Biomarker composition for diagnosis of degenerative brain disease

Abstract

The present invention relates to a biomarker composition for the diagnosis of degenerative brain diseases. Specifically, to distinguish between normal individuals, patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD), the levels of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), Tau, NSE (neuron-specific enolase), GFAP (glial fibrillary acidic protein), UCH-L1 (ubiquitin carboxy-terminal hydrolase L1), PIN1 (peptidyl-prolyl isomerase), CRMP2 (collapsing response mediator protein-2), Phospho-Tau231, and OMP (olfactory marker protein) are measured as blood biomarkers, and by identifying their phylogenetic relationships, normal individuals, patients with mild cognitive impairment (MCI), and The purpose is to provide a biomarker composition for distinguishing Alzheimer's (AD) patients.

Description

Technology Field

[0001] The present invention relates to a biomarker composition for the diagnosis of degenerative brain diseases. Background Technology

[0002] Degenerative brain diseases refer to diseases that occur in the brain among degenerative diseases that occur with aging. They can be classified based on major symptoms and the affected brain regions, and representative examples include Alzheimer's disease, frontotemporal dementia, Lewy body dementia, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Degenerative brain diseases are known to be caused by the aggregation of specific proteins due to age-related neurodegeneration and genetic and environmental factors, leading to the death of nerve cells. However, as the exact pathological mechanisms have not yet been elucidated, basic research to clarify them is actively underway.

[0003] Alzheimer's disease begins with a gradual decline in memory and progresses to a progressive and widespread loss of cognitive functions, such as loss of visuospatial ability, language impairment, and executive function, resulting in dementia symptoms that make it impossible to perform independent daily activities. Although the exact pathological mechanism has not yet been precisely identified, it is reported to be caused by the interaction between aging, genetic risk factors, and environmental factors.

[0004] According to an analysis by dementia-related agencies including the Ministry of Health and Welfare, the National Health Insurance Service, the Health Insurance Review and Assessment Service, the National Social Security Information Service, and Statistics Korea, the number of dementia patients was estimated to be over 700,000 as of the end of 2017. The annual management cost per patient is approximately 20.74 million won, and the national dementia management cost amounts to about 14.6 trillion won. Currently, the prevention, diagnosis, and management of dementia rely on questionnaires such as clinical interviews or expensive screenings like PET-CT scans. Furthermore, since biological samples such as cerebrospinal fluid are required for diagnosis and management after the onset of the disease, the suffering experienced by patients is compounded by the burden of management costs. Moreover, with the aging society, the number of patients and costs are expected to continue rising; considering the additional social costs resulting from this, it poses a significant burden on the nation.

[0005] However, existing blood-based dementia diagnostic technologies have academically been conducted using laser analyzers in pg / ml units within serum or plasma, but they do not utilize blood components. Existing related diagnostic kits rely on the potential difference of limited dementia markers, employing simple methods with low sensitivity; consequently, accurate diagnosis and differentiation of dementia are difficult. Therefore, there is a growing need for effective blood-based dementia diagnostic kits to prevent and detect dementia early and manage its progression, thereby securing individuals' right to health and reducing both individual and national management costs. Prior art literature

[0006] Korean Patent Publication No. 10-2020-0097636 (Published Aug. 19, 2020) The problem to be solved

[0007] The present invention relates to a biomarker composition for the diagnosis of degenerative brain diseases, and specifically, to distinguish between normal individuals, patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD), the invention provides a biomarker composition that distinguishes between normal individuals, patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD) by measuring the levels of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), Tau, NSE (neuron-specific enolase), GFAP (Glial fibrillary acidic protein), UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), PIN1 (peptidyl-prolyl isomerase), CRMP2 (collapsing response mediator protein-2), Phospho-Tau231 (Phospho-Tau231), and OMP (olfactory marker protein) as biomarkers in the blood and identifying their relationships. means of solving the problem

[0008] The present invention provides a diagnostic biomarker composition for distinguishing between normal individuals and mild cognitive impairment (MCI), comprising Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), CRMP2 (collapsing response mediator protein-2), and phosphorylated-tau231 (Phospho-Tau231) as active ingredients.

[0009] In addition, the present invention provides a diagnostic biomarker composition for distinguishing between normal individuals and Alzheimer's disease stages 1, 2, and 3, comprising Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), phosphorylated-tau231 (Phospho-Tau231), and OMP (olfactory marker protein) as active ingredients.

[0010] In addition, the present invention provides a diagnostic biomarker composition for distinguishing mild cognitive impairment (MCI) and Alzheimer's disease stages 1, 2, and 3, comprising Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), Tau, GFAP (Glial fibrillary acidic protein), and Phospho-Tau231 as active ingredients.

[0011] In addition, the present invention provides a method for providing information to distinguish between a normal person and mild cognitive impairment (MCI), comprising the steps of: measuring the level of a biomarker composed of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), CRMP2 (collapsing response mediator protein-2), and Phospho-Tau231 from a biological sample separated from a subject; measuring the level of one or more markers selected from Tau, UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), and OMP (olfactory marker protein) from the biological sample; and distinguishing between a normal person and mild cognitive impairment (MCI) by comparing the levels of the markers.

[0012] In addition, the present invention provides a method for providing information to distinguish between a normal person and Alzheimer's disease stages 1, 2, and 3, comprising: a step of measuring the level of a biomarker composed of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), Phospho-Tau231, and OMP (olfactory marker protein) from a biological sample separated from a subject; a step of measuring the level of a biomarker composed of Tau, NSE (neuron-specific enolase), GFAP (Glial fibrillary acidic protein), UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), and PIN1 (peptidyl-prolyl isomerase) from the biological sample; and a step of distinguishing between a normal person and Alzheimer's disease stages 1, 2, and 3 by comparing the levels of the markers.

[0013] In addition, the present invention provides a method for providing information to distinguish between mild cognitive impairment (MCI) and Alzheimer's disease stages 1, 2, and 3 by comparing the levels of the biomarkers, comprising Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), Tau, GFAP (Glial fibrillary acidic protein), and Phospho-Tau231, from a biological sample separated from a subject; a step of measuring the levels of biomarkers comprising BDNF (brain-derived neurotrophic factor), CRMP2 (collapsing response mediator protein-2), and OMP (olfactory marker protein) from the biological sample; and a step of measuring the levels of the biomarkers. Effects of the invention

[0014] According to the present invention, to distinguish between patients with mild cognitive impairment (MCI) and patients with Alzheimer's disease (AD), a biomarker composition can be provided for distinguishing between normal individuals, patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD) by measuring the levels of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), Tau, NSE (neuron-specific enolase), GFAP (Glial fibrillary acidic protein), UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), PIN1 (peptidyl-prolyl isomerase), CRMP2 (collapsing response mediator protein-2), Phospho-Tau231, and OMP (olfactory marker protein) as biomarkers in the blood and identifying their phylogenetic relationships. Brief explanation of the drawing

[0015] Figure 1 shows the results of evaluating Aβ40 (amyloid beta 40), a biomarker for diagnosing degenerative brain diseases. Figure 2 shows the results of evaluating Aβ42 (amyloid beta 42), a biomarker for diagnosing degenerative brain diseases. Figure 3 shows the results of evaluating BDNF (brain-derived neurotrophic factor), a biomarker for diagnosing degenerative brain diseases. Figure 4 shows the results of evaluating Tau, a biomarker for diagnosing degenerative brain diseases. Figure 5 shows the results of evaluating NSE (neuron-specific enolase), a biomarker for diagnosing degenerative brain diseases. Figure 6 shows the results of evaluating GFAP (Glial fibrillary acidic protein), a biomarker for diagnosing degenerative brain diseases. Figure 7 shows the results of evaluating UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), a biomarker for diagnosing degenerative brain diseases. Figure 8 shows the results of evaluating PIN1 (peptidyl-prolyl isomerase), a biomarker for diagnosing degenerative brain diseases. Figure 9 shows the results of evaluating CRMP2 (collapsing response mediator protein-2), a biomarker for diagnosing degenerative brain diseases. Figure 10 shows the results of evaluating phosphorylated-tau231 (Phospho-Tau231), a biomarker for diagnosing degenerative brain diseases. Figure 11 shows the results of evaluating OMP (olfactory marker protein), a biomarker for diagnosing degenerative brain diseases. Figure 12 is a heatmap result for analyzing the association of biomarkers for diagnosing degenerative brain diseases. Specific details for implementing the invention

[0016] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0017] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0018] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

[0019] The present invention will be described in more detail below.

[0020] The present invention provides a diagnostic biomarker composition for distinguishing between normal individuals and mild cognitive impairment (MCI), comprising Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), CRMP2 (collapsing response mediator protein-2), and phosphorylated-tau231 (Phospho-Tau231) as active ingredients.

[0021] The above diagnostic biomarker composition for distinguishing between normal individuals and mild cognitive impairment (MCI) may additionally include one or more selected from Tau, UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), and OMP (olfactory marker protein).

[0022] In addition, the present invention provides a diagnostic biomarker composition for distinguishing between normal individuals and Alzheimer's disease stages 1, 2, and 3, comprising Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), phosphorylated-tau231 (Phospho-Tau231), and OMP (olfactory marker protein) as active ingredients.

[0023] The diagnostic biomarker composition for distinguishing between normal individuals and Alzheimer's disease stages 1, 2, and 3 may further include one or more selected from Tau, NSE (neuron-specific enolase), GFAP (Glial fibrillary acidic protein), UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), and PIN1 (peptidyl-prolyl isomerase).

[0024] In addition, the present invention provides a diagnostic biomarker composition for distinguishing mild cognitive impairment (MCI) and Alzheimer's disease stages 1, 2, and 3, comprising Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), Tau, GFAP (Glial fibrillary acidic protein), and Phospho-Tau231 as active ingredients.

[0025] The diagnostic biomarker composition for distinguishing between mild cognitive impairment (MCI) and Alzheimer's disease stages 1, 2, and 3 may additionally include one or more selected from BDNF (brain-derived neurotrophic factor), CRMP2 (collapsing response mediator protein-2), and OMP (olfactory marker protein).

[0026] In addition, the present invention provides a diagnostic kit for distinguishing between normal individuals and mild cognitive impairment (MCI), comprising a preparation for measuring the level of a diagnostic biomarker composition for distinguishing between normal individuals and mild cognitive impairment (MCI).

[0027] In addition, the present invention provides a diagnostic kit for distinguishing between normal people and mild cognitive impairment (MCI), comprising a preparation for measuring the level of a diagnostic biomarker composition for distinguishing between normal people and Alzheimer's disease stages 1, 2, and 3.

[0028] In addition, the present invention provides a diagnostic kit for distinguishing mild cognitive impairment (MCI) and Alzheimer's disease stages 1, 2, and 3, comprising a preparation for measuring the level of a diagnostic biomarker composition for distinguishing mild cognitive impairment (MCI) and Alzheimer's disease stages 1, 2, and 3.

[0029] The agent used to measure the level of the above-mentioned biomarker composition is an antibody that specifically binds to each marker.

[0030] In addition, the present invention provides a method for providing information to distinguish between a normal person and mild cognitive impairment (MCI), comprising the steps of: measuring the level of a biomarker composed of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), CRMP2 (collapsing response mediator protein-2), and Phospho-Tau231 from a biological sample separated from a subject; measuring the level of one or more markers selected from Tau, UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), and OMP (olfactory marker protein) from the biological sample; and distinguishing between a normal person and mild cognitive impairment (MCI) by comparing the levels of the markers.

[0031] The above biological sample may be one or more selected from blood, serum, tissue, urine, saliva, and cerebrospinal fluid.

[0032] In the biological sample, if the level of Aβ40 (amyloid beta 40) is 5.00 pg / ml to less than 30.00 pg / ml, the level of Aβ42 (amyloid beta 42) is 0.2 pg / ml to less than 0.88 pg / ml, the level of CRMP2 (collapsing response mediator protein-2) is 3.00 ng / ml to less than 6.00 ng / ml, and the level of Phospho-Tau231 is 15.00 pg / ml to less than 40.00 pg / ml, it can be determined that the subject of the biological sample may have mild cognitive impairment (MCI).

[0033] In the above biological sample, if the level of Tau is 3.30 pg / ml to less than 10.00 pg / ml, the level of UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1) is 60.00 pg / ml to less than 90.00 pg / ml, and the level of OMP (olfactory marker protein) is greater than 0.5 ng / ml to less than 0.80 ng / ml, it can be determined that the subject of the biological sample may have mild cognitive impairment (MCI).

[0034] In addition, the present invention provides a method for providing information to distinguish between a normal person and Alzheimer's disease stages 1, 2, and 3, comprising: a step of measuring the level of a biomarker composed of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), Phospho-Tau231, and OMP (olfactory marker protein) from a biological sample separated from a subject; a step of measuring the level of a biomarker composed of Tau, NSE (neuron-specific enolase), GFAP (Glial fibrillary acidic protein), UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), and PIN1 (peptidyl-prolyl isomerase) from the biological sample; and a step of distinguishing between a normal person and Alzheimer's disease stages 1, 2, and 3 by comparing the levels of the markers.

[0035] In the biological sample, if the level of Aβ40 (amyloid beta 40) is 30.00 pg / ml to less than 91.00 pg / ml, the level of Aβ42 (amyloid beta 42) is less than 0.2 pg / ml, the level of BDNF (brain-derived neurotrophic factor) is 200.00 pg / ml to less than 600.00 pg / ml, the level of Phospho-Tau231 is 40.00 pg / ml to less than 102.00 pg / ml, and the level of OMP (olfactory marker protein) is 0.50 ng / ml or less, it can be determined that the subject of the biological sample may have Alzheimer's disease stage 1, stage 2, or stage 3.

[0036] In the biological sample, if the level of Tau is less than 10.00 pg / ml to 32.00 pg / ml, the level of NSE (neuron-specific enolase) is less than 1000.00 pg / ml to 1400.00 pg / ml, the level of GFAP (Glial fibrillary acidic protein) is less than 20.00 pg / ml to 90.00 pg / ml, the level of UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1) is less than 60.00 pg / ml, and the level of PIN1 (peptidyl-prolyl isomerase) is less than 4.0 pg / ml, it can be determined that the subject of the biological sample may have Alzheimer's disease stage 1, stage 2, or stage 3.

[0037] In addition, the present invention provides a method for providing information to distinguish between mild cognitive impairment (MCI) and Alzheimer's disease stages 1, 2, and 3 by comparing the levels of the biomarkers, comprising Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), Tau, GFAP (Glial fibrillary acidic protein), and Phospho-Tau231, from a biological sample separated from a subject; a step of measuring the levels of biomarkers comprising BDNF (brain-derived neurotrophic factor), CRMP2 (collapsing response mediator protein-2), and OMP (olfactory marker protein) from the biological sample; and a step of measuring the levels of the biomarkers.

[0038] In the biological sample, if the level of Aβ40 (amyloid beta 40) is 5.00 pg / ml to less than 30.00 pg / ml, the level of Aβ42 (amyloid beta 42) is 0.2 pg / ml to less than 0.88 pg / ml, and the level of Tau is 3.3 pg / ml to less than 10.00 pg / ml, if the level of GFAP (Glial fibrillary acidic protein) is 6.2 pg / ml to less than 20.00 pg / ml, and the level of Phospho-Tau231 is 15.00 pg / ml to less than 40.00 pg / ml, it can be determined that the subject of the biological sample may have mild cognitive impairment (MCI).

[0039] In the above biological sample, if the level of Aβ40 (amyloid beta 40) is less than 30.00 pg / ml to 91.00 pg / ml, the level of Aβ42 (amyloid beta 42) is less than 0.2 pg / ml, the level of Tau is less than 10.00 pg / ml to 32.00 pg / ml, the level of GFAP (Glial fibrillary acidic protein) is less than 20.00 pg / ml to 90.00 pg / ml, and the level of Phospho-Tau231 is less than 40.00 pg / ml to 102.00 pg / ml, it can be determined that the subject of the biological sample may have Alzheimer's disease stage 1, stage 2, or stage 3.

[0040] In the above biological sample, if the level of BDNF (brain-derived neurotrophic factor) is 600.00 pg / ml to less than 900.00 pg / ml, the level of CRMP2 (collapsing response mediator protein-2) is 3.00 ng / ml to less than 6.00 ng / ml, and the level of OMP (olfactory marker protein) is greater than 0.5 ng / ml to less than or equal to 0.80 ng / ml, it can be determined that the subject of the above biological sample may have mild cognitive impairment (MCI).

[0041] If, in the biological sample, the level of BDNF (brain-derived neurotrophic factor) is 200.00 pg / ml to less than 600.00 pg / ml, the level of CRMP2 (collapsing response mediator protein-2) is 1.00 ng / ml to less than 3.00 ng / ml, and the level of OMP (olfactory marker protein) is 0.50 ng / ml or less, it can be determined that the subject of the biological sample may have Alzheimer's disease stage 1, stage 2, or stage 3.

[0042] Hereinafter, experimental examples and embodiments will be described in detail to aid in understanding the present invention. However, the following experimental examples and embodiments are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following experimental examples and embodiments. The experimental examples and embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0043] <Example>

[0044] According to the present invention, an indirect immunometric ELISA was used to measure the levels of main markers and sub-markers for the diagnosis of degenerative brain diseases.

[0045] To measure the levels of main or sub-markers, antibodies specifically binding to Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), Tau, NSE (neuron-specific enolase), GFAP (glial fibrillary acidic protein), UCH-L1 (ubiquitin carboxy-terminal hydrolase L1), PIN1 (peptidyl-prolyl isomerase), CRMP2 (collapsing response mediator protein-2), Phospho-Tau231, and OMP (olfactory marker protein) were used. The antibodies and experimental methods used to measure the levels of each biomarker were based on commercially available kits.

[0046] Specifically, the Human Amyloid Beta 42 ELISA Kit (Ca. ab193692) is used for Aβ40 (Amyloid Beta 40), the Human Aβ42 Ultrasensitive ELISA Kit (Ca. KHB3544) is used for Aβ42 (Amyloid Beta 42), the Human BDNF ELISA Kit (Ca. ab212166) is used for BDNF (Brain-Derived Neurotrophic Factor), the Human Tau ELISA Kit (Ca. ab210972) is used for Tau (Tau), the Human Neuron-Specific Enolase ELISA Kit (Ca. ab217778) is used for NSE (Neuron-Specific Enolase), the Human GFAP DuoSet ELISA (Ca. DY2594-05) is used for GFAP (Glial Fibrillary Acidic Protein), and UCH-L1 (Ubiquitin For carboxy-terminal hydrolase L1, the Human UCH-L1 / PGP9.5 DuoSet ELISA (Ca. DY6007-05) was used; for PIN1 (peptidyl-prolyl isomerase), the Human Protein (peptidylprolyl cis / trans isomerase) NIMA-Interacting 1 (PIN1) ELISA Kit (Ca. CSB-E11308h) was used; for CRMP2 (collapsing response mediator protein-2), the Human DPYSL2 / CRMP2 ELISA Kit (Ca. LS-F37929) was used; for Phosphorylated-Tau231 (Phospho-Tau231), the Human Phosphorylated Tau 231 (PTAU231) ELISA kit (Ca. MBS724296) was used; and for OMP (olfactory marker protein), the Human Olfactory Marker Protein (OMP) An ELISA Kit (Ca. MBS9379411) was used.

[0047] Samples to be analyzed were prepared to measure the levels of main or sub-markers. Samples were prepared by storing them at 2–8°C within 5 days of collection. The prepared samples were coagulated for 2 hours using a serum separator tube (SST) and then centrifuged at 1000 xg for 15 minutes. Plasma was collected using plasma EDTA or heparin as an anticoagulant. Within 30 minutes of collection, the samples were centrifuged at 1000 xg at 2–8°C for 15 minutes. For cell cultures, particulates were removed by centrifugation at 1000 xg at 2–8°C for 15 minutes before use. For tissue homogenates, 100 mg of tissue was rinsed with 1X PBS, homogenized in 1 ml of 1X PBS, and subjected to two freeze-thaw cycles. The supernatant was then removed by centrifugation at 5000 xg at 2–8°C for 5 minutes before use.

[0048] (1) Aβ40 (amyloid beta 40)

[0049] 100 μl of the standard sample provided by the ELISA kit or each serum sample was loaded into the wells. Detection antibody solution was added to all wells except the blank, mixed well, and incubated at room temperature for 3 hours. After washing four times with wash buffer, the secondary antibody, anti-Rabbit IgG HRP, was loaded into all wells except the blank and incubated at room temperature for 30 minutes. After the reaction was complete, the wells were washed four times with wash buffer, stabilized chromogen was added, and the reaction was incubated at room temperature for 30 minutes under light blockage. Once the induction of the HRP-substrate color reaction was confirmed, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured.

[0050] (2) Aβ42 (amyloid beta 42)

[0051] 100 μl of the standard sample provided by the ELISA kit or each serum sample was loaded into the wells. Detection antibody solution was added to all wells except the blank, mixed well, and incubated at room temperature for 3 hours or overnight at 40°C. After washing four times with wash buffer, secondary antibody solution was loaded into all wells except the blank and incubated at room temperature for 30 minutes. After the reaction was complete, the wells were washed four times with wash buffer, stabilized chromogen was added, and the reaction was incubated at room temperature for 30 minutes under light blockage. Once the induction of the HRP-substrate color reaction was confirmed, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured.

[0052] (3) BDNF (Brain-Derived Neurotrophic Factor)

[0053] 100 μl of the standard sample provided by the ELISA kit or each serum sample was loaded into each well, and the antibody cocktail was added to each well. The reaction was then carried out at room temperature for 1 hour with shaking at 400 rpm. After the reaction was complete, the samples were washed three times with wash buffer, then treated with TMB development solution, and reacted at room temperature for 30 minutes with shaking at 400 rpm under light blockage. Once the HRP-substrate color development was confirmed, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured.

[0054] (4) Tau

[0055] 100 μl of the standard sample provided by the ELISA kit or each serum sample was loaded into each well, and the antibody cocktail was added to each well. The reaction was then carried out at room temperature for 1 hour with shaking at 400 rpm. After the reaction was complete, the samples were washed three times with wash buffer, then treated with TMB development solution, and reacted at room temperature for 30 minutes with shaking at 400 rpm under light blockage. Once the HRP-substrate color development was confirmed, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured.

[0056] (5) NSE (neuron-specific enolase)

[0057] 100 μl of the standard sample provided by the ELISA kit or each serum sample was loaded into each well, and the antibody cocktail was added to each well. The reaction was then carried out at room temperature for 1 hour with shaking at 400 rpm. After the reaction was complete, the samples were washed three times with wash buffer, then treated with TMB development solution, and reacted at room temperature for 30 minutes with shaking at 400 rpm under light blockage. Once the HRP-substrate color development was confirmed, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured.

[0058] (6) GFAP (Glial fibrillary acidic protein)

[0059] Capture antibodies were loaded into a 96-well plate and incubated overnight at room temperature to immobilize the capture antibodies on the well surfaces. After three washes, reagent diluent was loaded into each well, and the blocking step was carried out by incubating at room temperature for at least one hour. After the reaction was complete, the plates were washed three times with wash buffer. 100 μl of the standard samples provided by the ELISA kit or each serum sample was loaded into the wells and incubated at room temperature for two hours. After washing three times with wash buffer, biotinylated detection antibodies were loaded into each well and incubated at room temperature for two hours. After washing three times with wash buffer, streptavidin-HRP was loaded into each well and incubated at room temperature for 20 minutes under light blockage. After the reaction was completed, the sample was washed three times with wash buffer, then the substrate solution was added and the reaction was carried out at room temperature for 30 minutes under light blockage. After confirming that the HRP-substrate color reaction had been induced, the reaction was stopped by adding a stop solution, and the absorbance at 450 nm was measured.

[0060] (7) UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1)

[0061] Capture antibodies were loaded into a 96-well plate and incubated overnight at room temperature to immobilize the capture antibodies on the well surfaces. After three washes, reagent diluent was loaded into each well, and the blocking step was carried out by incubating at room temperature for at least one hour. After the reaction was complete, the plates were washed three times with wash buffer. 100 μl of the standard samples provided by the ELISA kit or each serum sample was loaded into the wells and incubated at room temperature for two hours. After washing three times with wash buffer, biotinylated detection antibodies were loaded into each well and incubated at room temperature for two hours. After washing three times with wash buffer, streptavidin-HRP was loaded into each well and incubated at room temperature for 20 minutes under light blockage. After the reaction was completed, the sample was washed three times with wash buffer, then the substrate solution was added and the reaction was carried out at room temperature for 30 minutes under light blockage. After confirming that the HRP-substrate color reaction had been induced, the reaction was stopped by adding a stop solution, and the absorbance at 450 nm was measured.

[0062] (8) PIN1 (peptidyl-prolyl isomerase)

[0063] 100 μl of the standard sample provided by the ELISA kit or each serum sample was loaded into wells and incubated at 37°C for 2 hours. After removing the reaction solution, 1X biotinylated detection antibody was loaded into each well and incubated at 37°C. After the reaction was complete, the wells were washed three times with wash buffer, then 1X HRP-avidin was loaded into each well and incubated at 37°C for 1 hour. After washing five times with wash buffer, TMB substrate was added, and the reaction was incubated at 37°C for 30 minutes under light blockage. Once the HRP-substrate color development was confirmed, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured.

[0064] (9) CRMP2 (collapse response mediator protein-2)

[0065] 100 μl of the standard sample provided by the ELISA kit or each serum sample was loaded into wells and incubated at 37°C for 2 hours. After removing the reaction solution, Detection Reagent A was loaded into each well and reacted at 37°C for 1 hour. After removing the reaction solution and washing three times with wash buffer, Detection Reagent B was loaded into each well and reacted at 37°C for 1 hour. After washing five times with wash buffer, TMB substrate was added and reacted at 37°C for 30 minutes under light blockage. Once the HRP-substrate color development was confirmed, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured.

[0066] (10) Phospho-Tau231

[0067] 100 μl of the standard sample provided by the ELISA kit or each serum sample was loaded into the wells, and enzyme conjugate was applied to all wells except the blank. The mixture was then incubated at 37°C for 1 hour. After washing all wells, including the blank wells, five times with wash buffer, Substrate A and Substrate B were sequentially added to all wells, and the mixture was incubated at 37°C for 30 minutes under light blockage. Once the HRP-substrate color development was confirmed, the reaction was stopped by adding stop solution, and the absorbance at 450 nm was measured.

[0068] (11) OMP (olfactory marker protein)

[0069] 100 μl of each serum sample or standard sample provided in the ELISA kit was loaded into the wells, and HRP-conjugate Reagent was applied to all wells except the blank. The mixture was incubated at 37°C for 1 hour. After the reaction, all wells, including the blank wells, were washed four times using wash buffer. Subsequently, Chromogen Solutions A and B were sequentially applied to all wells, and the mixture was incubated at 37°C for 30 minutes under light blockage. Once the HRP-substrate color development was confirmed, the reaction was stopped by adding a stop solution, and the absorbance at 450 nm was measured.

[0070] The concentration of each marker protein in all samples was calculated using standard curves obtained from the corresponding experiment.

[0071] <Test Example>

[0072] (1) Comparison of expression levels of main marker and sub-marker

[0073] To identify the main marker and sub-marker for diagnosing degenerative brain disease according to the present invention, 65 serum samples were obtained from normal volunteers, patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD) recruited from Dx Biosamples, LLC, a human biospecimen contract procurement organization, and the levels of the main marker and sub-marker present in each serum were measured.

[0074] The above patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD) are patients diagnosed using a diagnostic method subdivided into seven grades for Alzheimer's diagnosis. The seven grades of Alzheimer's diagnosis are as follows.

[0075] AD Stage 1: Cases where cognitive function appears normal, but pathological changes occur in the brain.

[0076] AD Stage 2: Mild cognitive impairment. Feeling of memory problems, such as forgetting familiar words, names, or the location of objects. Pathological changes appear in the brain.

[0077] AD Stage 3: Mild Cognitive Impairment (MCI). Difficulty recalling words or names. Getting lost or unable to find the correct words. Losing or misplacing valuables. Reading sentences and barely remembering them. Pathological changes appearing in the brain.

[0078] AD Stage 4: Moderate cognitive decline. Early stage of Alzheimer's (Stage 1). Short-term memory declines, and parts of the past are not remembered. Difficulty performing complex tasks.

[0079] AD Stage 5: Severe cognitive decline. Intermediate stage of Alzheimer's (Stage 2). Cognitive function continues to decline, requiring assistance from others for daily living. During interviews, the individual cannot recall key details such as current address, phone number, or alma mater. Confusion regarding current location, date, day of the week, or season. Inability to perform simple mental arithmetic.

[0080] AD Stage 6: Very severe cognitive decline. Severe Alzheimer's (Stage 3). Memory problems worsen, and significant personality changes appear. Significant assistance is required for routine daily activities. Remembers one's own name but cannot remember the names or faces of others. Sleep-wake cycles are disrupted. Frequent incontinence occurs. Suspicion, forgetfulness, hallucinations, or obsessive symptoms appear.

[0081] AD Stage 7: Terminal Alzheimer's. A stage imminent to death, characterized by a decline in motor function and an inability to communicate.

[0082] In the present invention, the mild cognitive impairment (MCI) patient refers to a patient in AD stages 1 to 3 of the 7-stage Alzheimer's disease classification. Additionally, in the present invention, the Alzheimer's disease (AD) patient refers to a patient in AD stages 4 to 6 of the 7-stage Alzheimer's disease classification, and includes patients in Alzheimer's disease stages 1, 2, and 3.

[0083] Using the method of the above example, the levels of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), Tau, NSE (neuron-specific enolase), GFAP (Glial fibrillary acidic protein), UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), PIN1 (peptidyl-prolyl isomerase), CRMP2 (collapsing response mediator protein-2), Phospho-Tau231, or OMP (olfactory marker protein) were measured from blood samples of each classified control group and experimental group, and the expression levels of each marker were compared using a bar graph.

[0084] Biomarker Normal MCI AD(1,2,3 stage) Aβ40 (pg / ml) 3.54±0.41 10.23±4.69 57.86±22.49 Aβ42 (pg / ml) 1.22±0.31 0.55±0.31 0.04±0.03 BDNF (pg / ml) 969.19±53.59 763.53±106.25 406.55±136.81 Tau (pg / ml) 2.63±0.44 4.85±1.47 23.25±8.61 NSE (pg / ml) 730.00±84.65 973.48±106.00 1204.59±184.54 GFAP (pg / ml) 5.56±0.68 7.30±0.92 55.06±33.32 UCH-L1 (pg / ml) 107.90±9.39 77.32±10.89 45.38±13.27 PIN1 (pg / ml) 5.83±0.43 4.85±0.47 3.10±0.41 CRMP2 (ng / ml) 1.19±0.11 4.86±0.79 2.00±0.50 p-Tau231 (pg / ml) 2.24±0.45 28.15±8.86 74.58±26.80 OMP (ng / ml) 1.11±0.24 0.70±0.27 0.47±0.22

[0085] As shown in Table 1 and Figures 1 to 11 above, there were significant differences in the expression levels of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), Tau, NSE (neuron-specific enolase), GFAP (Glial fibrillary acidic protein), UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), PIN1 (peptidyl-prolyl isomerase), CRMP2 (collapsing response mediator protein-2), Phospho-Tau231, and OMP (olfactory marker protein) in the serum of normal volunteers, patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD).

[0086] As shown in Figure 1, the level of Aβ40 (amyloid beta 40) was 3.54±0.41 pg / ml in the serum of normal volunteers, 10.23±4.69 pg / ml in the serum of patients with mild cognitive impairment (MCI), and 57.86±22.49 pg / ml in the serum of patients with Alzheimer's disease (AD).

[0087] As shown in Figure 2, the level of Aβ42 (amyloid beta 42) was 1.22±0.31 pg / ml in the serum of normal volunteers, 0.55±0.31 pg / ml in the serum of patients with mild cognitive impairment (MCI), and 0.04±0.03 pg / ml in the serum of patients with Alzheimer's disease (AD).

[0088] As shown in Figure 3, the level of BDNF (brain-derived neurotrophic factor) was 969.19±53.59 pg / ml in the serum of normal volunteers, 763.53±106.25 pg / ml in the serum of patients with mild cognitive impairment (MCI), and 406.55±136.81 pg / ml in the serum of patients with Alzheimer's disease (AD).

[0089] As shown in Figure 4, the level of Tau was 2.63±0.44 pg / ml in the serum of normal volunteers, 4.85±1.47 pg / ml in the serum of patients with mild cognitive impairment (MCI), and 23.25±8.61 pg / ml in the serum of patients with Alzheimer's disease (AD).

[0090] As shown in Figure 5, the level of NSE (neuron-specific enolase) was 730.00±84.65 pg / ml in the serum of normal volunteers, 973.48±106.00 pg / ml in the serum of patients with mild cognitive impairment (MCI), and 1204.59±184.54 pg / ml in the serum of patients with Alzheimer's disease (AD).

[0091] As shown in Figure 6, the level of GFAP (Glial fibrillary acidic protein) was 5.56±0.68 pg / ml in the serum of normal volunteers, 7.30±0.92 pg / ml in the serum of patients with mild cognitive impairment (MCI), and 55.06±33.32 pg / ml in the serum of patients with Alzheimer's disease (AD).

[0092] As shown in Figure 7, the level of UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1) was 107.90±9.39 pg / ml in the serum of normal volunteers, 77.32±10.89 pg / ml in the serum of patients with mild cognitive impairment (MCI), and 45.38±13.27 pg / ml in the serum of patients with Alzheimer's disease (AD).

[0093] As shown in Figure 8, the level of PIN1 (peptidyl-prolyl isomerase) was 5.83±0.43 pg / ml in the serum of normal volunteers, 4.85±0.47 pg / ml in the serum of patients with mild cognitive impairment (MCI), and 3.10±0.41 pg / ml in the serum of patients with Alzheimer's disease (AD).

[0094] As shown in Figure 9, the level of CRMP2 (collapsing response mediator protein-2) was 1.19±0.11 ng / ml in the serum of normal volunteers, 4.86±0.79 ng / ml in the serum of patients with mild cognitive impairment (MCI), and 2.00±0.50 ng / ml in the serum of patients with Alzheimer's disease (AD).

[0095] As shown in Figure 10, the levels of phosphorylated-tau231 (Phospho-Tau231) were found to be 2.24±0.45 pg / ml in the serum of normal volunteers, 28.15±8.86 pg / ml in the serum of patients with mild cognitive impairment (MCI), and 74.58±26.80 pg / ml in the serum of patients with Alzheimer's disease (AD).

[0096] As shown in Figure 11, the level of OMP (olfactory marker protein) was 1.11±0.24 ng / ml in the serum of normal volunteers, 0.70±0.27 ng / ml in the serum of patients with mild cognitive impairment (MCI), and 0.47±0.22 ng / ml in the serum of patients with Alzheimer's disease (AD).

[0097] (2) Analysis of the association between the main marker and the sub-marker

[0098] The expression levels of Aβ40, Aβ42, BDNF, Tau, NSE, GFAP, UCH-L1, Pin1, CRMP2, p-Tau231, and OMP in the serum of normal volunteers, patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD) were quantified, and the average of each quantified value was calculated. In this case, the ratio of the results of the remaining experimental groups was calculated when the lowest average value among the experimental groups for each marker was set to 1. Results for a total of 11 markers were calculated using the same method, and heat maps were constructed using these values.

[0099] For Aβ40, the mean expression level of Aβ40 in the serum of the Normal experimental group was set to 1, and the ratio of the mean expression levels of Aβ40 in the serum of the MCI and AD experimental groups was calculated. For Aβ42, the mean expression level of Aβ42 in the serum of the AD experimental group was set to 1, and the ratio of the mean expression levels of Aβ42 in the serum of the Normal and MCI experimental groups was calculated. For BDNF, the mean expression level of BDNF in the serum of the AD experimental group was set to 1, and the ratio of the mean expression levels of BDNF in the serum of the Normal and MCI experimental groups was calculated. For Tau, the mean expression level of Tau in the serum of the Normal experimental group was set to 1, and the ratio of the mean expression levels of Tau in the serum of the MCI and AD experimental groups was calculated. For NSE, the mean expression level of NSE in the serum of the Normal experimental group was set to 1, and the ratio of the mean expression levels of NSE in the serum of the MCI and AD experimental groups was calculated. For GFAP, the average expression level of GFAP in the serum of the Normal experimental group was set to 1, and the ratio of the average expression levels of GFAP in the serum of the MCI and AD experimental groups was calculated. For UCH-L1, the average expression level of UCH-L1 in the serum of the AD experimental group was set to 1, and the ratio of the average expression levels of UCH-L1 in the serum of the Normal and MCI experimental groups was calculated. For Pin1, the average expression level of Pin1 in the serum of the AD experimental group was set to 1, and the ratio of the average expression levels of Pin1 in the serum of the Normal and MCI experimental groups was calculated. For CRMP2, the average expression level of CRMP2 in the serum of the Normal experimental group was set to 1, and the ratio of the average expression levels of CRMP2 in the serum of the MCI and AD experimental groups was calculated.For Phospho-Tau231 (p-Tau231), the mean value of the expression level of p-Tau231 in the serum of the Normal experimental group was set to 1, and the ratio of the mean value of the expression level of p-Tau231 in the serum of the MCI and AD experimental groups was calculated. For OMP, the mean value of the expression level of OMP in the serum of the AD experimental group was set to 1, and the ratio of the mean value of the expression level of OMP in the serum of the Normal and MCI experimental groups was calculated.

[0100] Biomarkers were selected to distinguish between normal volunteers (Normal), patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD) by comparing the differences in expression levels shown in Figure 12.

[0101] When comparing normal volunteers and patients with mild cognitive impairment (MCI), BDNF, NSE, GFAP, and Pin1, which showed similar trends among 11 biomarkers, were excluded. Among the remaining 8 markers, distinct differences in expression levels were observed in Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), CRMP2 (collapsing response mediator protein-2), and Phospho-Tau231, and differences in expression levels were also observed in Tau, UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), and OMP (olfactory marker protein).

[0102] When comparing normal volunteers and Alzheimer's (AD) patients, CRMP2, which showed a similar trend among 11 biomarkers, was excluded. Among the remaining 10 markers, distinct differences in expression levels were observed in Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), Phospho-Tau231, BDNF (brain-derived neurotrophic factor), and OMP (olfactory marker protein), and differences in expression levels were also observed in Tau, NSE (neuron-specific enolase), GFAP (glial fibrillary acidic protein), UCH-L1 (ubiquitin carboxy-terminal hydrolase L1), and PIN1 (peptidyl-prolyl isomerase).

[0103] When comparing patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD), NSE, UCH-L1, and Pin1, which showed similar trends among 11 biomarkers, were excluded. Among the remaining 8 markers, distinct differences in expression levels were observed in Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), Tau, GFAP (Glial fibrillary acidic protein), and Phospho-Tau231, as well as in BDNF (brain-derived neurotrophic factor), CRMP2 (collapsing response mediator protein-2), and OMP (olfactory marker protein).

[0104] Based on the heatmap results above, biomarkers for distinguishing normal volunteers, patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD) are classified as shown in Table 2 below, and it has been proven that normal volunteers, patients with mild cognitive impairment (MCI), and patients with Alzheimer's disease (AD) can be distinguished using the biomarkers below.

[0105] division Normal vs MCI Normal vs AD MCI vs AD Main marker Aβ40, Aβ42, CRMP2, p-Tau Aβ40, Aβ42, BDNF, p-Tau, OMP Aβ40, Aβ42, Tau, GFAP, p-Tau Sub-marker Tau, UCH-L1, OMP Tau, NSE, GFAP, UCH-L1, Pin1 BDNF, CRMP2, OMP

[0106] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A diagnostic biomarker composition for distinguishing between normal individuals and Alzheimer's disease stages 1, 2, and 3, comprising Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), phosphorylated-tau231 (Phospho-Tau231), and OMP (olfactory marker protein) as active ingredients. Claim 2 A diagnostic biomarker composition for distinguishing between normal individuals and Alzheimer's disease stages 1, 2, and 3, characterized in that, in claim 1, the composition further comprises one or more selected from Tau, NSE (neuron-specific enolase), GFAP (Glial fibrillary acidic protein), UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), and PIN1 (peptidyl-prolyl isomerase). Claim 3 A diagnostic kit for distinguishing between normal individuals and Alzheimer's disease stages 1, 2, and 3, comprising a preparation that measures the level of the biomarker composition of claim 1 or 2. Claim 4 A diagnostic kit for distinguishing between normal people and Alzheimer's disease stages 1, 2, and 3, characterized in that, in paragraph 3, the preparation for measuring the level of the biomarker composition is an antibody that specifically binds to each marker. Claim 5 A method for providing information to distinguish between a normal person and Alzheimer's disease stages 1, 2, and 3, comprising: a step of measuring the level of a biomarker composed of Aβ40 (amyloid beta 40), Aβ42 (amyloid beta 42), BDNF (brain-derived neurotrophic factor), Phospho-Tau231, and OMP (olfactory marker protein) from a biological sample isolated from a subject; a step of measuring the level of a biomarker composed of Tau, NSE (neuron-specific enolase), GFAP (Glial fibrillary acidic protein), UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1), and PIN1 (peptidyl-prolyl isomerase) from the biological sample; and a step of distinguishing between a normal person and Alzheimer's disease stages 1, 2, and 3 by comparing the levels of the biomarkers. Claim 6 In claim 5, the provision of information for distinguishing between normal individuals and Alzheimer's disease stages 1, 2, and 3 is characterized by determining that the subject of the biological sample may have Alzheimer's disease stage 1, 2, or 3 if, within the biological sample, the level of Aβ40 (amyloid beta 40) is at a concentration of 30.00 pg / ml to 91.00 pg / ml, the level of Aβ42 (amyloid beta 42) is at a concentration of 0.2 pg / ml, the level of BDNF (brain-derived neurotrophic factor) is at a concentration of 200.00 pg / ml to 600.00 pg / ml, the level of Phospho-Tau231 is at a concentration of 40.00 pg / ml to 102.00 pg / ml, and the level of OMP (olfactory marker protein) is at a concentration of 0.50 ng / ml or less. method. Claim 7 Information for distinguishing between a normal person and Alzheimer's disease stages 1, 2, and 3, characterized in that, in the biological sample, if the level of Tau is 10.00 pg / ml to 32.00 pg / ml, the level of NSE (neuron-specific enolase) is 1000.00 pg / ml to 1400.00 pg / ml, the level of GFAP (Glial fibrillary acidic protein) is 20.00 pg / ml to 90.00 pg / ml, the level of UCH-L1 (Ubiquitin carboxy-terminal hydrolase L1) is 60.00 pg / ml, and the level of PIN1 (peptidyl-prolyl isomerase) is 4.0 pg / ml or less. Method of provision.

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