KR20190038257A - Composition and kit for distinguishing between mild Alzheimer's disease and moderate Alzheimer's disease using nasal fluid - Google Patents

Abstract

The present invention relates to a composition and kit for screening for disease progressions of Alzheimer′s disease, comprising an agent measuring the expression levels of a beta-amyloid oligomer and S100A9 protein in a nasal fluid sample; and a method of screening for disease progressions of Alzheimer′s disease, comprising a step of measuring the expression levels of a beta-amyloid oligomer and S100A9 protein in a nasal fluid sample. Furthermore, the present invention relates to a method of screening for a therapeutic agent for mild Alzheimer′s disease, comprising a step of measuring the expression levels of a beta-amyloid oligomer and S100A9 protein in a nasal fluid sample; and a screening kit for a therapeutic agent for mild Alzheimer′s disease, comprising an agent measuring the expression levels of a beta-amyloid oligomer and S100A9 protein in a nasal fluid sample.

Description

TECHNICAL FIELD The present invention relates to a composition and kit for distinguishing between hardness and mild Alzheimer's disease using a nasal sample and a kit for identifying the mild Alzheimer's disease and moderate Alzheimer's disease using nasal fluid.

The present invention relates to a composition for screening a progressive stage of Alzheimer's disease using a runny nose sample and a method of screening the progressive stage of Alzheimer's disease using the composition. More particularly, the present invention relates to a method for screening a progressive stage of Alzheimer's disease The present invention relates to a method for screening a progressive stage of Alzheimer's disease, which comprises measuring the level of expression of beta amyloid oligomer and S100A9 protein in a composition for screening a composition for Alzheimer's disease, a kit and a sample of a runny nose. The present invention also includes a method for screening a therapeutic agent for a mild Alzheimer's disease comprising measuring the level of beta amyloid oligomer and S100A9 protein in a nasal sample and a method for measuring the level of expression of beta amyloid oligomer and S100A9 protein in a nasal sample To a screening kit for a therapeutic agent for mild Alzheimer's disease.

Degenerative brain disease is a disease that occurs in the brain as it gets older. Until now, it is known that some brain cells gradually lose its function in the brain, the death of brain cells which is most important for the transmission of brain nervous system, the formation of synapses or functional problems that transmit information between brain cells and neurons It is known to be caused by an ideal increase or decrease in the electrical activity of the cranial nerve. Alzheimer's disease (AD) is a disease characterized by disease-specific protein expression, the death of specific cell populations, and specificity through its pathogenesis, in comparison with other brain diseases Symptoms can be identified.

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

For example, there is a brain imaging method using a high-resolution brain imaging apparatus as a conventional method for diagnosing Alzheimer's disease. A method for early diagnosis of Alzheimer's disease using brain imaging is to measure the degree of accumulation of beta amyloid protein by performing brain imaging on patients with suspected Alzheimer's disease, Study the accuracy of the imaging device. However, this image-based diagnostic method not only requires a high cost to the patient, but also the detection of the disease is delayed because the discrimination is made in the state where the brain contraction or damage has already proceeded.

Another representative diagnostic method is to diagnose spinal fluid and measure the amount of beta amyloid protein in cerebrospinal fluid. However, the CSF method itself is known to be too painful for the patient, and it is pointed out as a disadvantage that the risk is accompanied by the examination.

Under these circumstances, the present inventors have developed a method for diagnosing a brain nerve disease using a nasal sample collected by a non-invasive method and disclosed in Korean Patent Laid-Open Publication No. 10-2015-0144283. Specifically, the patent discloses a method of diagnosing brain disease by measuring the level of beta amyloid oligomer expression in a nasal sample.

However, a noninvasive diagnostic method that not only can confirm the onset of neurological disease but also can accurately diagnose the progress of neurological disease is still required.

Accordingly, the present inventors have identified a protein specifically expressed in mild Alzheimer's disease (Mild AD), which is an early stage of Alzheimer's disease, in a runny nose sample, and screening the progression stage of Alzheimer's disease Thereby completing the present invention.

Accordingly, an object of the present invention is to provide an agent for measuring the level of beta amyloid oligomer expression in a nasal sample; And an agent for measuring the expression level of the S100A9 protein in the separated nasal sample, and a kit for screening the progressive stage of Alzheimer's disease.

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

Another object of the present invention is to provide a method for treating a mild Alzheimer ' s disease, comprising the steps of: a) treating a mild Alzheimer ' s disease therapeutic agent candidate in a nasal sample separated from a patient suffering from Mild Alzheimer ' s disease; b) measuring the level of beta amyloid oligomer expression in the sample of step a); And c) measuring the expression level of the S100A9 protein in the sample of the step b). The present invention also provides a screening method for a therapeutic agent for a mild Alzheimer's disease.

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

In order to solve the above-mentioned problems, the present invention provides an agent for measuring the expression level of a beta amyloid oligomer in a separated nasal sample; And an agent for measuring the expression level of the S100A9 protein in the separated nasal sample. The present invention also provides a composition for screening an advanced stage of Alzheimer's disease.

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 an aptamer that specifically binds to the beta amyloid oligomer, and the agent for measuring the S100A9 protein expression level is S100A9 protein Lt; RTI ID = 0.0 > and / or < / RTI >

According to another preferred embodiment of the present invention, the composition for screening the advanced stage of Alzheimer's disease may further comprise an agent for measuring the level of expression of HYOU1 protein in a separated nasal 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 an aptamer which specifically binds to the HYOU1 protein.

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

The present invention also provides a method for detecting beta amyloid oligomer comprising the steps of: a) measuring the level of beta amyloid oligomer expression in a separated nasal sample; And b) measuring the expression level of the S100A9 protein in the sample of step a).

According to a preferred embodiment of the present invention, when β-amyloid oligomer is expressed in step a) and S100A9 protein is expressed in step b), diagnosis is made as Mild Alzheimer's disease; If the beta amyloid oligomer is expressed in the step a) and the S100A9 protein is not expressed in the step b), it can be diagnosed as Moderate Alzheimer's disease.

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

According to another preferred embodiment of the present invention, when the β-amyloid oligomer is expressed in the step a), the S100A9 protein is expressed in the step b), and the HYOU1 protein is confirmed to be expressed in the step c) Diagnosed with Mild Alzheimer ' s disease; If the β-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), the disease is diagnosed as Moderate Alzheimer's disease .

According to another preferred embodiment of the present invention, the measurement of steps a), b) and c) may be performed by Western blot, enzyme linked immunosorbent assay (ELISA), immunoprecipitation assay, (Complement Fixation Assay), Fluorescence Activated Cell Sorter (FACS), or protein chip.

The present invention also provides a method for treating Alzheimer's disease, comprising the steps of: a) treating a mild Alzheimer's disease therapeutic agent candidate in a nasal sample separated from a patient suffering from Mild Alzheimer's disease; b) measuring the level of beta amyloid oligomer expression in the sample of step a); And c) measuring the expression level of the S100A9 protein in the sample of step b). ≪ IMAGE >

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

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

According to another preferred embodiment of the present invention, the expression level of the beta amyloid oligomer is decreased before the candidate substance is treated in the step b), and the expression level of the S100A9 protein in the step c) And when the expression level of the HYOU1 protein in the step d) is lower than that before the candidate substance is treated, the hardness may be selected as a therapeutic agent for Alzheimer's disease.

The present invention also provides an agent for measuring the level of beta amyloid oligomer expression in a separated nasal sample; And a preparation for measuring the expression level of the S100A9 protein in the separated nasal sample. The present invention also provides a screening kit for a therapeutic agent for a mild Alzheimer's disease.

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

The screening method for the progressive stage of Alzheimer's disease of the present invention can accurately and rapidly screen the Alzheimer's progressing step in a noninvasive manner by simultaneously identifying proteins representing two or more different steps in a runny sample. The existing diagnostic method for Alzheimer's disease was tried to secure the accuracy of diagnosis by using two or more methods together, but there was a problem in terms of cost and accessibility, and there was a problem in confirming the organic connectivity between diagnosis methods . However, the method of the present invention can screen the diagnosis and progress of Alzheimer's disease at once without using other diagnostic methods, thereby overcoming the problems of the conventional Alzheimer's method.

FIG. 1 is a schematic diagram showing a procedure for confirming biomarker verification using proteomics.
FIG. 2 is a graph comparing the results of proteomics using mucus samples from a mild Alzheimer's disease patient group and a normal group, wherein (A) is a gel image of a runny nose protein and (B) is a graph of analyzing a gel band concentration. The bar represents the average value including the standard error of the mean, the gray bar represents the control group, and the red bar represents the mild Alzheimer's disease group (* P < 0.05).
FIG. 3 is a graph comparing changes in proteomes detected in the mucus samples of patients with mild Alzheimer's disease and normal groups.
FIG. 4 is a result of quantitative analysis of biomarker proteins S100A9, HYOU1 and SAA2 selected from mucus samples from patients with mild Alzheimer's disease and normal group in nasal samples collected from normal group, mild Alzheimer's group and middle Alzheimer's group (A) is a graph showing protein expression levels using S100A9, HYOU1 and SAA2 antibodies through immunoblotting, and (B) is a graph showing quantitation of expression levels of each protein ( ^*** P <0.001, * P &lt; 0.01, * P &lt; 0.05, a = 0.0622).
FIG. 5 is a graph showing the results of a biomarker protein S100A9 selected from beta amyloid oligomers, mild Alzheimer's disease patients and normal nasal samples of all Alzheimer's patients in a nasal sample collected from a normal group, a mild Alzheimer's group and a middle Alzheimer's group As a result of quantitative analysis, (A) shows the amount of protein expressed using the antibodies A11 and S100A9 through immunoblotting, and (B) shows the graphs showing quantitation of the expression level of each protein (**) P &lt; 0.01, *** P &lt; 0.001).
FIG. 6 shows a schematic diagram of a diagnostic kit capable of screening the diagnosis and progression stages of Alzheimer's disease in a runny nose sample.

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

As described above, non-invasive diagnostic methods capable of not only detecting the onset of cranial nerve disease but also diagnosing precisely the progress of cranial nerve disease are still required. Accordingly, the present inventors have identified a protein specifically expressed in mild Alzheimer's disease (AD), which is an early stage of Alzheimer's disease, in a nasal sample, and screening the progress of Alzheimer's disease using the protein And developed a solution to the above-mentioned problems.

The screening method for the progressive stage of Alzheimer's disease of the present invention can accurately and rapidly screen the Alzheimer's progressing step in a noninvasive manner by simultaneously identifying proteins representing two or more different steps in a runny sample.

Accordingly, the present invention provides an agent for measuring the level of beta amyloid oligomer expression in a separated nasal sample; And an agent for measuring the expression level of the S100A9 protein in the separated nasal sample. The present invention also provides a composition for screening an advanced stage of Alzheimer's disease.

The term " nasal sample " in the present invention may include a nasal washing liquid, for example, a sample collected by a nasal aspirator after spraying saline into the nose, a sample obtained by collecting nasal water flowing after passing saline through the nose, and the like.

As described above, the present inventors have demonstrated through the Korean Patent Laid-Open No. 10-2015-0144283 that the level of beta amyloid oligomer expression in the nasal sample can be measured to diagnose cranial nerve disease. Therefore, the present inventors firstly examined the level of beta amyloid oligomer expression in the nasal sample of Alzheimer's patients according to the progression of Alzheimer's disease. In each sample, a protein specific to Alzheimer's progress through the proteomics method Respectively.

FIG. 2 (A) is a graph showing the protein distribution according to the size through protein staining by electrophoresis on the gel of the protein of the nasal sample. FIG. 2 (B) , And it is confirmed that there is a significant difference in the band numbers 7 and 20. Proteins were extracted from the 7th, 8th, and 20th bands to which the band number 8 close to the significant difference including the target band was added, and the candidate protein group was identified through LC-MS / MS analysis. As shown in Fig.

As shown in FIG. 3, the number of secreted proteins, which is classified as involved in the immune system process in the nasal mucosa of patients with Alzheimer's disease, is the second largest increase, and the protein of this group is specific for Alzheimer's disease Lt; / RTI &gt; Based on these results, representative proteins of the group related to the immune system processes present in the nasal samples of Alzheimer's patients were classified as shown in Table 1, and these proteins were selected as biomarker candidates. Specifically, the proteins selected by the candidate biomarker are: S100A9, HYOU1, SAA2, S100A8, DAPK1, LACRT and LF.

Next, nasal samples were taken from the normal group, the mild Alzheimer's disease group and the middle Alzheimer's disease group, and protein representing the progression stage of Alzheimer's disease was quantitated by immunoblotting.

Specifically, the present inventors selected beta amyloid oligomers, proteins expressed by all Alzheimer's patients based on the results of previous studies, and quantitatively analyzed them using the A11 antibody. In the present invention, the candidate biomarker protein specific to mild Alzheimer's disease selected through proteomics was quantitatively analyzed using an antibody specifically binding to each protein.

As a result, as shown in FIG. 4, S100A9 was not expressed in the middle-aged Alzheimer's disease group but was specifically expressed in the mild Alzheimer's disease group. Thus, in the progress of Alzheimer's disease, the S100A9 protein is used as a biomarker protein representing mild Alzheimer's disease Respectively.

In addition, the HYOU1 protein showed an irregular pattern that was expressed in some normal controls and not expressed in other normal controls. However, a certain level of expression was observed in all cases of mild Alzheimer's disease. HYOU1 protein was also found to be an additional It can be used as a biomarker protein.

The expression level of SAA2 protein, one of the candidate biomarkers, was not significantly different in the normal group, the mild Alzheimer's group and the middle Alzheimer's group. Since candidate biomarkers detected in runny samples, such as SAA2 protein, are not all significant, it is essential to conduct a quantitative analysis of proteins individually for each candidate biomarker.

FIG. 5 is a graph showing the results of a biomarker protein S100A9 selected from beta amyloid oligomers, mild Alzheimer's disease patients and normal nasal samples of all Alzheimer's patients in a nasal sample collected from a normal group, a mild Alzheimer's group and a middle Alzheimer's group Quantitative analysis revealed that beta amyloid oligomers were expressed in all the Alzheimer's patients, although they were characteristic of different lanes depending on the stage of Alzheimer's disease progression. In contrast, S100A9 was not expressed in the middle-aged Alzheimer's group but was specifically expressed in the Alzheimer's group. Thus, it is possible to confirm the diagnosis and progress of Alzheimer's disease by confirming the expression of the protein in the nasal sample using the agent for measuring the expression level of beta amyloid oligomer and the agent for measuring the expression level of S100A9 protein Able to know.

In one embodiment of the present invention, the composition for screening the progressive stage of Alzheimer's disease may further include an agent for measuring the level of expression of HYOU1 protein in a separated nasal sample.

The term " protein expression level " in the present invention refers to a level at which a gene is expressed as a protein in a cell. By observing the expression level of the protein, mRNA level indicating the direct correlation between the level of mRNA in the cell and the level of protein expressed in the cell Of the study. In the present invention, the level of protein expression of beta amyloid oligomer in the runny nose sample is examined to diagnose the onset of Alzheimer's disease, and the level of protein expression of S100A9 and / or HYOU1 is confirmed, thereby screening the progress of Alzheimer's disease.

The term " agent for measuring the protein expression level " of the present invention includes, but is not limited to, an antibody or an aptamer that specifically binds to a target protein.

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

Polyclonal antibodies can be produced by methods well known in the art for obtaining sera containing antibodies by injection of the target protein antigen into an animal and blood sampling from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies can be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: 1992, Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991). The antibody prepared by the above method can be separated and purified by 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 with 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 an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

Methods for assaying the amount of the target protein bound to the antibody using the antibody include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Chip Protein (FACS), Immunoprecipitation Assay, Immunoprecipitation Assay, Ouchterlony Immunoprecipitation, Rocket Immunoelectrophoresis, chip, but are not limited thereto.

Also, in the present invention, "aptamer" refers to single stranded DNA (ssDNA) or RNA having high specificity and affinity for a specific substance. Since aptamers can be synthesized in a relatively stable, relatively simple manner with a high affinity for a specific substance, various modifications can be made to increase binding force, and even cells, proteins, and small organic substances can be target substances, Its specificity and stability are very high compared to antibodies that have already been developed.

The aptamer of the present invention is not limited as long as it is capable of specifically binding to the beta amyloid oligomer, and the base used for the aptamer is not limited to those of A, G, C and U, Lt; / RTI &gt;

In order to improve the stability, the aptamer may further include polyethylene glycol (PEG), inverted deoxythymidine (IDT), and locked nucleic acid (LNA) at the 5 'end, middle, , 2'-methoxynucleoside, 2'-amino nucleoside, 2'F-nucleoside, amine linker, thiol linker and cholesterol may be combined and modified.

The above-mentioned idT (inverted deoxythymidine) is one of the molecules used to prevent degradation by a nuclease of aptamer, which is generally resistant to nuclease, wherein the nucleic acid unit comprises 3'-OH of the former unit and 5'-OH, but idT binds the 3'-OH of the first unit and the 3'-OH of the next unit to form an artificial change so that 5'-OH, not 3'-OH, is exposed. (3 'exonuclease), which is a kind of nuclease, which inhibits the degradation by the 3' exonuclease.

The present invention also relates to a kit for screening a progressive stage of Alzheimer's disease, which comprises the aforementioned composition for screening of advanced stages of Alzheimer's disease.

The kit of the present invention can be used as an agent for measuring the level of expression of HYOU1 protein as well as an agent capable of measuring the expression level of beta amyloid oligomer and S100A9 protein in a nasal sample, Device. For example, the kit may comprise a substrate, a suitable buffer solution, a secondary antibody labeled with a detection label, and a chromogenic substrate for immunological detection of the antibody.

The "agent capable of measuring the level of expression of the β-amyloid oligomer and the S100A9 protein" and the "agent capable of measuring the level of expression of the HYOU1 protein" are antibodies capable of specifically binding to the target protein, An aptamer, and the like, and may be included in the kit in the form of a protein chip containing the agent.

The secondary antibody may further include a secondary antibody capable of detecting the antibody or the aptamer. The secondary antibody may be a beta amyloid oligomer capable of binding to the protein chip, a S100A9 protein and / Or HYOU1 protein, and may be labeled with a fluorescent substance, a radioactive isotope, or the like.

Specifically, the kit for diagnosing neuropsychiatric diseases may be a kit that includes essential elements necessary for performing ELISA in order to implement various ELISA methods such as an ELISA kit, a sandwich ELISA, and the like. ELISA kits may include antibodies or aptamers specific for these proteins. The antibody may be a monoclonal antibody, a polyclonal antibody or a recombinant antibody, which has high specificity and affinity for the beta amyloid oligomer, the S100A9 protein and the HYOU1 protein, and has little cross-reactivity to the other proteins. The ELISA kit may also include antibodies or aptamers specific for the control protein. Other ELISA kits may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromopores, enzymes and other substrates capable of binding to the substrate or antibody.

In addition to the above, the kit may be used in a Western blot, an enzyme linked immunosorbent assay (ELISA), an immunoprecipitation assay, a complement fixation assay, a fluorescence activated cell sorter (FACS) A protein chip, and the like, and may further include an additional configuration suitable for each analysis method. Through these analytical methods, the diagnosis and progression stages of Alzheimer's disease can be accurately screened by comparing the formation amounts of antigen-antibody complexes, and a patient-customized treatment method can be provided according to progressing stages.

According to a preferred embodiment of the present invention, there is provided a kit comprising a protein chip capable of detecting β-amyloid oligomer and S100A9 protein from a separated nasal sample as shown in FIG. As described above, the kit implementing the protein chip may include an additional structure for detecting the HYOU1 protein, thereby improving the accuracy of the diagnosis. You can also see the progression of the Alzheimer's progression (mid-to-long). Based on the contents in FIG. 5, screening results of the kit implementing the protein chip of FIG. 6 are analyzed as follows. First, when a normal nasal sample is administered to a protein chip kit, signals are not present in both the A and B lanes since the A11 and S100A9 proteins are not expressed. In the case of mild Alzheimer's patients, A11, S100A9 And HYOU1 proteins are expressed, signals appear in all of the A, B and C lanes. Finally, in patients with mild Alzheimer's disease, only the A11 protein is expressed in the runny nose, so signals appear only in the A lane.

As described above, the present invention provides a diagnostic method and a kit for identifying the two or more markers so as to cross-confirm the Alzheimer's disease, thereby improving the accuracy of the Alzheimer's diagnosis and continuously checking the progression stage.

Also, the present invention provides a method of detecting a beta-amyloid oligomer, comprising: a) measuring the level of beta amyloid oligomer expression in a separated nasal sample; And b) measuring the expression level of the S100A9 protein in the sample of step a).

Specifically, when the β-amyloid oligomer is expressed in step (a) and the S100A9 protein is identified to be expressed in step (b), the disease is diagnosed as Mild Alzheimer's disease; If the beta amyloid oligomer is expressed in the step a) and the S100A9 protein is not expressed in the step b), it can be diagnosed as Moderate Alzheimer's disease.

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

Specifically, when the β-amyloid oligomer is expressed in step a), the S100A9 protein is expressed in step b), and the HYOU1 protein is expressed in step c), the disease is diagnosed as Mild Alzheimer's disease and; If the β-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), the disease is diagnosed as Moderate Alzheimer's disease .

In the method of screening the progressive stage of Alzheimer's disease according to the present invention, the nasal sample may be separated from an individual to be diagnosed or progressed in Alzheimer's disease, and the process of separating the protein from the nasal sample may be performed using a known process , And the protein level can be measured by the above-mentioned various assay methods of antigen-antibody complexes.

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

In the screening method for the progressive stage of Alzheimer's disease according to the present invention, a method for detecting beta amyloid oligomer, S100A9 protein and / or HYOU1 protein from a nasal sample is exemplified by Western blotting, enzyme linked immunosorbent assay (ELISA) But are not limited to, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), or protein chip.

The present invention also provides a method for treating Alzheimer's disease, comprising the steps of: a) treating a mild Alzheimer's disease therapeutic agent candidate in a nasal sample separated from a patient suffering from Mild Alzheimer's disease; b) measuring the level of beta amyloid oligomer expression in the sample of step a); And c) measuring the expression level of the S100A9 protein in the sample of step b). [0002] The present invention relates to a method for screening a therapeutic agent for a mild Alzheimer's disease.

Specifically, when the expression level of the beta amyloid oligomer is decreased before the candidate substance is treated in step b) and the level of S100A9 protein expression in the step c) is decreased before the candidate substance is treated, You can choose.

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

Specifically, the level of expression of the beta amyloid oligomer is decreased before the candidate substance is treated in step b), the level of expression of the S100A9 protein in the step c) is decreased before the candidate substance is treated, and the step d) May be selected as a therapeutic agent for a mild Alzheimer's disease when the expression level of the HYOU1 protein is decreased before the candidate substance is treated.

In the screening method for a therapeutic agent for mild Alzheimer's disease of the present invention, the decrease in the expression level of the target protein may be reduced to the level of protein expression of the normal control or the expression may be completely inhibited.

The present invention also provides an agent for measuring the level of beta amyloid oligomer expression in a separated nasal sample; And a preparation for measuring the expression level of the S100A9 protein in the separated nasal sample. [0002] The present invention relates to a screening kit for a therapeutic agent for a mild Alzheimer's disease.

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

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

1-1. Subject selection

A normal control group was selected for persons aged 60 years or older who had normal cognitive ability on the neuropsychiatric test (SNSB) and who did not fall under any of the other exclusion criteria. Alzheimer's patients with Alzheimer's disease according to the NINCDS / ADRDA diagnostic criteria , A person with a deterioration in two or more cognitive domains including memory, a person who has not been cognitively affected by neuropsychological tests, has been identified as having an impairment in daily life due to cognitive impairment, .

Exclusion criteria include those who have been confirmed to have no brain structural problems through brain imaging (MRI or CT) examinations, those without other degenerative brain diseases, those without cerebrovascular disease that can cause cognitive dysfunction, People without metabolic brain disease, people without alcohol or drug addiction, and people without head trauma or psychiatric disorders.

1-2. The subject's body fluid collection protocol

At the outpatient visit, neuropsychological tests and imaging tests were performed at the first visit to normal persons and patients, and the nasal mucosa of the selected subjects was collected after confirming the test results. The selected subjects were visited twice in total. In addition, the research was carried out by thoroughly managing the information by random allocation and blind method so that the information of the normal person and the patient is not exposed to the person who takes the sample of the subject and the person who conducts the experiment.

The subjects' nasal secretions were collected using the following protocol. First, saline was sprayed into the nose, nasal mucus was collected using a nasal aspirator and stored in a 50 ml tube. When the method failed, the saline was poured into the eye, and the runny nose was collected and stored in a 50 ml tube. And stored at -20 ° C in the refrigerator until analysis.

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

Biomarker discovery using patient's runny nose

The proteomics-based biomarker analysis is an advantageous method for strategically developing biomarkers by dividing the biomarkers into epidemiological, diagnostic, and predictive types by directly reflecting the physiological state of the sample collected from the patient. More recently, more accurate and sensitive new technologies and mass analyzers have been developed to identify protein biomarkers, and low-abundance proteins, which are considered to be a true sign of disease (high probability of disease-specific biomarkers Protein) in the human body.

Thus, in this embodiment, biomarker verification using proteomics was performed using the nasal mucus collected from Example 1 (Fig. 1).

50 μl of nasal sample from 3 normal and 3 patients with mild Alzheimer's disease were sampled according to the protocol described in Example 1-1, sonicated for 10 seconds, proteins were separated using SDS-PAGE, and SYPRO Ruby dye Limit 2 to 10 ng) was used to stain the band of protein implemented in the gel. Thereafter, an increase or decrease in expression was confirmed by analyzing and comparing the scanned gel images, and the difference in expression was analyzed. Protein bands determined to have different expression patterns by image analysis were collected and digested with trypsin to obtain peptides. MALDI-TOF MS (Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry) To confirm the identity of the protein by comparing it with a database on the web (peptideSearch: http://www.mann.emblheidelberg.de/Services/ PeptideSearch / FR_sequenceOnlyForm.html).

FIG. 2 (A) is a representative image of a band of a protein implemented in a gel, and FIG. 2 (B) is a result of quantifying the protein. As shown in FIG. 2 (B), it was confirmed that when the protein was aligned in size, significant differences were observed in the band numbers 7, 8, and 20. Proteins were extracted from the target band, and the candidate protein groups were identified by LC-MS / MS analysis and analyzed by function.

Figure 3 compares proteome changes detected in nasal samples from patients with mild Alzheimer &apos; s disease and those from normal group through gel band analysis and is shown to be associated with cellular component organization or biogenesis in the mild Alzheimer &apos; The number of secreted proteins classified as the most increased, but the number of proteins secreted by cell death as well as Alzheimer 's disease is increasing, so it is excluded from biomarker candidates. Next, the number of secreted proteins classified as involved in the immune system process in the nasal mucosa of patients with Alzheimer's disease was the second largest increase, indicating that this group of proteins was expressed specifically in Alzheimer's disease .

Based on the above results, the representative proteins of the group related to the immune system process present in the nasal sample of Alzheimer's patients are shown in Table 1.

Protein ID Characteristic S100-A9
(protein S100-A9) Calcium binding protein, migration inhibitory factor related protein,
Inflammation-related protein HYOU1
(hypoxia up-regulated protein 1) Thermal shock, oxidative stress, chemical agents, hypoxia,
Stimulated by cytotoxicity or protein toxicity such as viral infection and inflammation SAA2
(anti-serum amyloid A) Similar properties to cytokines that regulate several cellular responses
And synthesized mainly by hepatocytes during an acute reaction
S100-A8
(protein S100-A8) Calcium and zinc binding proteins, inflammatory processes and modulation of immune responses,
Neutrophil chemotaxis and adhesion induction DAPK1
(death-associated protein kinase 1) Fas, gamma-interferon, and TNF-alpha.
Promoting actin filament-related, calcium / calmodulin dependent
Serine / threonine kinase LACRT
(extracellular glycoprotein lacritin) Plays a role as a modulator of myelogenous or inflammatory immune response
Plays an important role in regulating iron LF
(lactotransferrin) Involved in immune and inflammatory processes and defense against pathogens

candidate Biomarker  Protein quantification

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

Protein quantification was performed using a known quantification method of BCA. Protein concentration was determined using only 1 μl of the sample, and quantification was carried out through the same.

As a result, as shown in FIG. 4, S100A9 was not expressed in the middle-aged Alzheimer's disease group but was specifically expressed in the mild Alzheimer's disease group. Thus, in the progress of Alzheimer's disease, the S100A9 protein is used as a biomarker protein representing mild Alzheimer's disease Respectively.

In addition, the HYOU1 protein showed an irregular pattern that was expressed in some normal controls and not expressed in other normal controls. However, a certain level of expression was observed in all cases of mild Alzheimer's disease. HYOU1 protein was also found to be an additional It can be used as a biomarker protein.

The expression level of SAA2 protein, one of the candidate biomarkers, was not significantly different in the normal group, the mild Alzheimer's group and the middle Alzheimer's group.

From the above results, it can be deduced that the S100A9 protein and the HYOU1 protein are proteins representing mild Alzheimer's disease, and thus can be used for screening the progress of Alzheimer's disease.

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

In the present example, beta amyloid oligomers were further quantitatively analyzed in the nasal samples of the normal control group, the mild Alzheimer's group, and the severe Alzheimer's disease group of Example 3 above.

As a result, as shown in FIG. 5, the beta amyloid oligomers were distinguished in different lanes depending on the stage of progression of Alzheimer's disease, but they were confirmed to be expressed in all Alzheimer's patients. As a result, beta amyloid oligomers can be used as biomarkers for diagnosing Alzheimer's disease since they are expressed in all Alzheimer's patients regardless of the stage of Alzheimer's disease.

Therefore, when the expression levels of the S100A9 and HYOU1 proteins and the expression levels of the beta amyloid oligomers tested in Example 3 are measured together, it is possible to accurately diagnose not only the diagnosis of Alzheimer's disease but also the progression of the disease.

Claims (5)

An agent for measuring the expression level of beta amyloid oligomer in a separated nasal sample; And
An agent for measuring the expression level of S100A9 protein in a separated nasal sample;
(Mild Alzheimer ' s disease) and Moderate Alzheimer ' s disease. The method according to claim 1, wherein the agent for measuring the expression level of the beta amyloid oligomer is an antibody or an aptamer that specifically binds to beta amyloid oligomer, and the agent for measuring the S100A9 protein expression level specifically binds to S100A9 protein (Mild Alzheimer ' s disease) and Moderate Alzheimer ' s disease, which is an antibody or an aptamer for diagnosing Alzheimer &apos; s disease. The composition according to claim 1, further comprising an agent for measuring the expression level of HYOU1 protein in a separated nasal sample, and a composition for distinguishing between mild Alzheimer's disease and Moderate Alzheimer's disease . 4. The method according to claim 3, wherein the agent for measuring the expression level of the HYOU1 protein is selected from the group consisting of Mild Alzheimer ' s disease and Moderate Alzheimer ' s disease, which are antibodies or aptamers specifically binding to 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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