plate (ThermoFisher, 442404), 100 μL of a solution containing a capture antibody against claudin-5 (R9 antibody from Patent Document 1, 2B12 antibody from Patent Document 2) adjusted to 5 μg / mL was added and allowed to react at 25°C for 1 hour to immobilize the capture antibody. After removing the reaction solution, 300 μL of a blocking agent (Blocking One (Nacalai, 03953-95)) was added and allowed to react overnight at 4°C. After removing the blocking agent, the plate was washed three times with 300 μL of washing solution 1 (TBS-0.05% Tween 20-1 mM DTT). After washing, 100 μL of reaction solution 1 (0.5% BSA-TBS-1 mM DTT) prepared to a concentration of 10 ng / mL claudin-5 micelles or 100 ng / mL claudin-1–claudin-6 micelles was added and incubated at 25°C for 1.5 hours. After removing the reaction solution, the plate was washed three times with 200 μL of washing solution 1 and twice with 200 μL of washing solution 2 (TBS-1 mM DTT). Next, 50 μL of reaction solution 1, prepared to a concentration of 0.5 μg / mL of detection antibodies against claudin-5 (ab53765, EPR7583 (Abcam), SAB4502981 (Sigma-Aldrich)), was added and incubated at 25°C for 1 hour. After removing the reaction solution, the plate was washed five times with 200 μL of washing solution 2. Next, 50 μL of reaction solution 1, prepared with a biotin-labeled secondary antibody (Biotin-SP-AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson, 711-065-152)) at a concentration of 50 ng / mL, was added, and the mixture was incubated at 25°C for 1 hour. After removing the reaction solution, the mixture was washed five times with 200 μL of washing solution 2. After washing, 50 μL of reaction solution 1, prepared with HRP-labeled streptavidin (Pierce High Sensitivity Streptavidin-HRP (ThermoFisher, 21130)) at a concentration of 50 ng / mL, was added, and the mixture was incubated at 25°C for 30 minutes. After removing the reaction solution, the mixture was washed five times with 200 μL of washing solution 3 (TBS).Finally, 100 μL of substrate (ABTS peroxidase substrate system (KPL, 50-62-00)) was added and the mixture was incubated at 25°C for 10 to 60 minutes, after which the absorbance at a wavelength of 405 nm was measured using a plate reader TriStar LB941 (Berthold Technologies).

[0034] The R9 antibody used above has a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6. The 2B12 antibody used above has a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 9, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 12.

[0035] As a result, we confirmed that 10 ng / mL claudin-5 micelles could be detected using either the monoclonal antibodies R9 or 2B12, which recognize the extracellular domain of claudin-5, as the capture antibody, in combination with the polyclonal antibody SAB4502981, monoclonal antibody EPR7583, or polyclonal antibody ab53765 (epitope unknown), which recognize the intracellular domain, as the detection antibody (Figure 1). In subsequent studies, we used monoclonal antibodies (R9 or 2B12 as the capture antibody and EPR7583 as the detection antibody), which are thought to have less lot-to-lot variation.

[0036] To evaluate the specificity of the ELISA method for claudin-5, we performed an ELISA test using a reaction solution containing any of claudin-1 to claudin-6 as a sample. Regardless of the capture antibody used, a signal was observed only when claudin-5 micelles were used (Figure 2). In other words, it was confirmed that the immunoassay method we developed using the antigen-antibody reaction can specifically detect claudin-5.

[0037] 2. Analysis of claudin-5 in clinical samples using immunoassay Next, the amount of claudin-5 in serum from patients with various central nervous system diseases was measured using commercially available clinical specimens. For the calibration curve, pooled serum from healthy individuals was added with claudin-5 micelles to a final concentration of 0.03125-8 ng / mL. A 96-well ELISA plate (Nunc Maxisorp) was used. TMTo each well of a flat-bottom plate (ThermoFisher, 439454), 100 μL of a solution containing a 5 μg / mL capture antibody (R9) against claudin-5 was added and incubated at 25°C for 1 hour to immobilize the capture antibody. After removing the reaction solution, 300 μL of blocking agent (Blocking One) was added and incubated overnight at 4°C. After removing the blocking agent, the plate was washed three times with 300 μL of washing solution 1 (TBS-0.05% Tween 20-1 mM DTT). After washing, 250 μL of a sample prepared by adding 0.0078125–2 ng of claudin-5 micelles to pooled human serum from healthy volunteers (BioIVT) or a sample prepared by diluting human serum samples from various disease patients (BioIVT, ProteoGenex) to 10% with Reaction Solution 1 (0.5% BSA-TBS-1 mM DTT) was added and incubated for 1.5 hours with shaking at 25°C. After removing the reaction solution, the plate was washed three times with 300 μL of Wash Solution 1 and twice with 300 μL of Wash Solution 2 (TBS-1 mM DTT). Next, 50 μL of Reaction Solution 1, prepared with a detection antibody against claudin-5 (EPR7583) at 0.5 μg / mL, was added and incubated for 1 hour with shaking at 25°C. After removing the reaction solution, the plate was washed five times with 200 μL of Wash Solution 2. Next, 50 μL of reaction solution 1, which contained a biotin-labeled secondary antibody (Biotin-SP-AffiniPure Donkey Anti-Rabbit IgG (H+L)) adjusted to 50 ng / mL, was added, and the reaction was carried out at 25°C for 1 hour with shaking. After removing the reaction solution, the plate was washed five times with 200 μL of washing solution 2. After washing, 50 μL of reaction solution 1, which contained HRP-labeled streptavidin (Pierce High Sensitivity Streptavidin-HRP) adjusted to 50 ng / mL, was added, and the reaction was carried out at 25°C for 30 minutes with shaking. After removing the reaction solution, the plate was washed five times with 200 μL of TBS. Finally, 100 μL of substrate (TMB Substrate Kit (ThermoFisher, 34021)) was added and the mixture was incubated at 25°C for 30 minutes. The reaction was stopped by adding 100 μL of 10% sulfuric acid (Nacalai, 13089-25), and the absorbance at 450 nm was measured using a TriStar LB941 plate reader.Based on the absorbance obtained from samples containing claudin-5 micelles, a calibration curve was created using a four-parameter logistic model, and the amount of claudin-5 in the serum from patients with each disease was calculated.

[0038] The above immunoassay method was also able to detect claudin-5 in human biological samples, and it was confirmed that claudin-5 could be quantified in the range of 0.0625–4 ng / mL (Figure 3). Furthermore, claudin-5 levels were measured in serum samples from healthy individuals (Normal, n = 4), patients with Parkinson's disease (PD, n = 2), multiple sclerosis (MS, n = 2), Alzheimer's disease (AD, n = 7), mild cognitive impairment (MCI, n = 4), obsessive-compulsive disorder (OCD, n = 1), and bipolar disorder (BD, n = 2). Interestingly, as shown in Table 1 below, claudin-5 was detected in four out of four serum samples from MCI patients, with concentrations ranging from 0.886–1.520 ng / mL. In contrast, claudin-5 levels were below the detection limit in other clinical samples from healthy individuals or patients with other disorders.

[0039] [Table 1]

[0040] As described above, the immunoassay system we developed can specifically detect claudin-5, and claudin-5 was detected in the blood only in patients with MCI, which is before the onset of dementia. These results indicate that the presence or amount of claudin-5 in body fluids, especially in the blood, can be an indicator of MCI, which is a pre-disease stage. It is interesting that blood claudin-5 showed a significant correlation only with mild cognitive impairment, not with central nervous system or psychiatric disorders. This suggests that claudin-5 in body fluids, especially in the blood, is a specific biomarker for mild cognitive impairment and an effective means of identifying subjects who are likely to develop dementia in the future.

Claims

1. A biomarker for testing mild cognitive impairment consisting of claudin 5, Claudin 5 is claudin 5 in the blood, a biomarker.

2. 2. The biomarker of claim 1, wherein the mild cognitive impairment test comprises distinguishing mild cognitive impairment from a group of diseases consisting of Parkinson's disease, multiple sclerosis, Alzheimer's disease, obsessive-compulsive disorder, and bipolar disorder.

3. A method for assisting in testing a subject for mild cognitive impairment using claudin 5 in blood collected from the subject as an indicator, comprising: A method in which a subject is indicated to have mild cognitive impairment if the subject's blood level of claudin 5 is significantly higher than the blood level of claudin 5 in a healthy subject.

4. 4. The method of claim 3, wherein the testing comprises distinguishing mild cognitive impairment from a group of diseases consisting of Parkinson's disease, multiple sclerosis, Alzheimer's disease, obsessive-compulsive disorder, and bipolar disorder.

5. The method of claim 3 or 4, comprising measuring the amount of claudin 5 in a body fluid collected from the subject.

6. The method of claim 5, wherein the amount of claudin 5 in the body fluid is measured using an anti-claudin 5 monoclonal antibody.

7. The method according to any one of claims 3 to 6, wherein the subject is a subject suspected of having cognitive impairment.

8. The method according to any one of claims 3 to 7, further characterized by being combined with one or more diagnoses selected from the group consisting of a medical interview, brain image analysis, a phosphorylated tau test, an amyloid beta test, an electrophysiological test, a cerebrospinal fluid test, a urine test, a blood test, a chest X-ray test, a cerebral blood flow SPECT test, a cerebral glucose metabolism PET test, and a genetic test.

9. A reagent comprising an anti-claudin-5 antibody for carrying out the method according to any one of claims 3 to 8.

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