WO2023200281A1 - Unagglomerated dissolving composition for diagnosis of alzheimer's disease or mild cognitive impairment, and diagnostic method using same - Google Patents

Abstract

The present invention relates to an unagglomerated dissolving composition for the diagnosis of Alzheimer's disease or mild cognitive impairment, and a diagnostic method using same, wherein the present invention is useful in tracking pathological changes through the measurement of total-tau, phosphorylated tau, and beta-amyloid in plasma of Alzheimer's disease patients. In particular, it is possible to measure total-tau, phosphorylated tau, and beta-amyloid in blood without additionally requiring expensive special equipment and by using commonly used antibodies and equipment without additional processing for amyloid antibodies. In addition, the present invention enables the analysis of changes in representative proteins of Alzheimer's disease through a buffer treatment-concentration-lipase treatment presented in the present invention by using blood plasma itself, without a specially processed treatment step for amyloid-specific antibodies. Therefore, the composition and diagnostic method of the present invention have high diagnostic accuracy and reproducibility, and are also very simple so as to be able to be very effectively used for early diagnosis of Alzheimer's disease.

Description

Non-cohesive dissolved composition for diagnosing Alzheimer's disease or mild cognitive impairment and diagnostic method using the same

The present invention detects Alzheimer's dementia or mild cognitive impairment by quantitatively analyzing changes in beta-amyloid (Aβ) and phosphorylated tau present in patient body fluids such as blood before clinical symptoms of Alzheimer's dementia appear or develop. It relates to a diagnostic composition capable of making a diagnosis and a diagnostic method using the same.

Alzheimer's disease is a disease caused by functional abnormalities in the brain that causes gradual deterioration of memory, leading to dementia, which causes loss of intellectual functions (thinking, memory, reasoning) severe enough to cause difficulties in daily life. According to the Central Dementia Center, the estimated number of dementia patients aged 65 or older in Korea is 750,488 (as of 2018). 10.2% of the elderly population suffers from dementia. It is estimated that the number will increase to 1 million in 2024, 2 million in 2039, and 3 million in 2050. 15.3 trillion won (0.8% of gross domestic product) is invested annually, including 2.5 trillion won in treatment costs and 4 trillion won in care costs from long-term care insurance for the elderly. The Central Dementia Center estimates the world's dementia population to be approximately 50 million (data from Alzheimer's Disease International).

The main pathological characteristics of Alzheimer's disease are cerebral accumulation of beta-amyloid (Aβ) peptide plaques and neurofibrilary tangles (NFTs) of tau, which toxically affect neurons and synapses during the onset of Alzheimer's disease, leading to neuronal death. A regression mechanism occurs.

A non-invasive method for pathologically diagnosing Alzheimer's disease is positron emission tomography (PET) imaging for beta-amyloid (Aβ) or tau and phosphorylated tau protein. After injecting antibodies that can emit radiation against beta-amyloid or tau and phosphorylated tau protein into the body, brain beta-amyloid aggregation areas are monitored using positron emission tomography imaging. As an invasive method, there is a method of diagnosis by extracting cerebrospinal fluid and detecting beta-amyloid (Aβ) or tau and phosphorylated tau protein transferred from brain tissue to the cerebrospinal fluid. However, the positron emission tomography imaging method is only possible where expensive PET-CT is installed. Since periodic diagnosis is required to diagnose Alzheimer's disease and monitor the degree of pathological progression, the positron emission tomography imaging method not only has cost issues, but also has risks due to periodic radiation exposure. The method of detecting Alzheimer's-specific proteins by extracting cerebrospinal fluid using a non-invasive method also has the disadvantage of the risk of general anesthesia during the extraction process and the pain it causes to the patient after extraction.

Therefore, recently, methods have been developed to pathologically diagnose Alzheimer's disease and measure the course of the disease by detecting Alzheimer's-specific proteins using human derivatives such as blood, saliva, and nasal discharge. Among these, methods for detecting beta-amyloid, tau, and phosphorylated tau in the blood are mainly being developed. This is because by measuring the content of Alzheimer's-specific proteins such as beta-amyloid in the blood, the progress of Alzheimer's can be determined. Specifically, according to recent research results, beta-amyloid is not found to be limited to brain tissue, but beta-amyloid is also produced in blood and other tissues, and the produced protein flows back into brain tissue, contributing to the pathology of Alzheimer's disease. It has been revealed that the severity can be aggravated. However, the concentration of beta-amyloid in blood is 50 to 100 times lower than that in cerebrospinal fluid, and in addition, many other substances in the blood exist in greater quantities than Alzheimer's-specific proteins such as beta-amyloid, tau, and phosphorylated tau. It is difficult to measure the amount of Alzheimer's-specific protein in the blood.

As one of the methods to solve this problem, a method of detection through antigen-antibody binding using specific antibodies against beta-amyloid, tau, or phosphorylated tau has been developed. Most of the developed methods have a step of concentrating Alzheimer's-specific proteins present in trace amounts in plasma using specially treated antibodies so that they can be analyzed. Representative examples include immunomagnetic reduction (IMR), which detects and measures changes in the reaction that occurs in magnetic current when an antibody is attached to a magnetic particle and reacts with blood plasma; after an antibody is covalently linked to a microbead, it reacts with blood to distal Simgle-molecule array (SIMOA) analyzed using ELISA method, Immu-infrared sensor that analyzes the specific molecular structure of beta-amyloid, and Multimer that measures the amount of oligomerization with Aβ in plasma by adding Aβ peptide synthesized in plasma. There is a detection system, etc. However, methods using antigen-antibody binding also require an antibody engineering process that can bind to Alzheimer's-specific proteins in plasma. Additionally, it has the disadvantage of requiring separate expensive analysis equipment and analysis technology to measure antigen-antibody binding.

The purpose of the present invention is to provide a method for pathologically diagnosing Alzheimer's disease by effectively separating beta-amyloid, tau, and phosphorylated tau present in trace amounts in the blood plasma of Alzheimer's patients.

In order to achieve the above object, the present invention provides a biological sample pretreatment composition for diagnosing Alzheimer's disease or mild cognitive impairment containing lipase as an active ingredient.

Additionally, the present invention provides a composition and diagnostic kit for diagnosing Alzheimer's disease or mild cognitive impairment, including the biological sample pretreatment composition and diagnostic reagent.

Additionally, the present invention provides a biological sample pretreatment method for diagnosing Alzheimer's disease or mild cognitive impairment, which includes treating the biological sample with lipase to remove lipids.

In addition, the present invention includes the steps of 1) adding a surfactant and urea mixed solution to a biological sample and dissolving it; 2) adding methanol and chloroform to the solution to precipitate the protein required for analysis; and 3) treating the precipitate with lipase to remove lipids.

In addition, the present invention includes the steps of 1) treating a biological sample with lipase to remove lipids; 2) adding methanol and chloroform to the biological sample from which the lipids have been removed, thereby precipitating proteins required for analysis; and 3) adding a surfactant and urea mixed solution to the precipitate to dissolve it. It provides a biological sample pretreatment method for diagnosing Alzheimer's disease or mild cognitive impairment.

In addition, the present invention 1) measures the concentration of beta-amyloid (Aβ), tau (Tau), or phosphorylated tau (pTauS396) from biological samples pretreated and control biological samples not pretreated by the method described above. steps; 2) calculating the concentration ratio of beta-amyloid (Aβ), Tau, or phosphorylated tau (pTauS396) in the pretreated biological sample to the non-pretreated control biological sample; and 3) if the concentration ratio calculated in step 2) above is high compared to the concentration ratio calculated in the normal control group, providing information necessary for diagnosing Alzheimer's disease or mild cognitive impairment, including determining that it is Alzheimer's disease or mild cognitive impairment. Provides a method to provide.

The present invention relates to a non-cohesive dissolved composition for diagnosing Alzheimer's disease or mild cognitive impairment and a diagnostic method using the same. It tracks pathological changes by measuring total-tau, phosphorylated tau, and beta-amyloid in the plasma of Alzheimer's patients. useful. In particular, additional expensive special equipment is not required, and total-tau, phosphorylated tau, and beta-amyloid in blood can be measured using commonly used antibodies and equipment without additional processing for amyloid antibodies. In addition, the present invention makes it possible to analyze changes in representative proteins of Alzheimer's disease through the buffer treatment-concentration-lipase treatment proposed in the present invention using blood plasma itself, without special processing steps for these amyloid-specific antibodies. do. It can be easily analyzed by applying Western blot and unilaterally commercialized ELISA, and in addition, the sample prepared by the method presented in the present invention can be analyzed without a concentration process using a specially processed antibody in a device currently developed as a method for measuring beta-amyloid in blood plasma. It is expected that the same application will apply. Therefore, the composition and diagnostic method of the present invention not only have high diagnostic accuracy and reproducibility, but are also very simple and can be very useful for early diagnosis of Alzheimer's disease.

Figure 1 is a conceptual diagram of a method of treating plasma extracted from blood with urea/sodium dodecyl sulfate and lipase for diagnosing Alzheimer's disease in the present invention.

Figure 2 shows the results of detecting beta-amyloid in plasma extracted from the blood of an Alzheimer's patient through the method proposed in the present invention. When analyzed together with plasma extracted from the blood of normal people, beta-amyloid could not be detected in samples treated only with sodium dodecyl sulfate (SDS), regardless of whether lipase was treated or not. However, when the method proposed in the present invention - treatment with urea/sodium dodecyl sulfate and lipase treatment - was used, beta-amyloid, which was not detected in samples from normal people, was detected in the plasma of a patient diagnosed with Alzheimer's disease.

Figure 3 shows the results of testing the concentration of usable lipase using plasma from Alzheimer's patients (n = 2). In order to determine the lipase unit appropriate for separating and detecting beta-amyloid present in the blood, the same sample protein was treated with lipase in several units.

Figure 4 shows the results of measuring the change in the amount of beta-amyloid according to the lipase treatment proposed in the present invention after separating proteins from the plasma of normal people and Alzheimer's patients and quantifying them in the same amount. Beta-amyloid dissociated from plasma was confirmed due to lipase treatment. Compared to normal people, more beta-amyloid was detected in the plasma-derived protein of Alzheimer's patients, showing that Alzheimer's disease can be diagnosed through the present invention.

Figure 5 shows the results of measuring the contents of beta-amyloid-42 (Aβ-42) and beta-amyloid-40 (Aβ-40) in the plasma of normal people and Alzheimer's patients using the method of the present invention.

Figure 6 shows the results of measuring the contents of total-tau and phosphorylated tau (S396) in the plasma of normal people and Alzheimer's patients using the method proposed in the present invention.

Figure 7 shows the plasma of early Alzheimer's (asymptomatic AD, aAD) patients with amyloid beta confirmed through amyloid-PET imaging but no cognitive problems using the present invention and Alzheimer's (AD dementia, ADD) patients with cognitive impairment. This is the result of measuring the contents of Amyloid 1-42, Amyloid 1-40, and phosphorylated tau protein (pTauS396) using the method of the present invention and ELISA.

Figure 8 shows the results of the present invention showing the difference between normal people and cognitively impaired dementia patients in terms of Receiver operating characteristic (ROC) curve, AUC (Area under the ROC curve) value, and p-value.

Figure 9 shows the results showing accuracy, sensitivity, and specificity according to the method proposed in the present invention.

The present invention seeks to develop a method for processing samples to quickly and accurately separate, analyze, and identify beta-amyloid, tau, and phosphorylated tau in the plasma of Alzheimer's patients.

Accordingly, in the present invention, before the conventional procedure, a special method is used to change the hydrophobicity of Alzheimer's-specific markers in plasma, such as beta-amyloid, tau, and phosphorylated tau, into analyzable properties and to remove related lipids that reduce the sensitivity of detection. The composition of the dissolution buffer and the separation, purification, and concentration methods based on it were developed. In addition, through the present invention, Alzheimer's patients can be diagnosed regardless of time, place, or cost without specific equipment or expensive immunochemical binding antibodies (e.g., antibodies linked to magnetic particles or micro-beads). was confirmed and the present invention was completed.

The present invention provides a biological sample pretreatment composition for diagnosing Alzheimer's disease or mild cognitive impairment containing lipase as an active ingredient.

Preferably, the composition may further include a surfactant and urea mixed solution, but is not limited thereto.

Additionally, the present invention provides a composition for diagnosing Alzheimer's disease or mild cognitive impairment, comprising the biological sample pretreatment composition and a diagnostic reagent.

Additionally, the present invention provides a diagnostic kit for Alzheimer's disease or mild cognitive impairment comprising the biological sample pretreatment composition and a diagnostic reagent.

Additionally, the present invention provides a biological sample pretreatment method for diagnosing Alzheimer's disease or mild cognitive impairment, which includes treating the biological sample with lipase to remove lipids.

In addition, the present invention includes the steps of 1) adding a surfactant and urea mixed solution to a biological sample and dissolving it; 2) adding methanol and chloroform to the solution to precipitate the protein required for analysis; and 3) treating the precipitate with lipase to remove lipids.

In addition, the present invention includes the steps of 1) treating a biological sample with lipase to remove lipids; 2) adding methanol and chloroform to the biological sample from which the lipids have been removed, thereby precipitating proteins required for analysis; and 3) adding a surfactant and urea mixed solution to the precipitate to dissolve it. It provides a biological sample pretreatment method for diagnosing Alzheimer's disease or mild cognitive impairment.

The term ‘Alzheimer’s disease’ used in the present invention refers to a neurodegenerative disorder and includes familial and sporadic Alzheimer’s disease. Typical symptoms of Alzheimer's include mild to severe cognitive impairment, progressive memory impairment (from mild amnesia to disorientation and severe amnesia), lack of visuospatial skills, personality changes, lack of impulse control, lack of judgment, and distrust of others. , increased stubbornness, restless behavior, lack of planning ability, lack of decision-making, and lack of social skills. Representative symptoms of brain tissue include extracellular beta-amyloid plaque formation, neurofibrillary tangles, neurofibrillary degeneration, synapse loss, and widespread neuronal death.

The Alzheimer's patient used in the present invention refers to a patient who has been clinically diagnosed with Alzheimer's through amyloid-PET (Position Emission Tomography), which is diagnosed by injecting a small amount into the body by linking antibodies to beta-amyloid with radiation.

The term 'beta-amyloid (Aβ)' used in the present invention is a peptide that is an important factor in the formation of amyloid plaques in Alzheimer's disease and is produced by protein hydrolysis from amyloid precursor protein. Alzheimer's disease is characterized by causing functional impairment by inducing neuronal death due to the pathological accumulation of insoluble proteins in vulnerable neuron areas.

The term 'Tau' used in the present invention is a protein that binds to an intracellular structure called a microtubule in brain nerve cells. It regulates the function of the microtubule to support axon growth and neurotransmitter transport in nerve cells, as well as mitochondrial function in nerve cells. It plays a regulating role. The function of tau protein occurs through structural changes within the protein, such as intraprotein phosphorylation. Phosphorylation is regulated flexibly in normal nerve cells, but when Alzheimer's disease begins, phosphorylation occurs within the tau protein, and this protein is phosphorylated at serine position 396 within the soma or Axon-specific region in nerve cells. This phosphorylation causes the tau protein to cleave and spread to the surrounding area, and its hydrophobic characteristics increase, causing a phenomenon called neurofibrillary tangle (NFT), which has a toxic effect on surrounding nerve cells. Along with beta-amyloid plaques, it induces the death of surrounding nerve cells and inhibits the function of brain nerve cells.

In the present invention, 'biological sample' refers to any sample containing protein and includes viruses, microorganisms, cells, animal or plant tissues, animal or plant organs, and their body fluids. Examples of such samples include the brain. It can be a variety of samples such as organs and other brain fluid components, tissues, etc., and samples such as specific disease brain tissue, brain tissue with biomarkers, blood, spinal fluid, tears, or urine related to brain tissue, and the samples. It may include all endoplasmic reticulum, cell rajitates, samples grown through cell culture, or samples from the natural world. The sample can be obtained using methods known in the art. In the present invention, it is preferable to use blood or plasma as a biological sample.

In the present invention, ‘plasma’ is plasma derived from blood and is a sample containing proteins. The sample can be collected using methods known in the art. It is desirable that the collected samples undergo homogenization and dilution processes. The sample collected in this way contains plasma proteins such as albumin and IgG in addition to beta-amyloid, tau, and phosphorylated tau to be analyzed. There is no particular limitation to remove these plasma proteins, but it is preferable to remove them using an immunoseparation method using resin, beads, antibodies, etc. The sample may be used as is, or may be subjected to the above immunoisolation method, sonicator, heat treatment, etc., but is not limited thereto.

In the present invention, a surfactant must be used to dissolve proteins from plasma. The type of surfactant used in the present invention is not particularly limited, but for example, a buffer solution containing sodium dodecyl sulfate (SDS) can be used. An appropriate amount of SDS removes lipid molecules, denatures proteins, and promotes dissolution. However, if used in excess of the appropriate concentration or used alone, it modifies beta-amyloid, tau, and phosphorylated tau present in plasma, making them undetectable. . SDS can be used within 2%, and is more preferably used together with urea (UREA). The mixed solution of SDS/UREA contains SDS/UREA as the main ingredient, as well as triethyl ammonium (TEAB), NaCl, EDTA, and phenylmethylsulfonyl fluoride (PMSF). Appropriate amounts are 50mM, 150mM, 1mM, and 1mM, respectively, but are not limited to these. The reaction temperature and time are 25 to 90 ℃, within 5 hours, but 70 ℃, 2 hours is preferable. The urea concentration is preferably 4 to 8M, so the ratio of the sample to the SDS/UREA mixed solution is 1:1 by volume. This is appropriate, but is not limited to this.

In the present invention, lipids bound to beta-amyloid, tau, and phosphorylated tau extracted from plasma are treated with lipase. Liapase uses high-purity lipase derived from the pancreas, but is not limited to this. In order for the enzyme to react optimally, the reaction temperature is preferably 25 to 50°C, the reaction concentration is 0.5 to 5 unit/μg (quantitative protein result), and the reaction time is preferably 2 hours, but is not limited thereto. Afterwards, lipase is inactivated through reaction at 70°C for 30 minutes. This is a sample to be analyzed by quantifying protein using methods such as BCA (Bicinchoninic acid assay), and is used to measure beta-amyloid, tau, and phosphorylated tau through Western blot and ELISA methods.

In the present invention, the methanol/chloroform protein precipitation method commonly used in the art is used as a method to precipitate the protein required for analysis, but is not limited to this. Based on the total volume of the solution, sequentially add 4 times the volume of 100% Methanol, 1 volume of Chloroform, and 3 times the volume of distilled water. Centrifuge at 14,000 g force and remove the supernatant. Based on the total volume of the sample mixed with 8M Urea/SDS buffer, add 4 times the volume of 100% methanol and mix well, then centrifuge at 20,000 g force to remove the supernatant. After dissolving the remaining precipitate in 50mM Triethyl ammonium (TEAB), the amount of protein is quantified using a method such as BCA (Bicinchoninic acid assay) to confirm the exact protein concentration.

In addition, the present invention includes the steps of 1) measuring the concentration of beta-amyloid (Aβ), tau, or phosphorylated tau (pTauS396) from biological samples pretreated by the above method and control biological samples not pretreated; ; 2) calculating the concentration ratio of beta-amyloid (Aβ), Tau, or phosphorylated tau (pTauS396) in the pretreated biological sample to the non-pretreated control biological sample; and 3) if the concentration ratio calculated in step 2) above is high compared to the concentration ratio calculated in the normal control group, providing information necessary for diagnosing Alzheimer's disease or mild cognitive impairment, including determining that it is Alzheimer's disease or mild cognitive impairment. Provides a method to provide.

Preferably, the beta-amyloid may be beta-amyloid-42 (Aβ-42) or beta-amyloid-40 (Aβ-40), but is not limited thereto.

The term 'diagnosis' used in the present invention refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or condition, and determining whether an object currently has a specific disease or condition. Includes determining the prognosis of an affected subject, or therametrics (e.g., monitoring the condition of a subject to provide information about treatment efficacy).

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

All animal experiment procedures within this specification were conducted in accordance with the regulations of the Animal Experiment Ethics Committee of the Korea Brain Research Institute and, in the case of human samples, the guidelines of the Bioethics Committee of Chosun University and the Bioethics Committee of the Korea Brain Research Institute.

<Example 1>

Subjects were recruited from Chosun University. Clinical dementia rating (CDR) and global deterioration scale (GDS), which are general criteria for judging Alzheimer's patients, were performed, and Alzheimer's patients were diagnosed through amyloid-PET imaging. The criteria for diagnosing Alzheimer's followed the American Psychiatric Association's DSM-IV. Cognitive defects are distinguished through mini-mental state examination (MMSE), and amyloid is confirmed in the amyloid-PET image results, but asymptomatic AD (aAD), which is an early stage of Alzheimer's disease with no cognitive function problems, is diagnosed with amyloid-PET images. As a result, amyloid was confirmed and the patient was selected as an Alzheimer's disease (AD dementia, ADD) patient with significantly reduced cognitive function. The test was approved by the Institutional Review Board of Chosun University, and all participants participated in the experiment with the consent of themselves or their families.

<Example 2>

1. Sample extraction

To separate plasma from blood collected from a patient, the blood was centrifuged at 400 g for 10 minutes in a centrifuge (5430R, Eppendorf), and the supernatant was sequentially centrifuged at 2,000 g for 10 minutes to obtain plasma without blood cells. was separated.

2. Urea/surfactant treatment

Add the above solution to a 0.5mL e-tube, then add 8M urea (U5128, Sigma Aldrich), 2% sodium dodecyl sulfate (SDS; L3771, Sigma Aldrich), and 40mM triethyl ammonium. TEAB; 90114, Thermo), 150 mM sodium chloride (NaCl, S3014, Sigma-Aldrich), 1mM EDTA (Ethylenediaminetetraacetic acid, 15575-038, Invitrogen), 1mM PMSF (phenylmethylsulfonyl fluoride, 93482, Sigma-Aldrich), EDTA-free protease A mixed solution consisting of (Halt™ Protease Inhibitor Cocktail (100X), 87786, Thermo) was added in an amount equal to the volume of the above solution. The reaction was performed at 72 degrees for 1 hour in a heating block (ThermoMixer® C, Eppendorf).

3. Methanol/chloroform protein precipitation method

400 μl of methanol (Methanol, S452-4, Fisher) was added to 100 μl of the treated sample solution and mixed well using a vortex. Again, 100 ㎕ of chloroform (Chloroform, 319988, Sigma Aldrich) was added and mixed using a vortex. Next, 300 ㎕ of ultrapure water solution was added and mixed in the same manner as before. The supernatant was removed by centrifugation at 14,000 g for 2 minutes using a centrifuge (5430R, Eppendorf). 400 ㎕ methanol was added to the remaining solution and mixed using votex. Centrifugation was performed at 14,000 g for 3 minutes to remove as much methanol in the supernatant as possible. After evaporation using a high-speed vacuum concentrator (SpeedVac; SPD1010, Thermo, USA), 50mM triethyl ammonium (Triethylammonium bicarbonate buffer, TEAB; 90114, Thermo) was added to dissolve.

4. Protein quantification

The amount of protein in the solution containing the extracted sample was measured using the Pierce BCA Protein Assay Kit (23225, Thermo Scientific™). BSA was prepared in a volume of 0.1 mL at a concentration of 25 μg/mL using a stepwise dilution method. The solution to be measured was also appropriately diluted or used as is to prepare 0.1 mL. 2.0 mL of reagent was added to the prepared solution containing 0.1 mL of standard or measurement target and mixed well. The mixed solutions were reacted at 37°C for 30 minutes and cooled to room temperature. All samples were measured on a Fast Kinetic Multi-Detection Microplate ReaderFlexStation 3 (Molecular Device) set at 562 nm. After subtracting the measured value of the blank standard solution from the measured value, an expected concentration curve was drawn. The amount of protein in the sample was estimated by comparing the curve and the measured value of the sample.

5. Lipid decomposition and removal

The sample was transferred to a 0.5 mL e-tube, and 3 units of lipase (Sigma Aldrich) per μg of protein were added according to the calculated amount of protein, and then reacted for 1 hour in a heat block (Lab Companion) set at 37°C. The activity of the lipase enzyme was stopped by placing it in a heat block at 70 degrees for 30 minutes.

6. Western blotting

The prepared gel was placed in an electrophoresis device and Tris/Tricine/SDS Running Buffer (1610744, Bio-Rad) was poured. The sample was placed in the groove of the gel, and the same amount of gel loading buffer solution as the sample was added to the unused groove. The electric field of the gel was applied to 70 V/m for 30 minutes, and when bromophenol blue reached the resolving gel, the voltage was raised to 120 V/m and electrophoresis was performed until bromophenol blue reached the end of the resolving gel (approximately 1 hour).

After electrophoresis, the gel was equilibrated with approximately 250 mL of tris/glycine transfer buffer (1016734, Bio-Rad) for 30 minutes. The polyvinylidene difluoride (Immun-Blot PVDF Membrane, 1620177, Bio-Rad) membrane was cut into appropriate sizes and soaked together with the gel in tris/glycine transfer buffer (1016734, Bio-Rad) for 30 minutes. The cassette for Western blot was placed in a solution containing tris/glycine transfer buffer (1016734, Bio-Rad), and a polyvinylidene difluoride (Immun-Blot PVDF Membrane, 1620177, Bio-Rad) membrane, filter paper, and sponge were placed on the gel. After attaching the fixation device to the cassette, transfer it to the blotting chamber, add tris/glycine transfer buffer (1016734, Bio-Rad), set up the electrodes so that the membrane side becomes the cathode and the gel side becomes the anode, and then protein transfer at 400 mA for 50 minutes. was moved to the membrane.

The membrane onto which the antigen (protein) has been transcribed is soaked in phosphorylated wash buffer solution (PBST, Phosphated-buffered saline with Tween-20) containing 5% skim milk, shaken, and left at room temperature for 1 hour. The portion of the membrane to which no protein was bound was blocked. After blocking treatment, it was washed three times with washing buffer (PBST).

Remove the washing buffer, add antigen-specific primary antibodies diluted 1:1,000 in washing buffer (PBST) containing 5% BSA at an appropriate concentration, enough to fully submerge the transfer membrane, and process for more than 8 hours at 37°C. did. After removing the washing buffer solution (PBST) diluted with the primary antibody, the transfer membrane was washed three times for 10 minutes each with the washing buffer solution. Enzyme-labeled secondary antibodies diluted 1:5,000 in wash buffer (PBST) were reacted at room temperature for 1 hour. After removing the secondary antibody dilution buffer, the transfer membrane was washed three times with washing buffer (PBST) solution. An appropriate amount of luminescent substrate solution (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, 34580, Thermo) was added to submerge the transfer film, and the reaction was allowed to occur while gently shaking. When the desired protein band appeared, the reaction was stopped by washing with 10mM EDTA solution. The desired protein was detected using an enhanced chemiluminescence (ECL, ImageQuantTM LAS 4000, GE Healthcare) meter.

<Example 3>

All processes are the same as those in Example 2. However, in step 5 of Example 2, lipase was divided into 0.8, 1.6, and 3.0 unit /μg.

<Example 4>

All processes are the same as those in Example 2. After quantifying the protein in the process of Example 2, the amount of protein in all samples was adjusted to 20 μg.

<Example 5>

All processes are the same as steps 1 to 5 of Example 2.

6. Measurement of β Amyloid 1-42 concentration

The Human Amyloid beta 42 Assay Kit (KHB3441, invitrogen) was used to measure the concentration of β Amyloid 1-42 (Amyloid beta 42 or Abeta 42) in the sample prepared through the above process.

To prepare a standard solution for Aβ 42, the synthesized Aβ 42 peptide was first dissolved in ultrapure water to reach a concentration of 2,000 pg/mL. The dissolved Amyloid beta 42 peptide was diluted 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.25 pg/ml, 15.63 pg/ml, 0 pg/ A standard solution was prepared in ml.

50 ㎕ of the prepared Amyloid 42 peptide standard solution and 50 ㎕ of the sample to be analyzed were added to each well of a 96-well plate. Then, 50 ㎕ of Aβ detection antibody was added to each well and mixed carefully. A cover was placed to prevent the reagent from drying out, and the reaction was allowed to occur while shaking for 3 hours at room temperature. When the reaction of the solution was completed, the solution was removed and washed four times with the provided washing solution.

Dilute the provided Anti-Rabbit IgG HRP by 1/100, add 100 ㎕ of Anti-Rabbit IgG HRP solution to each well containing the Amyloid beta 42 standard solution and the well containing the sample, and react for 30 minutes in the same manner as above. , the solution was removed and washed. Stabilized chromogen was added to each well and reacted at room temperature without light for 30 minutes. After the reaction, 100 ㎕ of Stop solution was added.

After the reaction was completed, the absorbance of the standard solution and sample was measured in a spectrometer (Fast Kinetic Multi-Detection Microplate ReaderFlexStation 3, Molecular Device) set to 450 nm after 2 hours. After subtracting the measured value of chromogen blanks empty from the measured value, an expected concentration curve was drawn. By comparing the curve and the measured value of the sample, the value of Amyloid beta42 present in the sample was calculated.

<Example 6>

All processes are the same as steps 1 to 5 of Example 2.

6. Measurement of β Amyloid 1-40 concentration

To measure the concentration of β Amyloid 1-40 (Amyloid beta 40 or Abeta 40) in the sample prepared through the above process, the Human Amyloid beta 40 Assay Kit (KHB3481, invitrogen) was used.

Standard solutions were prepared through the stepwise dilution method. 55 mM Sodium bicarbonate buffer (pH9.0) was added so that the standard Amyloid beta 40 solution had a concentration of 100 ng/mL. 900 μL Standard Diluent buffer solution was added to 100 μL of the standard solution prepared in this way to make a standard solution of 10,000 pg/mL. 100 μl of this solution was added to 1,900 μL Standard Diluent buffer solution to create a standard solution of 500 pg/mL. Again, standard solutions with 7 different concentrations - 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 pg/mL were created by adding 150 ㎕ standard solution and 150 ㎕ Standard Diluent buffer solution. The subsequent method is the same as Example 4.

<Example 7>

All processes are the same as steps 1 to 5 of Example 2.

6. Measurement of Tau concentration

To measure the concentration of Tau in the sample prepared through the above process, the Tau (Total) Human ELISA Kit (KHB0441, invitrogen) was used.

The Standard Dilution buffer solution was mixed so that the concentration of Hu Tau (Total) Standard was 2,000 pg/mL. By adding 300 μL solution and 300 μL Standard Diluent buffer solution to the standard solution prepared in this way, standard solutions with 7 types of concentrations - 500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 pg/mL were created. The subsequent method is the same as Example 4.

<Example 8>

All processes are the same as steps 1 to 5 of Example 2.

6. Measurement of concentration of Tau (S396 phosphorylation)

To measure the concentration of pTau in the sample prepared through the above process, the Human Tau [pS396] ELISA Kit (KHB7031, invitrogen) was used.

The Standard Dilution buffer solution was mixed so that the concentration of Hu Tau [pS396] Standard was 5,000 pg/mL. 120 ㎕ of this solution and 480 ㎕ of Standard Diluent buffer solution were added to create a standard solution of 1,000 pg/mL. The standard solution prepared in this way was prepared by adding 300 μL solution and 300 μL Standard Diluent buffer solution in the same manner as in Example 6 to obtain 7 different concentrations - 500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 pg/mL. Eggplant prepared a standard solution. The subsequent method is the same as Example 5.

<Comparative Example 1>

The processing process of the obtained patient's blood was the same as Example 2. However, lipase was not added in step 5, but the same volume of the solution used to dissolve lipase was added and reacted for 1 hour in a heat block (Lab Companion) set at 37°C. Then, it was left in the same heat block at 70 degrees for 30 minutes.

<Comparative Example 2>

The process of processing the obtained patient's blood was the same as Example 2 and Comparative Example 1. However, in this comparative example, the mixed solution of urea and surfactant was not treated, and sodium dodecyl sulfate (SDS; L3771, Sigma Aldrich) and 40mM triethyl ammonium were added in step 2 of Example 2. TEAB; 90114, Thermo), 150 mM sodium chloride (NaCl, S3014, Sigma-Aldrich), 1mM EDTA (Ethylenediaminetetraacetic acid, 15575-038, Invitrogen), 1mM PMSF (phenylmethylsulfonyl fluoride, 93482, Sigma-Aldrich), EDTA-free protease A mixed solution consisting of (Halt™ Protease Inhibitor Cocktail (100X), 87786, Thermo) was added in an amount equal to the volume of the solution containing the sample. The reaction was performed at 72 degrees for 1 hour in a heating block.

<Comparative Example 3>

The processing process of the obtained patient's blood was the same as Example 3. However, lipase was not added, but the same volume of the solution used to dissolve lipase was added and reacted for 1 hour in a heat block (Lab Companion) set at 37°C. Then, it was left in the same heat block at 70 degrees for 30 minutes.

<Comparative Example 4>

The processing process of the obtained patient's blood was the same as Example 4. However, lipase was not added in step 5, but the same volume of the solution used to dissolve lipase was added and reacted for 1 hour in a heat block (Lab Companion) set to 37°C. Then, it was left in the same heat block at 70 degrees for 30 minutes.

<Comparative Example 5>

The processing process of the obtained patient's blood was the same as Example 5 and Comparative Example 5. However, lipase was not added in step 5, but the same volume of the solution used to dissolve lipase was added and reacted for 1 hour in a heat block (Lab Companion) set to 37°C. Then, it was left in the same heat block at 70 degrees for 30 minutes.

<Comparative Example 6>

The processing process of the obtained patient's blood was the same as Example 6. However, lipase was not added in step 5, but the same volume of the solution used to dissolve lipase was added and reacted for 1 hour in a heat block (Lab Companion) set to 37°C. Then, it was left in the same heat block at 70 degrees for 30 minutes.

<Comparative Example 7>

The processing process of the obtained patient's blood was the same as Example 7. However, lipase was not added in step 5, but the same volume of the solution used to dissolve lipase was added and reacted for 1 hour in a heat block (Lab Companion) set to 37°C. Then, it was left in the same heat block at 70 degrees for 30 minutes.

<Comparative Example 8>

The processing process of the obtained patient's blood was the same as Example 8. However, lipase was not added in step 5, but the same volume of the solution used to dissolve lipase was added and reacted for 1 hour in a heat block (Lab Companion) set to 37°C. Then, it was left in the same heat block at 70 degrees for 30 minutes.

<Control Example 1>

In Example 1, blood from a normal person was obtained. The processing process of the obtained blood was the same as Example 2, Comparative Example 1, and Comparative Example 2.

<Control Example 2>

In Example 1, blood from a normal person was obtained. All subsequent processes are the same as those of Example 4 and Comparative Example 4. After adjusting the protein amount of all samples to 20 μg, Western blotting was performed.

<Control Example 3>

In Example 1, blood from a normal person was obtained. The processing process of the obtained blood was the same as Example 5 and Comparative Example 5.

<Control Example 4>

In Example 1, blood from a normal person was obtained. The processing process of the obtained blood was the same as Example 6 and Comparative Example 6.

<Control Example 5>

In Example 1, blood from a normal person was obtained. The processing process of the obtained blood was the same as Example 7 and Comparative Example 7.

<Control Example 6>

In Example 1, blood from a normal person was obtained. The processing process of the obtained blood was the same as Example 8 and Comparative Example 8.

<Experimental Example 1>

The primary antibodies used in Western blotting to analyze the results of Example 2, Comparative Example 1, Comparative Example 2, and Control Example 1 are as follows.

- Aβ (D54D2) (β-Amyloid (D54D2) XP ^ⓡ Rabbit mAb, 8243s, Cell Signaling)

- β-Actin Anti-beta-actin, Clone OTI1 (Origene, TA811000S)

The secondary antibodies used were Rabbit IgG-HRP (NA-934, GE Healthcare) for Aβ (D54D2) and mouse IgG-HRP (NA-931, GE Healthcare) for β-Actin.

β-Amyloid (D54D2) XP® Rabbit mAb is an antibody that binds primarily to human-derived Aβ-42, Aβ-40, Aβ-39, Aβ-38, and Aβ-37 peptides. These Aβ peptides aggregate to form a β sheet. Additionally, because all blood contains β-Actin, antibodies against β-Actin and Aβ, an Alzheimer's specific marker, were used in the Western blotting experiment.

To measure the expression level of the protein, plasma of Alzheimer's patients recruited for the experiment was analyzed. Specifically, Western blotting experiments were performed on blood obtained from the patients recruited in Example 1 in the same manner as Example 2, Comparative Example 2, and Control Example 2. As a result, as shown in Figure 2, it was confirmed that beta-amyloid peptide was clearly extracted from the plasma of Alzheimer's patients when treated with 2% sodium dodecyl sulfate and 8M urea, which is the method proposed by the present invention, and then treated with lipase. did. As a comparative example, when lyapase was not treated, almost no beta-amyloid peptide was found in the plasma of Alzheimer's patients. In addition, when only 5% sodium dodecyl sulfate was used as a control example, the expression level of beta-amyloid peptide extracted from all samples regardless of lipase treatment was almost insignificant.

<Experimental Example 2>

The primary antibodies used in Example 3 and Comparative Example 3 are as follows.

- Aβ (D54D2) (β-Amyloid (D54D2) XP® Rabbit mAb, 8243s, Cell Signaling)

- CD63 polyclonal antibody (AB0047-200, Sicgen)

The secondary antibodies used were Rabbit IgG-HRP (NA-934, GE Healthcare) for Aβ (D54D2) and mouse anti Goat-HRP (ab157532, Abcam) for CD63. Blood generally contains exosomes. Therefore, Western blotting was performed using antibodies against CD63, an exosome marker, and Aβ, an Alzheimer's specific marker.

An experiment was performed as in Example 3 to find an appropriate concentration of lipase capable of detecting beta-amyloid through the present invention. For comparison, an experiment was performed as in Comparative Example 3 without lipase. As can be seen from the results in Figure 3, it was confirmed that the more the lipase was treated with the plasma of the two patients, the greater the amount of beta-amyloid isolated and extracted. As the concentration of lipase became stronger, proteins such as CD63 in the same sample disappeared by lipase, and the efficiency of identifying beta-amyloid increased. Therefore, through Experimental Example 3, it was confirmed that lipase treatment of 0.1 to 3 units/μg was appropriate.

<Experimental Example 3>

The primary antibodies used in Example 4, Comparative Example 4, and Control Example 2 are as follows.

- Aβ (D54D2) (β-Amyloid (D54D2) XP® Rabbit mAb, 8243s, Cell Signaling)

- APOE (ApoE (pan) (D7I9N) Rabbit mAb, 13366, Cell signaling)

Rabbit IgG-HRP (NA-934, GE Healthcare) was used as the secondary antibody.

In general, beta-amyloid is detected in the blood of normal people. Proteins extracted from plasma extracted from blood obtained from Alzheimer's patients were quantified and an amount of 20 μg was prepared, and then treated with lipase (Example 4), or an equal volume of solution was added instead (Comparative Example 4). Similarly, the protein extracted from plasma extracted from the blood of a normal person is quantified and an amount of 20 μg is prepared, and then treated with lipase, or the same volume of solution is added instead (Control Example 2). As shown in Figure 4, APOE protein is disappeared by lyapase, but beta-amyloid peptide is confirmed in both plasma extracted proteins of normal people and Alzheimer's patients. In addition, the expression level of beta-amyloid peptide in Alzheimer's patients is higher and clearly differentiated compared to the expression level in normal people. Through this, it can be seen that the method of the present invention using lipase is meaningful as a diagnostic method by detecting beta-amyloid present in the blood of Alzheimer's patients.

<Experimental Example 4>

Both Aβ40 and Aβ42 are known to be increased 2-3 times in patients with familial Alzheimer's disease, which is caused by a mutation in the preseniln gene, compared to normal people or patients with sporadic Alzheimer's disease. Additionally, it has been reported that Aβ40 is increased in Alzheimer's disease patients with the ApoE ε4 allele. However, as Alzheimer's disease progresses, Aβ42 is deposited in the brain and the amount of Aβ42 in the blood decreases, but it is known that the increased amount of Aβ40 does not change.

The antibody used in the experiment was the antibody provided by the ELISA kit used in Example 5/6, Comparative Example 5/6, and Control Example 3/4.

The content of β Amyolid 1-42 in the plasma of Alzheimer's patients who donated blood to this experiment was measured using ELISA (Example 5, n = 7). For comparison, the content of β Amyolid 1-42 was measured in the same plasma without lipase treatment using ELISA (Comparative Example 5). In addition, β Amyolid 1-42 in the plasma of normal people was also measured (Control Example 5, n = 8). As shown in Figure 5, when the content of Aβ42 extracted from the plasma of an Alzheimer's patient was measured using the method of the present invention, it was confirmed that it significantly (p = 0.058) increased compared to the method not used. However, the amount of Aβ42 extracted from the plasma of normal people did not differ depending on whether the method of the present invention was used. When treated with the lipase proposed by the present invention, the amount of Aβ-42 isolated from the plasma of Alzheimer's patients was significantly increased compared to the plasma of normal people.

In order to confirm whether the present invention can be used to distinguish plasma from patients with Alzheimer's disease, the ratio of the amount of Aβ42 extracted when the method of the present invention was used in the plasma of normal people and the amount of Aβ42 when the method was not used was measured. As with plasma from patients with Alzheimer's disease, the ratio of the amount of Aβ42 extracted using this method and the amount of Aβ42 when not used was measured. As a result, as shown in Figure 5, the proportion of plasma from Alzheimer's disease patients was not only higher than that of normal people, but also showed a significant difference (p = 0.05). However, when lipase was not treated, there was no significant difference in the extract content of Aβ-42 in the plasma of normal people and Alzheimer's patients.

Plasma from Alzheimer's patients was treated using the method of the present invention, and the content of Amyloid 1-40 was measured through ELISA (Example 6, n=9). For comparison, the same plasma was not treated with lipase, and the content of Amyloid 1-40 was measured through ELISA (Comparative Example 6). In the same way, the content of Amyloid 1-40 in the plasma of normal people was measured using ELISA (Control Example 4, n = 8). As a result of the measurement, in the case of Aβ-40, as shown in Figure 5, when using the lipase treatment method proposed by the present invention, the amount extracted from the plasma of Alzheimer's patients was significantly higher than the method without lipase treatment (p<0.01 ) increased significantly. In particular, when comparing the extracted content of Aβ-40 in the plasma of normal people and Alzheimer's patients using the method proposed by the present invention, a significant (p = 0.086) difference was confirmed. However, when lipase was not treated, there was no significant difference in the Aβ-40 extract content present in the plasma of normal subjects and Alzheimer's patients.

As in Example 5, Comparative Example 5, and Control Example 3, in order to determine whether the present invention can be used to distinguish plasma from patients with Alzheimer's disease, the content of Aβ-40 extracted from plasma using the present invention and Aβ when not used were measured. The content ratio of -40 was measured. As a result, as shown in Figure 5, the ratio of Aβ-40 content extracted from the plasma of Alzheimer's patients was not only higher than the ratio of Aβ-40 extracted from the plasma of normal people, but also showed a significant difference (p = 0.03).

As in Example 5, Comparative Example 5, and Control Example 3, amyloid beta was confirmed through amyloid-PET imaging using the present invention, but it was used in patients with early Alzheimer's disease (asymptomatic AD, aAD) and cognitive impairment without cognitive problems. The contents of Amyloid 1-42 and Amyloid 1-40 were measured in plasma from patients with impaired AD dementia (ADD) using the method of the present invention and ELISA (Example 6, Asymptomatic Dementia total n = 44_ Female n = 22 , Male n = 22) (Example 6, cognitive impairment dementia total n = 44_ Female n = 26, Male n = 18). For comparison, the contents of Amyloid 1-42 and Amyloid 1-40 were measured using ELISA in the same plasma without lipase treatment (Comparative Example 6). In the same way, the content of Amyloid 1-40 in the plasma of normal people was measured through ELISA (Control Example 4, total n = 88_ Female n = 44, Male n = 44). As a result of the measurement, as shown in Figure 7, it was confirmed that when the method of treating lipase proposed by the present invention was used, there was a significant difference between normal people, patients with early dementia, and patients with cognitive impairment dementia in the male group compared to the method without treatment. . Even in the case of women, it was confirmed that cognitive impairment dementia patients were significantly different from normal subjects when analyzed using lipase treatment. In the female asymptomatic dementia patient group, only Amyloid 1-40 showed no difference from normal subjects regardless of lipase treatment, but Amyloid 1-42 showed a significant difference from normal subjects. When looking at the Abeta1-42/Abeta1-40 ratio, it was confirmed that there was a significant difference compared to normal people in both the asymptomatic dementia patient group and the cognitive impairment dementia patient group. These results can be seen in Figure 8 through the present invention technique, the difference between normal people and asymptomatic dementia patients or cognitive impairment patients can be seen through the Receiver operating characteristic (ROC) curve and AUC (Area under the ROC curve) value and p-value. Accuracy is specified through Figure 9.

<Experimental Example 5>

To measure the content of tau protein, known as another marker for Alzheimer's diagnosis, an ELISA experiment was performed as in Example 7, Comparative Example 7, and Control Example 5 (normal blood n = 8, Alzheimer's patient blood n = 8). As shown in Figure 6, it was confirmed that when lipase was treated using the method proposed by the present invention, tau protein was extracted significantly (p<0.01) compared to when it was not treated (Example 7, Comparative Example 7). However, when tau protein was extracted from the blood of normal people, the difference depending on the use of the present invention was not significant. As in Experimental Example 4 above, in order to confirm whether the present invention can be used to distinguish plasma from patients with Alzheimer's disease, the ratio of the tau protein content extracted from plasma using the present invention and the content of tau protein when not used was measured. As a result, as shown in Figure 6, the ratio of tau protein extracted from the plasma of Alzheimer's patients according to the method proposed by the present invention showed a significant difference (p = 0.04) compared to the ratio of protein extracted from the plasma of normal people. .

Therefore, the same sample was treated as in Example 8, Comparative Example 8, and Control Example 6, and the content of phosphorylated tau protein (pTauS396) was measured by ELISA (normal blood n = 8, Alzheimer's patient blood n = 8). It was confirmed that phosphorylated tau protein extracted from the plasma of Alzheimer's patients was significantly (p<0.001) increased when extracted using the method proposed by the present invention compared to when not used (Example 8 and Comparative Example 8). In addition, when treated with lipase, it was confirmed that the phosphorylated tau protein in Alzheimer's patients was significantly (p < 0.01) increased compared to the phosphorylated tau protein extracted from normal patients (Control Example 6). In order to check whether the present invention can be used to distinguish plasma from patients with Alzheimer's disease as in Experimental Example 4, the ratio of the content of phosphorylated tau protein extracted from plasma using the present invention was compared to the content of phosphorylated tau protein when not used. Measured. As a result, as shown in Figure 6, the ratio of tau protein extracted from the plasma of Alzheimer's patients according to the method proposed by the present invention showed a significant (p = 0.01) difference compared to the ratio of protein extracted from the plasma of normal people. .

Through Experimental Examples 4 and 5, this method of treating lipase as the main ingredient showed beta-amyloid peptides, especially Aβ-42, Aβ-40 and tau, and phosphorylated tau protein (pTauS396) in the blood of Alzheimer's patients. It was confirmed that effective separation and measurement can be performed. It was confirmed that Alzheimer's disease can be diagnosed by more effectively extracting Aβ-42, Aβ-40, and phosphorylated tau protein (pTauS396) using the method proposed by the present invention.

As in Example 8, Comparative Example 8, and Control Example 6, using the present invention, patients with early Alzheimer's disease (asymptomatic AD, aAD) and Alzheimer's disease with cognitive impairment in which amyloid beta is confirmed through amyloid-PET imaging but do not have cognitive impairment The content of phosphorylated tau protein (pTauS396) was measured in the plasma of patients with (AD dementia, ADD) using the method of the present invention and ELISA (Example 8, asymptomatic dementia total n = 33_ Female n = 16, Male n = 17 ), (Example 8, cognitive impairment dementia total n = 41_ Female n = 23, Male n = 18). For comparison, the same plasma was not treated with lipase, and the content of phosphorylated tau protein (pTauS396) was measured through ELISA (Comparative Example 8). In the same way, the content of phosphorylated tau protein (pTauS396) in the plasma of normal people was measured through ELISA (Control Example 4, total n = 74_ Female n = 42, Male n = 32). As a result of the measurement, as shown in FIG. 7, it was confirmed that when the method of treating lipase proposed by the present invention was used, there was a significant difference between normal people and cognitively impaired dementia patients in the female and male groups compared to the method without treatment. These results can be seen through the receiver operating characteristic (ROC) curve and AUC (Area under the ROC curve) value and p-value of the difference between normal people and cognitively impaired dementia patients through the present invention technique in Figure 8, but asymptomatic dementia patients It was confirmed that there was no difference from normal subjects in terms of phosphorylated tau protein (pTauS396) in the female and male groups.

Therefore, as shown in Figure 9, the analysis accuracy for each item, through these results, the present inventors used Alzheimer's disease markers, especially beta- In order to separate and identify amyloid peptides and phosphorylated proteins, treatment of the patient's plasma through the method of the present invention and treatment with a solution containing lipase as an active ingredient can be said to be effective methods.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (13)

1. A biological sample pretreatment composition for diagnosing Alzheimer's disease or mild cognitive impairment containing lipase as an active ingredient.
2. The biological sample pretreatment composition according to claim 1, further comprising a surfactant and urea mixed solution.
3. A composition for diagnosing Alzheimer's disease or mild cognitive impairment comprising the biological sample pretreatment composition of claim 1 or 2 and a diagnostic reagent.
4. A diagnostic kit for Alzheimer's disease or mild cognitive impairment comprising the biological sample pretreatment composition of claim 1 or 2 and a diagnostic reagent.
5. A biological sample pretreatment method for diagnosing Alzheimer's disease or mild cognitive impairment, comprising removing lipids by treating the biological sample with lipase.
6. 1) adding a surfactant and urea mixed solution to the biological sample to dissolve it;

   2) adding methanol and chloroform to the solution to precipitate the protein required for analysis; and

   3) A biological sample pretreatment method for diagnosing Alzheimer's disease or mild cognitive impairment, comprising the step of treating the precipitate with lipase to remove lipids.
7. 1) Removing lipids by treating a biological sample with lipase;

   2) adding methanol and chloroform to the biological sample from which the lipids have been removed, thereby precipitating proteins required for analysis; and

   3) A biological sample pretreatment method for diagnosing Alzheimer's disease or mild cognitive impairment, comprising the step of dissolving a mixed solution of surfactant and urea in the precipitate.
8. The method according to claim 6 or 7, wherein the surfactant and urea mixed solution is a mixture of 1 to 4% Sodium Dodecyl Sulfate (SDS) and 4 to 8M urea.
9. The method according to any one of claims 5 to 7, wherein the lipase treatment is performed with 0.5 to 5 unit/μg lipase at 25 to 50°C for 0.1 to 2 hours.
10. The method according to claim 6 or 7, wherein the protein required for the analysis is beta-amyloid (Aβ), tau, or phosphorylated tau (pTauS396).
11. The method according to any one of claims 5 to 7, wherein the biological sample is blood, plasma, tissue, cells, cerebrospinal fluid, tears, or urine.
12. 1) Beta-amyloid (Aβ), tau, or phosphorylated tau (pTauS396) from biological samples pretreated by the method according to any one of claims 5 to 7 and control biological samples without pretreatment. ) measuring the concentration of;

    2) calculating the concentration ratio of beta-amyloid (Aβ), tau, or phosphorylated tau (pTauS396) in the pretreated biological sample to the non-pretreated control biological sample; and

    3) If the concentration ratio calculated in step 2) above is high compared to the concentration ratio calculated in the normal control group, it provides information necessary for diagnosing Alzheimer's disease or mild cognitive impairment, including the step of determining Alzheimer's disease or mild cognitive impairment. How to.
13. The method of claim 12, wherein the beta-amyloid is beta-amyloid-42 (Aβ-42) or beta-amyloid-40 (Aβ-40).

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