KR102041115B1 - Composition and kit for distinguishing between mild Alzheimer's disease and moderate Alzheimer's disease using nasal fluid - Google Patents

Abstract

The present invention is a composition for screening progression stages of Alzheimer's disease, including kits for measuring beta amyloid oligomers and S100A9 protein expression levels in a runny nose samples, kits and runny nose expression levels of beta amyloid oligomers and S100A9 protein It relates to a method for screening progression of Alzheimer's disease comprising the step of measuring. The present invention also includes a method for screening a mild Alzheimer's disease therapeutic agent comprising the step of measuring the expression level of beta amyloid oligomer and S100A9 protein in a runny nose sample and an agent for measuring the expression level of beta amyloid oligomer and S100A9 protein in a runny nose sample. The present invention relates to a screening kit for treating a mild Alzheimer's disease.

Description

Composition and kit for distinguishing mild and moderate Alzheimer's disease using a runny nose sample {composition and kit for distinguishing between mild Alzheimer's disease and moderate Alzheimer's disease using nasal fluid}

The present invention relates to a composition for screening progression of Alzheimer's disease using a runny nose sample and a method for screening progression of Alzheimer's disease using the same, and more specifically, to the expression level of beta amyloid oligomer and S100A9 protein in a runny nose sample. The present invention relates to a method for screening progression of Alzheimer's disease, comprising measuring the expression level of beta amyloid oligomer and S100A9 protein in a composition, kit, and runny nose screening composition including an agent for measuring an amount thereof. The present invention also includes a method for screening a mild Alzheimer's disease therapeutic agent comprising the step of measuring the expression level of beta amyloid oligomer and S100A9 protein in a runny nose sample and an agent for measuring the expression level of beta amyloid oligomer and S100A9 protein in a runny nose sample. The present invention relates to a screening kit for treating a mild Alzheimer's disease.

Degenerative brain disease is a disease that occurs in the brain as you age. To date, unknown causes of certain brain cell populations in the brain slowly lose their function, the death of brain neurons, which is the most important for the transmission of information in the brain nervous system, the formation of synapses or functional problems that transmit information between brain nerve cells and brain nerve cells, or It is known to be caused by an ideal increase or decrease in electrical activity of the cranial nerve. Degenerative brain diseases can be distinguished by considering the main symptoms and the invading brain areas.Alzheimer's disease (AD) is associated with disease-specific protein expression, killing of specific cell populations, and specific outcomes compared to other brain diseases. Symptoms can be confirmed.

With the recent rapid increase in Alzheimer's disease, there is an urgent need for early diagnosis, prevention and treatment, but to date, the diagnosis of Alzheimer's disease is a neuropsychological test, for example, the Short Form of Depression. Only time-consuming and costly methods such as Geriatric Depression Scale (GDS) or mini-mental state examination (MMSE) or specialized MRI imaging were possible.

For example, the conventional Alzheimer's disease diagnosis method, there is a brain imaging technique using a high-resolution brain imaging apparatus. Early diagnosis of Alzheimer's disease using brain imaging techniques is performed by taking a brain scan of patients with suspected Alzheimer's disease, measuring the degree of abnormal accumulation of beta amyloid protein, and comparing the results with postmortem brain tissue tests. Study the precision of photographing equipment. However, the image-based diagnostic method not only requires a high cost to the patient, but also detects the disease because the discrimination is performed in a state where brain contraction or damage is already in progress.

Another representative diagnostic method is the diagnosis of spinal fluid, which measures changes in the amount of beta amyloid protein in the cerebrospinal fluid. However, the cerebrospinal fluid test itself is known to be a painful method for patients, and it is pointed out that it is accompanied by a risk during the test.

Under this background, the present inventors have developed a method for diagnosing cerebral neurological diseases using a runny nose sample collected by a non-invasive method and disclosed in Korean Patent Laid-Open No. 10-2015-0144283. Specifically, the patent discloses a method for diagnosing cranial nerve disease by measuring the expression level of beta amyloid oligomer in a runny nose sample.

However, there is still a need for a non-invasive diagnostic method that can not only confirm the onset of cerebral neuropathy but also to precisely diagnose the progression stage of cerebral neuropathy.

Accordingly, the present inventors identify a protein that specifically expresses only mild Alzheimer's disease (Mild AD) corresponding to the initial stage of Alzheimer's disease in a runny nose sample, and screens the progression of Alzheimer's disease using the same. The present invention has been completed by the development.

Accordingly, an object of the present invention is an agent for measuring the expression level of beta amyloid oligomer in a runny nose sample; It provides a composition and kit for screening the progress stage of Alzheimer's disease comprising; and an agent for measuring the expression level of the S100A9 protein in the isolated runny nose sample.

Another object of the present invention is to provide a method for screening progression of Alzheimer's disease, comprising measuring the expression levels of beta amyloid oligomer and S100A9 protein in a runny nose sample.

Still another object of the present invention is to provide a method for treating a mild Alzheimer's disease therapeutic agent in a runny nose sample isolated from a patient suffering from Mild Alzheimer's disease; b) measuring the expression level of beta amyloid oligomer in the sample of step a); And c) measuring the expression level of the S100A9 protein in the sample of step b) to provide a method for screening a mild Alzheimer's disease therapeutic agent comprising a.

Still another object of the present invention is to provide a screening kit of a mild Alzheimer's disease therapeutic agent comprising an agent for measuring the expression level of beta amyloid oligomer and S100A9 protein in a runny nose sample.

In order to solve the above problems, the present invention provides an agent for measuring the expression level of beta amyloid oligomer in isolated runny nose samples; It provides a composition for screening the progression stage of Alzheimer's disease comprising; and an agent for measuring the expression level of the S100A9 protein in the isolated runny nose sample.

According to a preferred embodiment of the present invention, the agent for measuring the expression level of the beta amyloid oligomer may be an antibody or aptamer specifically binding to the beta amyloid oligomer, the agent for measuring the S100A9 protein expression level is S100A9 protein It may be an antibody or aptamer that specifically binds to.

According to another preferred embodiment of the present invention, the composition for screening the progress stage of Alzheimer's disease may further comprise an agent for measuring the expression level of the HYOU1 protein in the isolated runny nose sample.

According to another preferred embodiment of the present invention, the agent for measuring the expression level of the HYOU1 protein may be an antibody or aptamer specifically binding to the HYOU1 protein.

The present invention also provides a kit for screening the stage of progression of Alzheimer's disease comprising the composition described above.

The present invention also provides a method for preparing a beta amyloid oligomer, the method comprising the steps of: And b) measuring the expression level of the S100A9 protein in the sample of step a) provides a method for screening progression of Alzheimer's disease comprising a.

According to a preferred embodiment of the present invention, when it is confirmed that the beta amyloid oligomer is expressed in step a) and the S100A9 protein is expressed in step b), the diagnosis is mild Alzheimer's disease; When the beta amyloid oligomer is expressed in step a) and the S100A9 protein is not expressed in step b), it may be diagnosed as moderate Alzheimer's disease.

According to another preferred embodiment of the present invention, the method for screening progression of Alzheimer's disease may further comprise c) measuring the expression level of HYOU1 protein in the sample of step b).

According to another preferred embodiment of the present invention, the beta amyloid oligomer is expressed in the step a), the S100A9 protein is expressed in the step b), the hardness is confirmed that the HYOU1 protein is expressed in the step c) Diagnosed as Alzheimer's disease; When the beta amyloid oligomer is expressed in step a), the S100A9 protein is not expressed in step b), and the HYOU1 protein is not expressed in step c), it is diagnosed as moderate Alzheimer's disease. Can be.

According to another preferred embodiment of the present invention, the measurement of steps a), b) and c) is Western blot, enzyme linked immunosorbent assay (ELISA), immunoprecipitation assay (Immunoprecipitation Assay), complement fixation assay (Complement Fixation Assay), Fluorecence Activated Cell Sorter (FACS), or a protein chip.

The present invention also provides a method for treating a mild Alzheimer's disease therapeutic agent in a runny nose sample isolated from a patient suffering from Mild Alzheimer's disease; b) measuring the expression level of beta amyloid oligomer in the sample of step a); And c) measuring the expression level of S100A9 protein in the sample of step b).

According to a preferred embodiment of the present invention, the expression level of the beta amyloid oligomer in step b) is lower than before treating the candidate, and in step c) the expression level of the S100A9 protein is lower than before treating the candidate. If decreased, it may be selected as a treatment for mild Alzheimer's disease.

According to another preferred embodiment of the present invention, the method for screening the mild Alzheimer's disease therapeutic agent may further comprise d) measuring the expression level of HYOU1 protein in the sample of step c).

According to another preferred embodiment of the present invention, the expression level of the beta amyloid oligomer in step b) is lower than before treatment with the candidate, and in step c) the expression level of the S100A9 protein treated with the candidate It can be selected as a therapeutic agent for mild Alzheimer's disease when the expression level of HYOU1 protein decreases in step d) than before treatment with the candidate material.

The present invention also provides an agent for measuring the expression level of beta amyloid oligomer in isolated runny nose samples; It provides a screening kit of a mild Alzheimer's disease therapeutic agent comprising; and an agent for measuring the expression level of S100A9 protein in the isolated runny nose sample.

According to one preferred embodiment of the present invention, the screening kit of the mild Alzheimer's disease therapeutic agent may further comprise an agent for measuring the expression level of the HYOU1 protein in the isolated runny nose sample.

The method of screening progression of Alzheimer's disease of the present invention can screen the Alzheimer's progression stage accurately and quickly in a non-invasive way by simultaneously identifying proteins representing two or more different stages in a runny nose sample. Conventional methods for diagnosing Alzheimer's disease have attempted to secure the accuracy of diagnosis by using two or more methods together, but this has problems in terms of cost and accessibility and problems in confirmation due to the organic linkage between the diagnostic methods. . However, the method of the present invention can screen the diagnosis and progression stages of Alzheimer's disease at a time without overcoming other diagnostic methods, thereby overcoming the problems of the existing Alzheimer's diagnosis method.

Figure 1 shows a schematic diagram of a biomarker verification confirmation procedure through proteomics.
Figure 2 is a comparison of the results of the proteomics using a runny nose sample of the mild Alzheimer's disease patients and the normal group, (A) is a gel image of the runny protein and (B) is a graph showing the analysis of the gel band concentration. Bars represent mean values including the standard error of the mean, gray bars represent control, and red bars represent mild Alzheimer's patients (* P <0.05).
Figure 3 shows a comparison of the proteome changes detected through the runny nose samples of patients with mild Alzheimer's disease.
4 is a result of quantitative analysis of the biomarker proteins S100A9, HYOU1 and SAA2 selected through the runny nose samples of the mild Alzheimer's disease patient group and the normal group, respectively, in the runny nose samples collected from the normal group, the mild Alzheimer patient group and the moderate Alzheimer patient group, ( A) shows the protein expression level using S100A9, HYOU1 and SAA2 antibodies, respectively, through immunoblotting, and (B) shows the graph quantifying the expression level of each protein ( ^*** P <0.001, * * P <0.01, * P <0.05, a = 0.0622).
Figure 5 is a protein expressed in all Alzheimer patients beta amyloid oligomers and mild Alzheimer's disease patients and normal runny nose samples selected from the runny nose samples collected from normal, mild Alzheimer's patients and moderate Alzheimer's patients group As a result of quantitative analysis, (A) shows the protein expression level using A11 and S100A9 antibodies through immunoblotting, and (B) shows the graph quantifying the expression level of each protein (** P <0.01, *** P <0.001).
6 shows a schematic of a diagnostic kit that can screen for the diagnosis and progression of Alzheimer's disease in a runny nose sample.

Hereinafter, the present invention will be described in more detail.

As described above, there is still a need for a non-invasive diagnostic method that can not only confirm the onset of cerebral neuropathy but also precisely diagnose the progression stage of cerebral neuropathy. Accordingly, the present inventors have identified a method of specifically expressing a protein specifically expressed in mild Alzheimer's disease (AD) corresponding to the initial stage of Alzheimer's disease in a runny nose sample, and screening the progression of Alzheimer's disease using the same. By developing, the solution to the above-mentioned problems was sought.

The method of screening progression of Alzheimer's disease of the present invention can screen the Alzheimer's progression stage accurately and quickly in a non-invasive way by simultaneously identifying proteins representing two or more different stages in a runny nose sample.

Accordingly, the present invention provides an agent for measuring the expression level of beta amyloid oligomer in an isolated runny nose sample; It provides a composition for screening the progression stage of Alzheimer's disease comprising; and an agent for measuring the expression level of the S100A9 protein in the isolated runny nose sample.

In the present invention, the term "nose sample" may include a sample taken with a nasal wash, for example, a sample taken with a runny nose inhaler after spraying saline into the nose, a sample taken with a runny nose flowing from the saline solution.

As mentioned above, the present inventors have demonstrated through the Republic of Korea Patent Publication No. 10-2015-0144283 to determine the level of beta amyloid oligomer in the runny nose sample can diagnose the neurological disease. Therefore, the present inventors first confirmed the expression level of beta amyloid oligomer in the runny nose samples of Alzheimer's patients by sampling according to Alzheimer's progression step, and in each sample, a protein specific to the Alzheimer's progression step was identified through a proteomic method. Confirmed.

FIG. 2 (A) shows the protein distribution according to size through protein staining by homologous electrophoresis of the protein of the runny nose sample, and FIG. 2 (B) shows the quantitative graph and aligns with the size of the protein. When the band number 7 and 20 it was confirmed that the significant difference. The candidate bands were identified through LC-MS / MS analysis by extracting proteins from the bands 7, 8 and 20 to which the band number 8 added to the target band, including the target band, was added. 3 is shown.

As shown in FIG. 3, the number of secreted proteins, which are classified as involved in the immune system process in the runny nose of mild Alzheimer's patients, increased by a second, with proteins in this group specific to Alzheimer's disease. It was confirmed to express. Based on these results, representative proteins of the group related to immune system processes present in the runny nose samples of Alzheimer's patients were classified as shown in Table 1, and these proteins were selected as biomarker candidates. Specifically, proteins selected as candidate biomarkers are as follows: S100A9, HYOU1, SAA2, S100A8, DAPK1, LACRT and LF.

Next, a runny nose sample was taken from a normal group, a mild Alzheimer's patient group, and a moderate Alzheimer's patient group to quantify proteins representing immunologic stages of Alzheimer's disease through immunoblotting.

Specifically, the present inventors selected beta amyloid oligomer, a protein expressed by all Alzheimer's patients, based on the results found in previous studies, and performed quantitative analysis using A11 antibody. In addition, in the present invention, candidate biomarker proteins specific for mild Alzheimer's disease selected through proteomics were also quantitatively analyzed using an antibody that specifically binds to each protein.

As a result, as shown in FIG. 4, S100A9 was not expressed in the patients with moderate Alzheimer's disease, but specifically expressed in the patients with mild Alzheimer's disease. Thus, S100A9 protein was used as a biomarker protein representing mild Alzheimer's disease during the progression of Alzheimer's disease. Confirmed that it can.

In addition, the HYOU1 protein was expressed in some normal controls and not in the other normal controls. However, in mild Alzheimer's disease, a certain level of expression was confirmed. The HYOU1 protein is also an additional representative of mild Alzheimer's disease. It has been confirmed that it can be used as a biomarker protein.

The expression level of SAA2 protein, one of the candidate biomarkers, did not show a significant difference in the normal group, the mild Alzheimer's patient group, and the moderate Alzheimer's patient group. Since candidate biomarkers detected in a runny nose sample, such as SAA2 protein, do not all show significance, an operation for verifying by performing a quantitative analysis of proteins for each candidate biomarker is required.

Figure 5 is a protein expressed in all Alzheimer patients beta amyloid oligomers and mild Alzheimer's disease patients and normal runny nose samples selected from the runny nose samples collected from normal, mild Alzheimer's patients and moderate Alzheimer's patients group As a result of each quantitative analysis, it was confirmed that beta amyloid oligomer is characterized in different lanes according to the stage of Alzheimer's disease but expressed in all Alzheimer's patient groups. In comparison, S100A9 was not expressed in the moderate Alzheimer's patient group, but specifically expressed in the mild Alzheimer's patient group. Through this, it is possible to confirm the diagnosis and progression stage of Alzheimer's disease by confirming the expression of the protein in the runny nose sample using the agent measuring the expression level of beta amyloid oligomer and the agent measuring the expression level of S100A9 protein. Able to know.

In one embodiment of the present invention, the composition for screening the progress stage of Alzheimer's disease may further comprise an agent for measuring the expression level of the HYOU1 protein in the isolated runny nose sample.

The term "protein expression level" in the present invention refers to the level of expression of the protein from the gene in the cell, by observing the expression level of the protein mRNA that does not show a direct correlation between the level of mRNA in the cell and the protein expressed in the cell This can complement the limitations of research that targets. In the present invention, by observing the protein expression level of beta amyloid oligomer in the runny nose sample to diagnose the onset of Alzheimer's disease and at the same time to check the protein expression level of S100A9 and / or HYOU1 to screen the progression stage of Alzheimer's disease.

The term "agent for measuring protein expression level" includes, but is not particularly limited to, an antibody or aptamer specifically binding to a target protein.

The term "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to beta amyloid oligomer, S100a9 and / or HYOU1 protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Generating antibodies that specifically bind to the target protein can be readily prepared using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for injecting the target protein antigen into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.

Monoclonal antibodies are known in the art by the hybridoma method (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991). Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.

Analytical methods for confirming the amount of the target protein bound thereto using the antibody include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and oukteroni (Ouchterlony) immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip (protein) chip) and the like, but is not limited thereto.

In addition, in the present invention, "aptamer" refers to single-stranded DNA (ssDNA) or RNA having high specificity and affinity for a specific substance. Aptamers have a very high affinity for certain substances, are stable, can be synthesized in a relatively simple manner, and can be modified in various ways to increase binding capacity, and can be targets for cells, proteins, and even small organics. Its specificity and stability are very high compared to antibodies already developed.

The aptamer of the present invention is not limited as long as it can specifically bind to beta amyloid oligomer, and bases used in the aptamer are composed of bases of A, G, C, and U deoxy forms thereof unless otherwise specified. It may be selected from the group.

In addition, the aptamer is a polyethylene glycol (PEG), inverted deoxythymidine (IDT), Locked Nucleic Acid (LNA) at the 5 'terminal, intermediate, 3' terminal, or both ends to improve stability , At least one selected from the group consisting of 2'-methoxy nucleosides, 2'-amino nucleosides, 2'F-nucleosides, amine linkers, thiol linkers and cholesterol may be modified.

The inverted deoxythymidine (idT) is one of the molecules generally used to prevent the degradation of nucleases of aptamers that are poorly resistant to nucleases, the nucleic acid unit is the 3'-OH of the previous unit and the next unit Although it forms a chain by combining with 5'-OH, idT combines 3'-OH of the previous unit and 3'-OH of the next unit to artificially change to expose 5'-OH instead of 3'-OH. Thus, it is a molecule that produces an effect of inhibiting degradation by 3'exonuclease, a kind of nuclease.

The present invention also relates to a kit for screening progression of Alzheimer's disease, comprising the composition for screening progression of Alzheimer's disease described above.

The kit of the present invention is not only an agent capable of measuring the expression level of beta amyloid oligomer and S100A9 protein in a runny nose sample, but also an agent capable of measuring the expression level of HYOU1 protein, one or more compositions, solutions or solutions suitable for expression level analysis, or It may include a device. For example, the kit may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a detection label, a chromogenic substrate, and the like for immunological detection of the antibody.

The "agent that can measure the expression level of beta amyloid oligomer and S100A9 protein" and "the agent that can measure the expression level of HYOU1 protein" as described above, the antibody that can specifically bind to the target protein, Aptamers and the like can be used, and can be included in the kit in the form of a protein chip containing the agent.

In addition, a secondary antibody capable of detecting the antibody or aptamer may be further included, but the secondary antibody is not particularly limited thereto, but is not limited to beta amyloid oligomer, S100A9 protein, and / or Alternatively, the compound may bind to the HYOU1 protein and may be labeled with a fluorescent substance, a radioisotope, or the like.

In detail, the kit for diagnosing a brain neurological disease may be a kit including an essential element necessary to perform an ELISA in order to implement various ELISA methods such as an ELISA kit and a sandwich ELISA. ELISA kits may comprise antibodies or aptamers specific for these proteins. The antibody is an antibody having high specificity and affinity for beta amyloid oligomer, S100A9 protein, and HYOU1 protein, and having little cross-reactivity to other proteins, respectively, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. The ELISA kit can also include antibodies or aptamers specific for the control protein. Other ELISA kits may include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromopores, enzymes, and substrates thereof, or other materials that can bind to antibodies.

In addition, the kit is Western blot, ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay (Immunoprecipitation Assay), Complement Fixation Assay, Fluorecence Activated Cell Sorter (FACS), or It may be a kit for implementing a protein chip (protein chip) and the like, it may further include an additional configuration suitable for each analysis method. Through these analytical methods, it is possible to accurately screen Alzheimer's disease diagnosis and progression steps by comparing antigen-antibody complex formation amounts, and to provide patient-specific treatment methods according to the progression steps.

According to a preferred embodiment of the present invention, as shown in Figure 6 provides a kit implementing a protein chip that can detect the beta amyloid oligomer and S100A9 protein from the separated runny nose samples. As described above, the kit implementing the protein chip may include an additional configuration to detect the HYOU1 protein, thereby improving the accuracy of the diagnosis. You can also check the progression of Alzheimer's progression (long to moderate). Based on the contents in FIG. 5, the screening results of the kit implementing the protein chip of FIG. 6 are interpreted as follows. First, when a normal runny nose sample is administered to a protein chip kit, since the A11 and S100A9 proteins are not expressed, the signals are not displayed in both A and B lanes, and in a mild Alzheimer's patient, the A11 and S100A9 samples are present. And since both HYOU1 protein is expressed, the signal appears in all of the lanes A, B and C. Lastly, in patients with moderate Alzheimer's disease, only the A11 protein is expressed in the runny nose sample so that the signal appears only in lane A.

As such, the present invention provides a diagnostic method and kit that can easily check the progression step while increasing the accuracy of Alzheimer's diagnosis by cross-checking the two or more markers.

In addition, the present invention comprises the steps of a) measuring the expression level of beta amyloid oligomer in the isolated runny nose sample; And b) measuring the expression level of the S100A9 protein in the sample of step a) relates to a method for screening progression of Alzheimer's disease comprising a.

Specifically, when it is confirmed that the beta amyloid oligomer is expressed in step a) and the S100A9 protein is expressed in step b), the diagnosis is mild Alzheimer's disease; When the beta amyloid oligomer is expressed in step a) and the S100A9 protein is not expressed in step b), it may be diagnosed as moderate Alzheimer's disease.

The method for screening progression of Alzheimer's disease of the present invention may further comprise the step of measuring the expression level of c) HYOU1 protein in the sample of step b).

Specifically, when it is confirmed that the beta amyloid oligomer is expressed in step a), the S100A9 protein is expressed in step b), and the HYOU1 protein is expressed in step c), it is diagnosed as mild Alzheimer's disease. and; When the beta amyloid oligomer is expressed in step a), the S100A9 protein is not expressed in step b), and the HYOU1 protein is not expressed in step c), it is diagnosed as moderate Alzheimer's disease. Can be.

In the screening method of progression of Alzheimer's disease of the present invention, the runny nose sample may be separated from the individual to check the diagnosis or progression stage of Alzheimer's disease, and the process of separating the protein from the runny nose sample using a known process The protein level can be measured by the various antigen-antibody complex measurement methods mentioned above.

Specifically, the measurement of the protein expression level can be performed using an antibody or aptamer specifically binding to beta amyloid oligomer, S100A9 protein and / or HYOU1 protein. More specifically, the antibody may be a monoclonal antibody or polyclonal antibody specific for the beta amyloid oligomer, and may be a functional fragment of an antibody that retains antigen binding activity, but is not limited thereto.

In the screening method of progression of Alzheimer's disease of the present invention, Western blot, ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay as a method for detecting beta amyloid oligomer, S100A9 protein and / or HYOU1 protein from a runny nose sample (Immunoprecipitation Assay), Complement Fixation Assay (Fluorecence Activated Cell Sorter, FACS), or protein chip (protein chip) may be exemplified, but is not limited thereto.

The present invention also provides a method for treating a mild Alzheimer's disease therapeutic agent in a runny nose sample isolated from a patient suffering from Mild Alzheimer's disease; b) measuring the expression level of beta amyloid oligomer in the sample of step a); And c) measuring the expression level of the S100A9 protein in the sample of step b) relates to a screening method for treating a mild Alzheimer's disease.

Specifically, if the expression level of the beta amyloid oligomer in step b) is lower than before treating the candidate, and in step c) the S100A9 protein expression level is lower than before treatment with the candidate, mild Alzheimer's disease treatment You can choose.

The method for screening a mild Alzheimer's disease therapeutic agent of the present invention may further comprise the step of d) measuring the expression level of HYOU1 protein in the sample of step c).

Specifically, in step b), the expression level of the beta amyloid oligomer is decreased than before treating the candidate, and in step c), the expression level of the S100A9 protein is decreased than before treating the candidate, and d) If the level of expression of the HYOU1 protein decreases before treatment with the candidate, it may be selected as a treatment for mild Alzheimer's disease.

In the screening method of the mild Alzheimer's disease therapeutic agent of the present invention, the decrease in the expression level of the target protein may mean that the expression level of the normal control is reduced or the expression is completely suppressed.

The present invention also provides an agent for measuring the expression level of beta amyloid oligomer in isolated runny nose samples; It relates to a screening kit of a mild Alzheimer's disease therapeutic agent comprising; and an agent for measuring the expression level of S100A9 protein in the isolated runny nose sample.

The screening kit of the mild Alzheimer's disease therapeutic agent of the present invention may further comprise an agent for measuring the expression level of the HYOU1 protein in the isolated runny nose sample.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

1-1. Subject Selection

The normal control group selected people over 60 years of age who did not meet the other exclusion criteria and had normal cognitive abilities on the neuropsychological test (SNSB). Alzheimer's dementia patients were diagnosed with Alzheimer's disease according to the NINCDS / ADRDA diagnostic criteria. , People with deterioration in two or more cognitive domains, including memory, those who have been identified as having cognitive impairment in their daily lives by other psychological tests, and who do not meet other exclusion criteria. Selected.

Exclusion criteria include those identified by brain imaging (MRI or CT) tests without structural structural problems, those without other degenerative brain diseases, those without cerebrovascular disease that may cause cognitive dysfunction, and thyroid disease. People who do not have metabolic brain diseases, including alcohol or drug addiction, and who have no head trauma or psychiatric illness.

1-2. Subject's fluid collection protocol

At an outpatient visit, a neuropsychological test and imaging test were performed on a single visit to a normal person and a patient. After confirming the test results, a runny nose of a selected subject was collected. The selected subjects visited a total of two times. In addition, the study was conducted with thorough management of information by randomization and blinding so that the information about the normal person and the patient was not exposed to the sampler and the experimenter.

Subject's runny nose was collected using the following protocol. First, saline was sprayed into the nose, and then a runny nose was collected using a runny nose inhaler and stored in a 50 ml tube. If the method failed, the saline solution was flushed to the eye, and then a runny nose was collected and stored in a 50 ml tube. It was stored in a -20 ℃ refrigerator until analysis.

During the experiment, 50 μl of the sample was sonicated for 10 seconds, and the target protein was detected by Western blot and ELISA, and diagnosed by quantitative comparison.

Discovery of biomarkers using the runny nose of the patient

Proteomics-based biomarker analysis is an advantageous method to strategically develop biomarkers into epidemiological, diagnostic, and predictive types by more directly reflecting the physiological state of samples taken from patients. In recent years, more accurate and sensitive new technologies and mass spectrometers have been developed to discover protein biomarkers, and low-abundance proteins (a high probability of disease specific biomarkers) are considered to be true signs of disease. Protein) can be identified and confirmed.

Thus, in the present embodiment, biomarker verification through proteomics was performed using the runny nose of the patient collected in Example 1 (FIG. 1).

According to the protocol described in Example 1-1, 50 μl of a runny nose sample of three normal persons and three mild Alzheimer's patients were sonicated for 10 seconds, and then the proteins were separated using SDS-PAGE, and the SYPRO Ruby dye (detected). Limit 2-10 ng) was used to stain bands of protein embodied in the gel. Thereafter, the scanned gel images were analyzed and compared to confirm the increase or decrease of expression, and the difference in expression was analyzed. Peptides were obtained by decomposing the protein bands determined by image analysis with differences in expression patterns and trypsin (Trypsin), and MALDI-TOF MS (Matrix assisted laser desorption / ionization time-of-flight mass spectrometry) After securing the sequence by confirming the mass of the peptide through, the identity of the protein was confirmed by comparison with a database on the web (peptideSearch: http://www.mann.emblheidelberg.de/Services/ PeptideSearch / FR_sequenceOnlyForm.html).

Figure 2 (A) is a representative picture staining the band of the protein implemented in the gel, Figure 2 (B) is the result of quantification. As shown in Figure 2 (B), when the protein is aligned in size it was confirmed that the significant difference in band number 7, 8 and 20. Proteins were extracted from the target bands, and candidate protein groups were identified through LC-MS / MS analysis.

FIG. 3 is a comparison of proteome changes detected in a runny nose sample of a mild Alzheimer's disease patient group with a normal Alzheimer's disease patient using a gel band analysis result, and related to cellular component organization or biogenesis in a mild Alzheimer's disease patient group. The number of secreted proteins, which are classified as most, increased, but this was excluded as a biomarker candidate because it increases the number of proteins secreted by cell death as well as Alzheimer's disease. Next, the number of secreted proteins that are classified as involved in the immune system process in the runny nose of patients with mild Alzheimer's disease increased for the second time, and the proteins in this group were specifically expressed in Alzheimer's disease. It became.

Based on the results, the representative proteins of the group related to immune system processes present in the runny nose samples of Alzheimer's patients are shown in Table 1.

Protein ID Characteristic S100-A9
(protein S100-A9) Calcium binding protein, migration inhibitor related protein,
Inflammation-related proteins HYOU1
(hypoxia up-regulated protein1) Heat shock, oxidative stress, chemical agents, hypoxia,
Stimulated by cytotoxicity or protein toxicity, such as viral infections and inflammation SAA2
(anti-serum amyloid A) Cytokine-like properties that regulate some cellular responses
Synthesized predominantly by hepatocytes during acute reactions
S100-A8
(protein S100-A8) Calcium and zinc binding proteins, inflammatory processes and immune response regulation,
Neutrophil chemotaxis and adhesion induction DAPK1
(death-associated protein kinase 1) Apoptosis in response to Fas, γ-interferon and TNF-α
Promoting Actin Filament Related, Calcium / Calmodulin Dependence
Serine / Threonine Kinase LACRT
(extracellular glycoprotein lacritin) Serves as a modulator of bone marrow formation or inflammatory immune response
Plays an important role in controlling iron LF
(lactotransferrin) Involved in the defense of immune and inflammatory processes and pathogens

candidate Biomarker  Protein Quantitation

Candidate biomarker proteins selected in Example 2 were quantified from runny nose samples of two normal (CTL), two mild Alzheimer's patients (Mild AD) and two moderate Alzheimer's patients (Moderate AD).

Protein quantification was performed using a known BCA quantitative method, and only 1 ul of the sample was used to confirm the protein concentration, thereby enabling the same amount quantification.

As a result, as shown in FIG. 4, S100A9 was not expressed in the patients with moderate Alzheimer's disease, but specifically expressed in the patients with mild Alzheimer's disease. Thus, S100A9 protein was used as a biomarker protein representing mild Alzheimer's disease during the progression of Alzheimer's disease. Confirmed that it can.

In addition, the HYOU1 protein was expressed in some normal controls and not in the other normal controls. However, in mild Alzheimer's disease, a certain level of expression was confirmed. The HYOU1 protein is also an additional representative of mild Alzheimer's disease. It has been confirmed that it can be used as a biomarker protein.

The expression level of SAA2 protein, one of the candidate biomarkers, did not show a significant difference in the normal group, the mild Alzheimer's patient group, and the moderate Alzheimer's patient group.

Through the above results, the S100A9 protein and HYOU1 protein can be used to screen the progression stage of Alzheimer's disease because it is a protein representing a mild Alzheimer's disease.

Identification of proteins representative of the stage of progression of Alzheimer's disease

In this example, beta amyloid oligomer was further quantitatively analyzed in the runny nose samples of the normal control group, the mild Alzheimer's patient group and the moderate Alzheimer's patient group of Example 3.

As a result, as shown in Figure 5, the beta amyloid oligomer is characterized in different lanes according to the progression stage of Alzheimer's disease, but it was confirmed that it is expressed in all Alzheimer's patients. Through this, beta amyloid oligomer is expressed in all Alzheimer's patients regardless of the stage of Alzheimer's disease, it can be derived that can be used as a biomarker for diagnosing Alzheimer's disease.

Therefore, when the expression level of the beta amyloid oligomer and the expression level of the S100A9 and HYOU1 protein verified in Example 3 are measured together, not only the diagnosis of Alzheimer's disease but also the disease progression stage can be accurately diagnosed.

Claims (5)

Agents for measuring the expression level of beta amyloid oligomer in isolated runny nose samples; And
Agents for measuring expression levels of S100A9 protein in isolated runny nose samples;
A composition for distinguishing and diagnosing mild Alzheimer's disease and Moderate Alzheimer's disease. The method of claim 1, wherein the agent for measuring the expression level of the beta amyloid oligomer is an antibody or aptamer specifically binding to beta amyloid oligomer, and the agent for measuring the S100A9 protein expression level specifically binds to S100A9 protein. A composition for distinguishing and diagnosing mild Alzheimer's disease and moderate Alzheimer's disease, which are antibodies or aptamers. The composition of claim 1 for distinguishing between mild Alzheimer's disease and moderate Alzheimer's disease, further comprising an agent for measuring the expression level of the HYOU1 protein in the isolated runny nose sample. . The method of claim 3, wherein the agent for measuring the expression level of the HYOU1 protein distinguishes between mild Alzheimer's disease and moderate Alzheimer's disease, which are antibodies or aptamers that specifically bind to the HYOU1 protein. Composition for diagnosis. A kit for distinguishing between mild Alzheimer's disease and Moderate Alzheimer's disease comprising the composition of any one of claims 1 to 4.

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