KR20180021440A - Potential autoantibody biomarkers for the detection and diagnosis of dementia - Google Patents

Abstract

The present invention relates to an autoantibody biomarker for dementia diagnosis and a dementia diagnosis kit including the same. More particularly, the present invention relates to an autoantibody biomarker selectively recognizing ATCAY (ataxia, cerebellar, cayman type), NME7 (NME/NM23 family member 7) and NOL3 (nucleolar protein 3) which are unusually increased in blood of a dementia patient than a normal person so as to be capable of dementia diagnosis, and a diagnosis kit for diagnosing dementia including an antigen recognizing the same. The kit for diagnosing dementia including autoantibodies of ATCAY, NME7 and NOL3, which can be used as the marker for dementia diagnosis of the present invention, can be used for early diagnosis of dementia by using blood of a patient to diagnose dementia very easily.

Description

TECHNICAL FIELD The present invention relates to an autoantibody biomarker for diagnosis of dementia,

The present invention relates to an autoantibody biomarker for dementia diagnosis and a method for diagnosing dementia using the same.

Alzheimer's disease is the most common degenerative brain disease that causes the most representative dementia among age-related neurodegenerative diseases. It is a disease that accounts for more than 70% of the dementia in elderly people. . Alzheimer's disease patients have generalized brain atrophy due to loss of neurons. Neuritic plaques and neurofibrillary tangles are characteristic lesions in brain tissue. In the early stages of the disease, these brain pathologies are confined mainly to the hippocampus, which is the main brain region responsible for memory, and the olfactory cortex, but gradually expand to the entire brain. It develops seriously as a result of various clinical symptoms such as mental disorder behavior and lowered thinking judgment ability and daily life performance ability.

The precise mechanisms and causes of Alzheimer's disease are not known precisely. It is thought that beta-amyloid is over-produced and is harmful to brain cells as it is deposited in the brain. It is thought that hyperphosphorylation, inflammation, oxidative damage of tau protein is also a cause . The nervous system (or the elderly) is involved in the deposition of beta amyloid protein, and the nerve fiber bundle is associated with tau protein hyperphosphorylation.

Mild Cognitive Impairment (MCI) refers to the pre-dementia stage in which cognitive function, especially memory, is reduced compared to the same age group, but the ability to perform everyday life is maintained. People with mild cognitive impairment are identified as being at high risk for progression to Alzheimer's disease. Thus, mild cognitive impairment is the stage in which Alzheimer's disease is the earliest discoverable stage. Recently, a new type of Alzheimer's disease drug has been shown to be more effective early in the term than Alzheimer's disease, Early detection has clinical significance. In addition, the prevalence of Alzheimer's disease in men over 65 years is 33% and in women it is 45%. Along with the increase in life expectancy due to population growth and medical advances, It is important to select a group that is likely to progress to Alzheimer's disease early in life and to establish a proper treatment system for it, as well as the life aspect of a healthy old age life, as well as the enormous social and economic costs Which can lead to savings.

Auto-antibodies are commonly found in all human blood, and it is known that the amount of autoantibody increases with age. Autoantibodies are known to be involved in adaptive debris-clearance mechanisms as self-reactive antibodies. For example, in the case of an autoimmune disease, it is known that the autoantibodies against the self-specific cells or tissue components cause or worsen the disease.

Recently, it is known that autoantibodies and Alzheimer's are closely related. For example, autoantibodies against PTCD2 and FRMD8 (FERM domain containing 8) are more present in the serum of Alzheimer's patients than in the normal group, and the antigen-bound autoantibodies are found in patients with Alzheimer's disease There are reports of increased serum and cerebrospinal fluid. Recently, autoantibodies have been attracting attention as biomarkers for diagnosis and prediction of Alzheimer's disease.

To date, the diagnosis of mild cognitive impairment or Alzheimer's disease has relied mainly on parental history and neuropsychological assessment, cognitive testing, and brain imaging, but it is possible to identify the onset and progression of these, The development of a biomarker is urgently needed. Ideally, biomarkers are easily identifiable from blood samples. Considering that the patient with the disorder or disease is generally old, the lumber puncture for obtaining cerebrospinal fluid is high in potential risk, and is not suitable to be performed in a normal small-sized hospital. Blood samples are usually obtained easily and safely at home or in a general hospital and can be inspected, and do not require other expensive equipment or surgical procedures.

Accordingly, the present inventors sought to find an autoantibody capable of predicting the diagnosis and onset of a cognitive impairment or Alzheimer's disease from the blood of a patient. The inventors of the present invention have found that a human proteome microarray can be used to detect normal autoimmune dementia patients and Alzheimer's patients The autologous antibody was profiling from the serum and the autoantibodies specific for Alzheimer's disease were screened and verified by ELISA.

It is an object of the present invention to provide a compound of the present invention which has a hardness composed of an autoantibody against one or more self antigens selected from the group consisting of ATCAY (Ataxia, Cerebellar, Cayman Type), NME7 (NME / NM23 Family Member 7) and NOL3 And to provide a biomarker for diagnosing cognitive disorders.

Another object of the present invention is to provide a dementia diagnosis biomarker composed of an autoantibody against ATCAY (Ataxia, Cerebellar, Cayman Type) autoantigen.

Yet another object of the present invention is to provide a kit for diagnosing a mild cognitive disorder comprising one or more proteins selected from the group consisting of ATCAY, NME7 and NOL3.

It is still another object of the present invention to provide a dementia diagnostic kit comprising the ATCAY protein.

It is still another object of the present invention to provide a mild cognitive impairment diagnostic strip comprising at least one protein antibody selected from the group consisting of ATCAY, NME7 and NOL3.

It is a further object of the present invention to provide a diagnostic strip for dementia comprising the ATCAY protein antibody.

In order to achieve the above object, an autoantibody against one or more autoantigens selected from the group consisting of ATCAY (Ataxia, Cerebellar, Cayman Type), NME7 (NME / NM23 Family Member 7) and NOL3 (Nucleolar Protein 3) And a diagnostic hardness cognitive disorder diagnostic biomarker.

In addition, the present invention provides a dementia diagnosis biomarker composed of an autoantibody against ATCAY (Ataxia, Cerebellar, Cayman Type) autoantigen.

Also, the present invention provides a kit for diagnosing a mild cognitive disorder comprising one or more proteins selected from the group consisting of ATCAY, NME7 and NOL3.

The present invention also provides a dementia diagnostic kit comprising the ATCAY protein.

The present invention also relates to a sample pad on which a biological sample is absorbed;

A conjugate pad binding to any one or more selected from the group consisting of ATCAY (Ataxia, Cerebellar, Cayman Type), NME7 (NME / NM23 Family Member 7) and NOL3 (Nucleolar Protein 3);

A reaction membrane on which a test line and a control line containing each antibody of the protein are treated;

An absorption pad on which a sample of the remaining amount is absorbed; And

The present invention provides a hardness cognitive diagnostic strip comprising a support.

The present invention also relates to a sample pad on which a biological sample is absorbed;

A conjugate pad coupled with ATCAY (Ataxia, Cerebellar, Cayman Type);

A reaction membrane on which a test line and a control line containing each antibody of the protein are treated;

An absorption pad on which a sample of the remaining amount is absorbed; And

A diagnostic strip for dementia comprising a support.

The present invention is based on the finding that the serum levels of anti-ATCAY IgG and anti-NME7 IgG are high in the Alzheimer's disease group, The level of anti-PAIP2 IgG was high in the cognitive impairment group, and the higher the detection rate of the two autoantibody positive antibodies combining anti-PAIP2 IgG and anti-ATCAY IgG, the higher the risk of Alzheimer's disease, We confirmed that the higher the positive detection frequency of the two autoantibodies combining the anti-NOL3 IgG and the anti-NME7 IgG, the higher the risk of the memory hardness cognitive impairment (aMCI), and the biomarker for the diagnosis of dementia was provided , And can be used to provide a basis for early diagnosis and treatment of Alzheimer's disease by screening in advance a high-risk group which is likely to develop Alzheimer's disease in the future.

FIG. 1 is a graph showing the distribution of autoantibodies present in serum of 10 Korean elderly people aged 60 years or older. Occurrence (frequency) means the number of positive autoantibodies among all 10 individuals.
Figure 2 compares the number of autoantibodies of 5 normal individuals and 5 Alzheimer's disease patients.
FIG. 3 is a graph showing the total IgG level in a serum sample in the normal group, the mild cognitive impairment group, and the Alzheimer's disease group. As a result of one-way ANOVA test, there was no difference in total IgG level between the three groups (p = 0.227). Represents a subject, and bar represents the mean standard deviation error (eroor).
FIG. 4 shows the results of normalization of the level of autoantibody to the total IgG level (total IgG level) and then comparing the difference in the levels of these autoantibodies between the normal cognitive group, the mild cognitive impairment group and the Alzheimer's disease group It is a degree. Each point represents one subject and bar represents the mean standard deviation error (eroor).
Figure 5 shows the correlation between the level of anti-ATCAY IgG and the MMSE score and the K-MoCA score:
A: Diagram showing correlation between anti-ATCAY IgG level and MMSE score; And
B: Diagram showing correlation between anti-ATCAY IgG level and K-MoCA score.

Hereinafter, the present invention will be described in detail.

The present invention provides a hardness cognitive disorder diagnosis kit comprising one or more proteins selected from the group consisting of ATCAY (Ataxia, Cerebellar, Cayman Type), NME7 (NME / NM23 Family Member 7) and NOL3 (Nucleolar Protein 3) .

Preferably, the ATCAY (Ataxia, Cerebellar, Cayman Type) includes the amino acid sequence of SEQ ID NO: 1, and the NME7 (NME / NM23 Family Member 7) includes the amino acid sequence of SEQ ID NO: 2 And NOL3 (Nucleolar Protein 3) includes the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

Preferably, the ATCAY (Ataxia, Cerebellar, Cayman Type) includes the nucleotide sequence of SEQ ID NO: 4, and the NME7 (NME / NM23 Family Member 7) includes the nucleotide sequence of SEQ ID NO: 5 And NOL3 (Nucleolar Protein 3) includes the nucleotide sequence of SEQ ID NO: 6, but is not limited thereto.

In addition, the present invention provides a dementia diagnostic kit comprising ATCAY (Ataxia, Cerebellar, Cayman Type) protein.

In addition, the diagnostic kit of the present invention is characterized in that it comprises a secondary antibody conjugated with a marker which is developed by a reaction with an antigenic substrate specific to an autoantibody specifically binding to any one or more proteins selected from the group consisting of ATCAY, NME7 and NOL3 Antibody conjugate (Conjugate); And a color developing substrate solution washing solution or an enzyme reaction stop solution to be color-developed with the label.

The solid substrate may be a nitrocellulose membrane, a PVDF membrane, a polyvinyl or a polystyrene, or a combination thereof. The solid substrate may be one selected from the group consisting of ATCAY, NME7, and NOL3. A well plate synthesized with a resin, and a slide glass made of glass. However, the present invention is not limited thereto.

It is preferable that the secondary antibody is labeled with a conventional coloring agent that performs a color development reaction. It is preferable that the secondary antibody is labeled with a coloring agent such as HRP (Horseradish peroxidase), Alkaline phosphatase, Coloid gold, Poly-Lysine-fluorescein isothiocyanate , Fluorescent material (Fluorescein) such as Rhodamine-B-isothiocyanate (RITC), and dye (Dye) can be used.

The substrate for inducing color development is TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD ), But the present invention is not limited thereto.

The kit may further include a washing solution for removing the remaining substances after the antigen-antibody binding reaction and the binding reaction of the detection body. Preferably, the wash liquor comprises a phosphate buffer solution, NaCl and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 But is not limited thereto. After the antigen-antibody binding reaction, the antibody-antibody conjugate is reacted with the detection antibody, and an appropriate amount of the antibody is added to the solid body and washed 3 to 6 times. The reaction stop solution is preferably, but not limited to, a sulfuric acid solution (H2SO4).

Also, the present invention provides a method for preparing an antigen-antibody reaction, comprising the steps of: 1) adding a sample derived from a subject to a solid body coated with at least one protein selected from the group consisting of ATCAY, NME7 and NOL3,

2) detecting the antigen-antibody reactant produced in step 1) using a chromogenic substrate solution; And

3) A method for detecting an autoantibody in order to provide information on the diagnosis of a mild cognitive impairment comprising the step of judging an increased test subject as compared with a normal autoantibody amount to a person having a mild cognitive impairment or a possibility of taking a mild cognitive impairment .

Also, the present invention provides a method for preparing an ATCAY protein comprising the steps of: 1) adding a sample derived from a subject to an immobilized body coated with ATCAY protein to perform an antigen-antibody reaction;

2) detecting the antigen-antibody reactant produced in step 1) using a chromogenic substrate solution; And

And 3) determining that the detected autoantibody amount is higher than that of the normal autoantibody, and determining the subject to be demented or possibly susceptible to dementia.

Also, the sample derived from the sample is selected from the group consisting of human serum, plasma and blood, and the fixture is synthesized by a nitrocellulose membrane, a PVDF membrane, a polyvinyl or a polystyrene resin Wherein the antigen-antibody reaction is selected from the group consisting of a well plate and a glass slide glass, wherein the antigen-antibody reaction is selected from the group consisting of enzyme immunoassay, radioactive immunoassay, sandwich assay, Western blotting, immunoprecipitation, Fluorescence immunoassay, enzyme substrate staining method, and antigen-antibody aggregation method. However, the present invention is not limited thereto.

The present invention also relates to a sample pad to which a biological sample is absorbed;

A conjugate pad binding to any one or more selected from the group consisting of ATCAY (Ataxia, Cerebellar, Cayman Type), NME7 (NME / NM23 Family Member 7) and NOL3 (Nucleolar Protein 3);

A reaction membrane on which a test line and a control line containing each antibody of the protein are treated;

An absorption pad on which a sample of the remaining amount is absorbed; And

The present invention provides a hardness cognitive diagnostic strip comprising a support.

The present invention also relates to a sample pad to which a biological sample is absorbed;

A conjugate pad coupled with ATCAY (Ataxia, Cerebellar, Cayman Type);

A reaction membrane on which a test line and a control line containing each antibody of the protein are treated;

An absorption pad on which a sample of the remaining amount is absorbed; And

A diagnostic strip for dementia comprising a support.

Wherein the biological sample is blood and the reaction membrane is nitrocellulose, cellulose, poly ethylene terephthalate (PVDF), polyethersufone (PES), glass fiber or nylon, and the absorbent pad is a cellulose, an anonymous or hydrophilic porous polymer. But is not limited thereto.

In a specific example of the present invention, the present inventors obtained sera from patients aged 60 years or older, mild cognitive impairment and dementia patients collected between 2009 and 2010 in urban areas (Ansan, Kyonggi-do) , CDR scores and apoE genotypes were analyzed. Educational and MMSE scores were lower in patients with mild cognitive impairment and in patients with Alzheimer 's disease. CDR scores were increased in patients with mild cognitive impairment and Alzheimer' s disease, The percentages of genes were found to be high in patients with mild cognitive impairment and Alzheimer's disease (see Table 1).

In addition, the inventors analyzed the autoantibody profile from the HuProt proteome microarray data to determine the type and distribution of autoantibodies present in the serum of 10 Korean elderly persons over 60 years old, Protein probes detected autoantibodies against a total of 434 proteins except for immunoglobulin molecules, and 269 (62%) of autoantibodies against 434 proteins were self-specific individual-specific) and 5 (1.2%) autoantibodies were detected in all sera (see Figure 1 and Table 2).

In order to search for an AD-abundant autoantibody (AD-abundant autoantibody), the present inventors compared and analyzed the autoantibody profiles of 5 healthy individuals and 5 Alzheimer's disease patients from HuProt proteome microarray data to determine whether or not Alzheimer's disease The number of autoantibodies in patients was 1.5 times that in normal subjects (see FIG. 2), confirming that the AD-abundant autoantigens present in patients with Alzheimer's disease were PAIP2, SURF5, HIST1H3F and C4orf40 (see Table 3) Functional enrichment analysis using DAVID (Database for Annotation, Visualization and Integrated Discovery) showed that most of the antigens present in patients with Alzheimer's disease were non-membrane bounded organelle and protein localization, And the antigenic protein associated with the ErbB signaling pathway Was confirmed (see Table 4 and Table 5).

In addition, the present inventors conducted a HuProt proteome microarray to select 12 protein probes among protein probes showing an autoantibody positive signal, and compared the 12 antigen proteins selected as above with respect to sex and age (± HIST1H3F, NME7, NOL3, and PAIP2 among these 12 antigenic proteins, using an indirect ELISA assay in a discovery set consisting of five individuals with normal and dementia matched to each other. (Table 7). The correlation between the AU value and the chip data was significant.

In addition, the present inventors have found that autoantibodies of ATCAY, HIST1H3F, NME7, NOL3 and PAIP2 5 antigens showing significant correlation between autoantibody ELISA AU value and chip data are biomarkers for Alzheimer's disease and hardness cognitive impairment (AMSI) and Alzheimer's disease group (44 patients), respectively, in a validation sample set. The results of the indirect enzyme immunoassay (ELISA assay), the levels of anti-ATCAY autoantibody IgG and anti-NME7 autoantibody IgG levels were statistically significant between normal, MCI, and Alzheimer's disease groups (See Table 8). In particular, the level of anti-ATCAY IgG was higher in the Alzheimer's disease group than in the normal group, and the level of anti-NME7 IgG was higher in the group of the hard cognitive impairment The level of anti-PAIP2 IgG was significantly higher in the hard cognitive impairment group than in the normal cognitive group.

 The present inventors also investigated whether the total IgG level in the normal group, the mild cognitive impairment group, and the Alzheimer's disease group affects the level of autoantibody, the total IgG level in the serum sample, The total IgG level in the serum was not different between the normal group, the mild cognitive impairment group, and the Alzheimer's disease group (see FIG. 3), but ATCAY , Anti-NME7 IgG levels were significantly correlated with levels of antibody to HIST1H3F, NME7, NOL3, and PAIP2, and the level of anti-NME7 IgG was positively correlated with the level of total IgG (total IgG level) (rs = 0.473, p < 0.001) (see Table 9).

In addition, the present inventors have normalized the level of autoantibodies to the total IgG level (total IgG level), and then compared the difference in the level of these autoantibodies between the normal cognitive group, the mild cognitive impairment group, and the Alzheimer's disease group , It was confirmed that the significance of anti-ATCAY IgG difference between the normal cognitive group and the Alzheimer's disease group was increased (see Fig. 4).

We also compared the correlation between the MMSE score and the K-MoCA and CDR scores with the MMSE score, the K-MoCA score and the CDR score, for the mild cognitive impairment test, and the Spearman rank correlation test Spearman rank correlation test), confirming that the level of anti-ATCAY IgG is related to cognitive function (see FIG. 5).

In addition, the present inventors confirmed the positive detection frequency of autoantibodies between the normal cognitive group, the mild cognitive impairment group, and the Alzheimer's disease group, and performed a risk assessment using the anti-PAIP2 IgG and anti-ATCAY The higher the positive detection frequency of two autoantibodies combining IgG, the greater the risk of Alzheimer's disease. The higher the positive detection frequency of two autoantibodies, which bind anti-NOL3 IgG and anti-NME7 IgG, And increased the risk of mild cognitive impairment (aMCI) (see Table 10).

Therefore, the level of anti-ATCAY IgG and anti-NME7 IgG was high in the Alzheimer's disease group, and the level of anti-PAIP2 IgG was high in the mild cognitive impairment group, and anti-PAIP2 IgG and anti- The higher the positive detection frequency of two autoantibodies combining ATCAY IgG, the higher the risk of Alzheimer's disease. The higher the positive detection frequency of the two autoantibodies that bind anti-NOL3 IgG and anti-NME7 IgG, (AMCI), or as a biomarker for the diagnosis of Alzheimer's disease, as the autoantibodies against the autoantigens of PAIP2, ATCAY, NOL3, NME7, and HIST1H3F were identified as having increased risk of type-A hardness cognitive impairment .

Normal people, Mild cognitive impairment  Patient, statistical data of Alzheimer's disease patients and apoE  Genotype analysis

A population-based geriatric cohort study was conducted in urban areas (Ansan, Gyeonggi-do, Korea). Serum samples were obtained from patients aged 60 years or older, mild cognitive impairment and dementia patients collected between 2009 and 2010, Sample) and validation set. The discovery set consisted of five normal and demented patients, each matching sex and age (± 2) for autologous antibody profiling using a human proteome microarray. validation set) was used for validation of AD-abundant autoantibodies in 44 groups of 3 groups including sex, age matched normal group, amnestic mild cognitive impairment (aMCI), and Alzheimer's group Respectively. The patient group was divided into three groups according to gender, age, education score, Mini-Mental State Examination (MMSE), Clinical Dementia Rating (CDR) and apo E (Apolipoprotein E) Were analyzed by the following method.

Specifically, questionnaires were collected for the patients' education scores. The questionnaire items were divided into the following categories: first (6th), middle (3y), high (3y), university (4y), and graduate And total number of years of education. Diagnosis of dementia followed the criteria set forth in the Diagnosis and Statistical Manual of Mental Disorders, fourth edition (DSM-IV). Mild cognitive impairment followed the Petersen / Winblad criteria. In the present study, we used the MMSE-KC (Mini-Mental State Examination, MMSE) and the Montreal Cognitive Assessment (MoCA) CERAD Assessment Packet) and K-MoCA. MMSE scores ranged from 0 to 19 for mild cognitive impairment, 20 to 23 for mild cognitive impairment, and 24 to 30 for normal Respectively. The Clinical Dementia Rating Scale (CDR) was used for the Korean version of the Expanded Clinical Dementia Rating scale developed by Choi, Sung Hye et al. (2001). The CDR score was 0 for normal and 0.5 for suspected dementia. Point, 1 point for dementia and 2 points for severe dementia. ApoE genotyping was performed according to the manufacturer's protocol using the SNaPshot system (Applied Biosystems, Forster City, CA, USA). ApoE is a risk gene that increases the risk of developing Alzheimer's disease. If the gene has E4 (ApoE4) genotype in Korea, it is about 2.7 times when it has one allele gene compared with a person without this genotype, and about 17.4 times when it has two It is reported that the risk of Alzheimer's disease increases. In brief, the PCR was performed by using an extension primer to remove the primers and dNTP that were not used in the synthesis, and the PCR products were purified. The product is again The SNaPshot ready Reaction Mix and SNaPshot primer were mixed with each other to perform a secondary PCR reaction, and the PCR product was sequenced to determine the genotype.

As a result, the scores of education and MMSE were statistically significant and were lower in MCI patients and Alzheimer's disease patients group. CDR scores were statistically significant and increased in patients with MCI and Alzheimer's disease. ApoE Genotype analysis showed that apo E4 allele ratio was higher in patients with MCI and Alzheimer's disease (Table 1).

[Table 1]

Statistical data and apoE genotyping of normal, MCI, and Alzheimer patients

Protein body Microarray ( proteome microarray ) Self-port Autoantibody analysis

To determine the type and distribution of autoantibodies present in the serum of 10 elderly Koreans aged 60 years or older, an autoantibody profile was analyzed from HuProt proteome microarray data as follows.

Specifically, a HuProt microarray (CDI Laboratories, Baltimore, Md., USA), in which about 19,000 protein antigens were spotted, was used to investigate the autoantibody profile of human serum. Proteins in the protein microarray chip are expressed in yeast (Saccharomyces cerevisiae) as GST-fusion protein, and each protein is duplicated. The microarray experiment method was performed according to the manual provided by the manufacturer, and the method is as follows. After adding 3 ml blocking buffer (5% BSA in TBST (TBS, 0.1% tween 20)) to the microarray chip for 2 hours at room temperature, the serum samples were blocked with blocking buffer at a ratio of 1: 500 , And 3 ml was added to the chip, followed by reaction at room temperature for 1 hour. Then, the chip was washed with shaking incubation for 10 minutes by adding 4 ml of TBST buffer to the chip. The chip was washed three times, and then a Cy5-conjugated goat anti-rabbit antibody diluted 1: 1000 in blocking buffer 3 ml of human IgG (H + L) (Abcam, Cambridge, UK) was washed 3 times with TBST, and the dried microarray chip was exposed to 635 nm wavelength using a GenePix 4000B Florescence scanner (Molecular Devices, Sunnyvale, Calif. And stored the images in TIFF format.

Scanned images were aligned and quantitated using the GAL file provided by GenePix Pro software 6.0 (Molecular Devices, Sunnyvale, Calif., USA) and Huprot proteome microarray manufacturer . The spot intensity signal value is defined as F635 median (the median of all the feature pixel intensities) - B635 (the median of all the background pixel intensities) and the signal intensity of a specific protein probe the probe was considered to be an autoantibody positive signal if the signal value was greater than three standard deviations of the intensity signal values of all the probes.

As a result, protein probes showing autoantibody positive signals showed autoantibodies against a total of 434 proteins except for immunoglobulin molecules, and there was an average of 82.0 ± 41.1 autoantibodies per person , 269 (62%) of the autoantibodies against 434 proteins were individual-specific, and 5 (1.2%) of autoantibodies were detected in all the sera (Fig. 1 and Table 2).

[Table 2]

Self-antibodies in serum of Korean elderly over 60 years old

Alzheimer's disease association Autoantibody  analysis

<3-1> Patients with Alzheimer's Disease Autoantibody  And antigen identification

To investigate AD-abundant autoantibody, which is common in patients with Alzheimer's disease, the autoantibody profiles of 5 normal subjects and 5 Alzheimer's disease patients were compared from the HuProt proteome microarray data by the same method as in Example 2 above And analyzed.

As a result, the average number of autoantibodies in patients with Alzheimer's disease was 98.0 ± 39.9, which was 1.5 times more than that in healthy individuals (66.0 ± 39.6) (FIG. 2) SURF5, HIST1H3F and C4orf40 (Table 3). In addition, functional enrichment analysis using DAVID (Database for Annotation, Visualization and Integrated Discovery) showed that many of the antigens present in patients with Alzheimer's disease had no membrane in the cellular component ontology (GO cellular component) It is highly related to non-membrane bounded organelle and is highly related to protein localization in the biological process ontology (GO biological process) that deals with various biological purposes. KEGG (Kyoto Encyclopedia of Genes and Genomes ) Antibody proteins related to the ErbB signaling pathway were significantly distributed in the biochemical pathway database (Table 4 and Table 5).

[Table 3]

Alzheimer's disease is a common autoantigen

[Table 4]

Enrichment analysis of autoantigen in patients with Alzheimer's disease using DAVID

[Table 5]

The KEGG pathway associated with autoantibodies in patients with Alzheimer's disease

<3-2> Verification of autoantibodies in a discovery sample set consisting of patients with normal and Alzheimer's disease

In order to verify the results of the HuProt proteome microarray, the autologous antibody was verified in a discovery set consisting of five individuals of normal and dementia matched with the sex and age (± 2) of the above <Example 1> .

Specifically, HuProt proteome microarray was performed to select twelve protein probes among protein probes showing autoantibody positive signals. In the first group of dementia consisting of 5 patients, the occurrence of autoantibodies was 3 or more and the occurrence was 2 or more in the normal group. The presence of autoantibodies is likely to be different between the control and patient groups. Secondly, we could distinguish between control group and patient group by difference of autoantibody level rather than presence of autoantibody. In the present study, we compared the results of immunohistochemistry and immunohistochemical staining for CHAC2, HIST1H3F, NOL3, PAIP2, RAB11FIP1 and SURF5 probes We selected ATCAY, CLC, GPBP1, SPANXN2 and TPM3 probes with an autoantibody occurrence of 7 or more without distinction between normal and control groups. Finally, they performed the functions related to Alzheimer's or neurodegeneration or other neuroimmunity Twelve protein probes, such as the putative NME7 probe, were screened (Table 6).

The autoantibody levels of the 12 antigen proteins selected as described above were measured by an indirect ELISA assay in the discovery sample set of Example 1, Were compared with each other. Twelve recombinant proteins (see Table 6) selected as described above were added to a 96-well plate (Thermo Scientific, Roskilde, Denmark) in a coating buffer (0.05M Carbonate / bicarbonate buffer, pH 9.6: SIGMA) (1 ug / ml), and then diluted recombinant protein (100 쨉 l / well) was added and reacted at 4 째 C overnight. The plate was then washed with 300 μl of PBST (prepared by adding 0.05% Tween 20 to PBS), then 200 μl blocking buffer (prepared by adding 5% skim milk powder to PBST) was added and reacted at room temperature for 2 hours . After 2 hours, the cells were washed with PBST, and 50 μl of a diluted serum sample diluted 1:50 in blocking buffer was added to the wells, followed by reaction at room temperature for 2 hours. After 2 hours, the plate was washed with PBST, and 100 μl of a secondary antibody (HRP-conjugated goat anti-human IgG H & L: Abcam, Cambridge, UK) with 1: 5000 diluted HRP was added to blocking buffer. For 1 hour. (Invitrogen, Frederick, MD, USA) was added to the wells, and the mixture was reacted at room temperature for 15 minutes. Then, 100 μl of stop solution (Invitrogen, The optical density (OD) was measured at 450 nm using an ELISA reader (Molecular Devices, Sunnyvale, Calif., USA). Human IgG (Human whole IgG: GenScript, Piscataway, NJ, USA) was used for internal variability test and normalization of autoantibody OD value. IgG), the normalized arbitrary unit (AU) was calculated and compared in the following manner.

Autoantibody AU =

[Autoantibody OD - background OD] / [human whole IgG OD - background OD].

※ background OD: OD value of well coated with protein except serum sample

The correlation between the thus obtained ELISA AU value and microarray chip data was analyzed by Spearman correlation analysis.

As a result, a significant correlation between the autoantibody ELISA AU values for ATCAY, HIST1H3F, NME7, NOL3, and PAIP2 and chip data was confirmed among these 12 antigen proteins (Table 7) Protein chip results were also reproduced in ELISA experiments.

[Table 6]

Twelve recombinant proteins selected

[Table 7]

Association of microarray data with ELISA data for selected 12 autoantibodies

<3-3> Normal cognitive groups  In a validation sample set consisting of memory-type hard cognitive impairment group and Alzheimer's disease group Autoantibody  Verification

The autoantibodies of ATCAY, HIST1H3F, NME7, NOL3, and PAIP2, which showed significant correlation between the autoantibody ELISA AU value and chip data in the above <Example 3-2>, were found in Alzheimer's disease and hardness cognitive disorder In a validation sample set consisting of 44 normal individuals, sex, age group, amnestic mild cognitive impairment (aMCI), and Alzheimer's disease group, (Indirect ELISA assay) as in Example 3-2 &gt;.

 As a result, there was a statistically significant difference between the levels of anti-ATCAY autoantibody IgG level and anti-NME7 autoantibody IgG level, normal cognitive impairment group (MCI), and Alzheimer's disease group (Table 8). Especially, the level of anti-ATCAY IgG was higher in the Alzheimer's disease group than in the normal group (p = 0.019) and the level of anti-NME7 IgG was higher in the hard cognitive impairment group (P = 0.046), and the level of anti-PAIP2 IgG was significantly higher in the hard cognitive impairment group than in the normal cognitive group.

[Table 8]

Comparison of the levels of autoantibodies in the normal cognitive group, the mild cognitive impairment group (MCI), and the Alzheimer's disease group

<3-4> Autoantibody  Normal group, Mild cognitive impairment group , Alzheimer's Disease  all IgG  Amount (total IgG  level)

  To determine whether the total IgG level of the normal group, the mild cognitive impairment group, and the Alzheimer's disease group affects the level of autoantibody, the total IgG level in the serum sample was directly measured Were measured by an indirect ELISA assay.

Specifically, a serum sample was diluted 1: 500,000 in a coating buffer, and then 100 μl of each sample was added to a 96-well plate in duplicate, followed by reaction at room temperature for 2 hours. After 2 hours, the plate was washed with 300 μl PBST three times, and 200 μl blocking buffer (1% BSA in PBS) was added thereto, followed by reaction at room temperature for 2 hours. After 2 hours, wash with PBST, add 100 μl of HRP-conjugated goat anti-human IgG H & L (Abcam, Cambridge, UK) diluted 1: 10,000 in blocking buffer and incubate for 1 hour at room temperature Lt; / RTI &gt; After 1 hour, the cells were washed and reacted with 100 μl of TMB at room temperature for 15 minutes. 100 μl of stop solution was added and the OD value was measured at 450 nm within 30 minutes.

As a result, the total IgG level in the serum was not different between the normal group, the mild cognitive impairment group and the Alzheimer's disease group (Fig. 3), but the ATCAY, The level of anti-NME7 IgG was significantly correlated with the total IgG level (total IgG level) (r), and the level of anti-NME7 IgG was positively correlated with the level of IgE antibody (HIST1H3F, NME7, NOL3, PAIP2) _s = 0.473, p &lt; 0.001) (Table 9).

Therefore, the level of autoantibody was normalized to total IgG level (total IgG level) in order to exclude the effect of total IgG level on the level of autoantibody, , Mild cognitive impairment group and Alzheimer's disease group. As a result, it was confirmed that the difference in the level of anti-ATCAY IgG between the normal cognitive group and the Alzheimer's disease group was increased (p = 0.010) (Fig. 4).

[Table 9]

Relationship between total IgG (total IgG) and autoantibodies

&Lt; 3-5 > ATCAY IgG  Relationship between level and cognitive function

In order to examine the relationship between the level of anti-ATCAY IgG and the cognitive function, the MMSE score obtained from the <Example 1> and the K-MoCA score and the CDR score for the mild cognitive impairment test were used to calculate the MMSE score And correlations with K-MoCA and CDR scores were confirmed by Spearman rank correlation test.

As a result, the MMSE score (r _s = -0.204, p = 0.019) and the K-MoCA score (r _s = -0.242, p = 0.005) were found to be significantly lower when the anti-ATCAY IgG level was higher , It was confirmed that the level of anti-ATCAY IgG was related to cognitive function (FIG. 5).

<3-6> Normal cognitive groups, Mild cognitive impairment group  And Alzheimer's disease group  The liver Autoantibody  Positive detection frequency ( occurence ) Confirm

We examined the positive detection frequency of autoantibodies between normal cognitive group, mild cognitive impairment group and Alzheimer 's disease group and performed risk assessment using them. The detection frequency of autoantibody positivity was calculated as the mean (normal) + 2 × standard deviation (SD) of this normalized value of normal (n = 44) normalized value of each autoantibody amount (AU) ) Were considered to be positive.

As a result, the risk of Alzheimer's disease was increased (p = 0.011, OR = 4.03, 95% CI: 1.32-12.37) as the two autoantibody positive detection incidence combining anti-PAIP2 IgG and anti- (P = 0.006, OR = 5.73, 95% CI) were associated with a higher positive detection frequency of the two autoantibodies that bound anti-NOL3 IgG and anti-NME7 IgG : 1.50-21.87) (Table 10), an autoantibody against the autoantigens of PAIP2, ATCAY, NOL3 and NME7 could be used as a biomarker for memory hardness cognitive impairment (aMCI) or Alzheimer's disease Respectively.

[Table 10]

The positive detection frequency of autoantibodies in the normal cognitive group, the mild cognitive impairment group, and the Alzheimer's disease group, and the risk assessment using them,

<110> Centers for Disease Control and Prevention <120> Potential autoantibody biomarkers for the detection and diagnosis          of dementia <130> 2016P-07-030 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 371 <212> PRT <213> Homo sapiens <400> 1 Met Gly Thr Thr Glu Ala Thr Leu Arg Met Glu Asn Val Asp Val Lys   1 5 10 15 Glu Glu Trp Gln Asp Glu Asp Leu Pro Arg Pro Leu Pro Glu Glu Thr              20 25 30 Gly Val Glu Leu Leu Gly Ser Pro Val Glu Asp Thr Ser Ser Pro Pro          35 40 45 Asn Thr Leu Asn Phe Asn Gly Ala His Arg Lys Arg Lys Thr Leu Val      50 55 60 Ala Pro Glu Ile Asn Ile Ser Leu Asp Gln Ser Glu Gly Ser Leu Leu  65 70 75 80 Ser Asp Phe Leu Asp Thr Pro Asp Asp Leu Asp Ile Asn Val Asp                  85 90 95 Asp Ile Glu Thr Pro Asp Glu Thr Asp Ser Leu Glu Phe Leu Gly Asn             100 105 110 Gly Asn Glu Leu Glu Trp Glu Asp Asp Thr Pro Val Ala Thr Ala Lys         115 120 125 Asn Met Pro Gly Asp Ser Ala Asp Leu Phe Gly Asp Gly Thr Thr Glu     130 135 140 Asp Gly Ser Ala Ala Asn Gly Arg Leu Trp Arg Thr Val Ile Ile Gly 145 150 155 160 Glu Gln Glu His Arg Ile Asp Leu His Met Ile Arg Pro Tyr Met Lys                 165 170 175 Val Val Thr His Gly Gly Tyr Tyr Gly Glu Gly Leu Asn Ala Ile Ile             180 185 190 Val Phe Ala Ala Cys Phe Leu Pro Asp Ser Ser Leu Pro Asp Tyr His         195 200 205 Tyr Ile Met Glu Asn Leu Phe Leu Tyr Val Ile Ser Ser Leu Glu Leu     210 215 220 Leu Val Ala Glu Asp Tyr Met Ile Val Tyr Leu Asn Gly Ala Thr Pro 225 230 235 240 Arg Arg Arg Met Pro Gly Ile Gly Trp Leu Lys Lys Cys Tyr Gln Met                 245 250 255 Ile Asp Arg Arg Leu Arg Lys Asn Leu Lys Ser Leu Ile Ile Val His             260 265 270 Pro Ser Trp Phe Ile Arg Thr Val Leu Ala Ile Ser Arg Pro Phe Ile         275 280 285 Ser Val Lys Phe Ile Asn Lys Ile Gln Tyr Val His Ser Leu Glu Asp     290 295 300 Leu Glu Gln Leu Ile Pro Met Glu His Val Gln Ile Pro Asp Cys Val 305 310 315 320 Leu Gln Tyr Glu Glu Glu Arg Leu Lys Ala Arg Arg Glu Ser Ala Arg                 325 330 335 Pro Gln Pro Glu Phe Val Leu Pro Arg Ser Glu Glu Lys Pro Glu Val             340 345 350 Ala Pro Val Glu Asn Arg Ser Ala Leu Val Ser Glu Asp Gln Glu Thr         355 360 365 Ser Met Ser     370 <210> 2 <211> 376 <212> PRT <213> Homo sapiens <400> 2 Met Asn His Ser Glu Arg Phe Val Phe Ile Ala Glu Trp Tyr Asp Pro   1 5 10 15 Asn Ala Ser Leu Leu Arg Arg Tyr Glu Leu Leu Phe Tyr Pro Gly Asp              20 25 30 Gly Ser Val Glu Met His Asp Val Lys Asn His Arg Thr Phe Leu Lys          35 40 45 Arg Thr Lys Tyr Asp Asn Leu His Leu Glu Asp Leu Phe Ile Gly Asn      50 55 60 Lys Val Asn Val Phe Ser Arg Gln Leu Val Leu Ile Asp Tyr Gly Asp  65 70 75 80 Gln Tyr Thr Ala Arg Gln Leu Gly Ser Arg Lys Glu Lys Thr Leu Ala                  85 90 95 Leu Ile Lys Pro Asp Ala Ile Ser Lys Ala Gly Glu Ile Ile Glu Ile             100 105 110 Ile Asn Lys Ala Gly Phe Thr Ile Thr Lys Leu Lys Met Met Met Leu         115 120 125 Ser Arg Lys Glu Ala Leu Asp Phe His Val Asp His Gln Ser Arg Pro     130 135 140 Phe Phe Asn Glu Leu Ile Gln Phe Ile Thr Thr Gly Pro Ile Ile Ala 145 150 155 160 Met Glu Ile Leu Arg Asp Asp Ala Ile Cys Glu Trp Lys Arg Leu Leu                 165 170 175 Gly Pro Ala Asn Ser Gly Val Ala Arg Thr Asp Ala Ser Glu Ser Ile             180 185 190 Arg Ala Leu Phe Gly Thr Asp Gly Ile Arg Asn Ala Ala His Gly Pro         195 200 205 Asp Ser Phe Ala Ser Ala Ala Arg Glu Met Glu Leu Phe Phe Pro Ser     210 215 220 Ser Gly Gly Cys Gly Pro Ala Asn Thr Ala Lys Phe Thr Asn Cys Thr 225 230 235 240 Cys Cys Ile Val Lys Pro His Ala Val Ser Glu Gly Leu Leu Gly Lys                 245 250 255 Ile Leu Met Ala Ile Arg Asp Ala Gly Phe Glu Ile Ser Ala Met Gln             260 265 270 Met Phe Asn Met Asp Arg Val Asn Val Glu Glu Phe Tyr Glu Val Tyr         275 280 285 Lys Gly Val Val Thr Glu Tyr His Asp Met Val Thr Glu Met Tyr Ser     290 295 300 Gly Pro Cys Val Ala Met Glu Ile Gln Gln Asn Asn Ala Thr Lys Thr 305 310 315 320 Phe Arg Glu Phe Cys Gly Pro Ala Asp Pro Glu Ile Ala Arg His Leu                 325 330 335 Arg Pro Gly Thr Leu Arg Ala Ile Phe Gly Lys Thr Lys Ile Gln Asn             340 345 350 Ala Val His Cys Thr Asp Leu Pro Glu Asp Gly Leu Leu Glu Val Gln         355 360 365 Tyr Phe Phe Lys Ile Leu Asp Asn     370 375 <210> 3 <211> 208 <212> PRT <213> Homo sapiens <400> 3 Met Gly Asn Ala Gln Glu Arg Pro Ser Glu Thr Ile Asp Arg Glu Arg   1 5 10 15 Lys Arg Leu Val Glu Thr Leu Gln Ala Asp Ser Gly Leu Leu Leu Asp              20 25 30 Ala Leu Leu Ala Arg Gly Val Leu Thr Gly Pro Glu Tyr Glu Ala Leu          35 40 45 Asp Ala Leu Pro Asp Ala Glu Arg Arg Val Arg Arg Leu Leu Leu Leu      50 55 60 Val Gln Gly Lys Gly Glu Ala Ala Cys Gln Glu Leu Leu Arg Cys Ala  65 70 75 80 Gln Arg Thr Ala Gly Ala Pro Asp Pro Ala Trp Asp Trp Gln His Val                  85 90 95 Gly Pro Gly Tyr Arg Asp Arg Ser Tyr Asp Pro Pro Cys Pro Gly His             100 105 110 Trp Thr Pro Glu Ala Pro Gly Ser Gly Thr Thr Cys Pro Gly Leu Pro         115 120 125 Arg Ala Ser Asp Pro Asp Glu Ala Gly Gly Pro Glu Gly Ser Glu Ala     130 135 140 Val Gln Ser Gly Thr Pro Glu Glu Pro Glu Pro Glu Leu Glu Ala Glu 145 150 155 160 Ala Ser Lys Glu Ala Glu Pro Glu Pro Glu Pro Glu Pro Glu Leu Glu                 165 170 175 Pro Glu Ala Glu Ala Glu Pro Glu Pro Glu Leu Glu Pro Glu Pro Asp             180 185 190 Pro Glu Pro Glu Pro Asp Phe Glu Glu Arg Asp Glu Ser Glu Asp Ser         195 200 205 <210> 4 <211> 5049 <212> DNA <213> Homo sapiens <400> 4 accccccacc ccctcctccc tgcacacaaa agcagcataa attaaccgtc ttcgggaagc 60 cgagcctctg ccagccctga gctgggaaga agcagctacc tcggaggcag ggcgcgcagg 120 cgggcggcga tgagaggggg cgcagccgca gccccgcgct ggggagccca ccgctaaccc 180 tgcaccccac ccacccctgc acaaaagagc tggcgggcgc tggccacgtc gccctgggtg 240 accttcctcg gatgcagaat ccgcccctgc gagcatcctc ttcctcctag gctctgaagg 300 cccggggagc gtgagcgatg cccagctgca cccgggcagg gctcgccttt gtttgccagt 360 aaggaggaga ggctgtctca gctgcagagg ggtcatccct gcttcaagcc agtgcctctt 420 cccagctccc atggggacca ccgaagccac gctccggatg gaaaacgtgg acgtgaagga 480 ggaatggcag gacgaagatc ttcccaggcc actcccagaa gagacggggg tggaactgct 540 tggcagcccg gtggaagaca catcctctcc tcccaacacg ctaaatttca acggagcgca 600 tcgtaagagg aagacgctgg tggccccaga gatcaacatt tctctggatc agagtgaggg 660 gtccctgctg tccgatgact tcttggatac ccctgatgac ctggatatta acgtggatga 720 catcgagacc cccgatgaga ccgactcgct ggagttcctg gggaatggca acgaactgga 780 gtgggaagac gacacccccg tggccaccgc caagaacatg cccggggaca gcgcggatct 840 atttggggac ggcacgacgg aggacggcag cgccgccaac gggcgcctgt ggcggacagt 900 gatcatcggg gagcaagagc accgtataga cctgcacatg atccggcctt acatgaaagt 960 ggtcacccac ggagggtact acggcgaagg cctcaacgcc atcatcgtct tcgcagcctg 1020 cttccttcca gacagcagcc tccccgacta ccactacatc atggagaacc tcttcctgta 1080 cgtcatcagc agcttagagc tcctggtggc tgaggactac atgatcgtgt acctgaacgg 1140 tgccacgccc cggcggagga tgcctggaat cggctggctg aagaagtgct accagatgat 1200 cgaccggagg ttgcggaaaa acctgaagtc cttgatcatc gtccacccct cgtggttcat 1260 tcggactgtg ctggccatct ctcgcccttt catcagcgtc aagttcatca acaagatcca 1320 gtacgtgcac agcttggaag acctggagca actcatccct atggaacacg tccagatccc 1380 agactgcgtc ctgcaatacg aagaggaaag actgaaggcc aggagggaga gcgcgaggcc 1440 ccagccggag tttgtgctgc ccaggtctga agagaagcca gaggtggcac cagtggaaaa 1500 caggtctgct ctggtctcag aagatcagga aacaagcatg tcctgaggcg acgtgagcat 1560 aacaaaggac atggaagaag attccagatg ccagaaaacc tctgtcagac gcccactggc 1620 cccagatctc atcctgcctc atcctgagtc ccaatcttcc aagggtgcca gcccctccgt 1680 tcatctctga aacccagcat ccttttcagc tgcttgaaaa cattgtattt ttttttttta 1740 acgatgcagt atttgtgcgt tccagaaaag ggcccagctc tgagcccctc acccttccac 1800 actcacgaac tctcagccga ggaaggcaag aagcgcaggg ggtggcccgc gtggcgtcgg 1860 tggcctccgc tcctgctcgc agcccctgtg gtcagagctg gatacaagat tcaagaccct 1920 tctcttgctt gtcacccgct ccaggttgga gccacagaca cccaccgcca ccccggctgg 1980 gtctgcgtcc tttcctgtgc ctttccctcc agaatgcggc ctcagaccta gaagctcaac 2040 ccccctatga gggccacgtc ctggggtagc tcctgacctc cgaccttatg tccaaatttc 2100 acacccatgg tttttcattt gacccgcccc cttctcgctc ataatgacac ccagctcctt 2160 tgagaggatc agagcccatt gcacaagaag agccgctgcc aaccatcctt gtcctccgat 2220 tgcaaaatga caccccagta atctagaaca ttctcaagcc cctttaactc agatgtcaag 2280 ccaccgggca aaccccgtca atacctccca ccaaggaatg agatatgtgg acctcactgc 2340 tcccccaacc cagcgtcagg ctgggacacg ccaacgctgt tccgggttgg aacagcagag 2400 gctcagaaac tggctctgaa ataggcagac ctagcaagag gaagatacag ggtatcgggc 2460 gtttgagtgt ttcagaagtc attcgggaag ataaatccag tgcgctggcc gcagccacct 2520 gcattcaaag cttggaccag cgggttcttg ttcgggaggc aaatttccct aggaaaaaga 2580 agacagactt ttctaatgtg gtccaaatgc ggatcactgg tcagatggac tctagaagca 2640 ctgagctccc tgtctctgga agtatttaag aaaaggctgg gccaggcacg atggctcacg 2700 cctgtaatcc cagactttgg gaggccgagg caggcggatc acctgaggtg aggagtttga 2760 gaacagcctg gccaacatgg tgaaacctca tctctactaa aaatacaaaa attagccagg 2820 cgtggtggca ggtgcctgta atcccagcta cttgggaggc tgaggcatga gaatcactta 2880 aacctgagag gcagaggtta cagtgagcca agatcgtgcc actgcattcc agcctgggcg 2940 acagagcaag actctgtctc aaaaaaaata aaaaataatc agggcacagt ggctcatgcc 3000 tgtaatccca gcactctggg aggctgaggt gggtggatca cctgaggtca ggagttcaag 3060 accagcctgg tgaacatggc gaaaccccgt ctctaataaa aatacaaaaa ttagccgggc 3120 atggtggtgc atgcctgtaa tcccagctac tcgggaggct gaggcaggag aactgcttga 3180 acccaggagg cagaggttgc agtgatccaa gatcatgcca ctgcactcca gcctgggcaa 3240 caagagcaaa actccgtctc aaaataaaaa gaaaagaaaa gaatggacaga tgtttgcaga 3300 gagttgctca cgagtttccc tctaatccta aatgtcttca tgtctatcag tctgagcaga 3360 cggtgagtag ggcgggcaca ttctccaggc ccttcttcct agctctgtgg ttgacctctc 3420 agcaagtgct atccaggctg ggccaaccag acccacaatt aactgagcct cagtgaaagc 3480 gtccagtgca tcttgacctg agacagcaag gaattgcatt tggggttatt ccaacgatga 3540 tggcagggaa ctggtggtat ttagtgctga ggggcagtga tacagaaaga tttgccctgt 3600 gggacagggt cctgcgcgag tcccatcccc aaaagccagc agctcctgcc atgaggaaga 3660 cggggtttct gagcaggctt atgcctgcag gttcctgtgg agccaccggc tgtgacggga 3720 cacctctggg tctcagcatt gccctgggga ggctgggaca tttagggaca tggtagggtt 3780 ttaacatttg tttcccaaat gtcaaatccc gggcacaggg gcaagaccct gtcccgaatt 3840 cccaccccag tgaatggtgt cgctgccaaa gccaacacaa gatgacaaaa gtggctgggt 3900 acggtggctc acgcctataa tcccagcact ttgggagacc gagacaggtg gatcacctga 3960 ggtcaggagt tcgagaccag gctggccaac atggtgaaac cccatctcta ctaaaaatac 4020 aaaaattagc tgggtgtggt ggcgcgcacc tgtagtccca gctactcagg aggctgaggt 4080 agaagaatag ctggaaccca ggaggcagag attgcagtca gccgagattg caccactgca 4140 ctccagcctg ggagacagag caagactgac tcaaaagaaa aaaaatgaca gaagcctgat 4200 tatcagactg cccggaggag acaggctcca gcagatagat gccagccagg cccagctgcc 4260 acgatttgtc ccaggtgacc aaaggcacgc agctccagca tgaatcgttc taacccaaca 4320 gtgacaagaa ctgctgggcc ttaaccgtca tggaagactg gggccgcttc caagtcacag 4380 acaggagacg gggacaggaa agaactcatt ccacccaatc ggacacctaa taattgagtg 4440 tctacagcag caatcaagtg acaagtgagg ccctacctga cccagaaggt gcctgccggc 4500 taaacattct gcccccacca gaaactccag ggggtccgcc cgttatgccg tggcccaccc 4560 acgccccttt ggatcaccag cagtcacaga caacaggcag gcgaaactga agaccccaac 4620 tcagccccag cggaccctcc agagcaaaag aggcccccgg cgaggccacc tgtcggcagg 4680 catgccgagg tcaaacagcc ggggccaccg ttcccagctg ggccacgacc tgcaccgtcc 4740 acagatgggc tttgagatgg atttgtatca gggtgggggg tgtggtttgg ccaaaatgca 4800 atggaccccg acccctcctc gtaaaaggat gttgggtttc cctctggtga cacatgggat 4860 gcgtcataaa ccctccccca aagtcctggt cagcagccca tccttccaac gatgagtttt 4920 gcggtttttc agaacagaaa tgatcactac gattgacgac ggtcgtgatg ttaagacgtc 4980 gtctccatga gctttggggg gacttttatg tggaataaag aaactatcac tgagaaaaaa 5040 aaaaaaaaa 5049 <210> 5 <211> 1656 <212> DNA <213> Homo sapiens <400> 5 gtaaaactgc cggaaacaga ataatggcgt ctcgtagccc caggcgacag cgtggagggg 60 cgggtctgtc gattggatga acgcagctga gattactccc agccactaag gacgaagagg 120 tggggcggtg gcgtcccacg cctcgtgcga cagtgggcgg ggctttgttg cctgagtaac 180 cgtatgatgg tggtggtggt ggtgtcttcc tgtctcaacg atacctattt tctagtgctg 240 agatcctgag acaatgaatc atagtgaaag attcgttttc attgcagagt ggtatgatcc 300 aaatgcttca cttcttcgac gttatgagct tttattttac ccaggggatg gatctgttga 360 aatgcatgat gtaaagaatc atcgcacctt tttaaagcgg accaaatatg ataacctgca 420 cttggaagat ttatttatag gcaacaaagt gaatgtcttt tctcgacaac tggtattaat 480 tgactatggg gatcaatata cagctcgcca gctgggcagt aggaaagaaa aaacgctagc 540 cctaattaaa ccagatgcaa tatcaaaggc tggagaaata attgaaataa taaacaaagc 600 tggatttact ataaccaaac tcaaaatgat gatgctttca aggaaagaag cattggattt 660 tcatgtagat caccagtcaa gacccttttt caatgagctg atccagttta ttacaactgg 720 tcctattatt gccatggaga ttttaagaga tgatgctata tgtgaatgga aaagactgct 780 gggacctgca aactctggag tggcacgcac agatgcttct gaaagcatta gagccctctt 840 tggaacagat ggcataagaa atgcagcgca tggccctgat tcttttgctt ctgcggccag 900 agaaatggag ttgttttttc cttcaagtgg aggttgtggg ccggcaaaca ctgctaaatt 960 tactaattgt acctgttgca ttgttaaacc ccatgctgtc agtgaaggac tgttgggaaa 1020 gatcctgatg gctatccgag atgcaggttt tgaaatctca gctatgcaga tgttcaatat 1080 ggatcgggtt aatgttgagg aattctatga agtttataaa ggagtagtga ccgaatatca 1140 tgacatggtg acagaaatgt attctggccc ttgtgtagca atggagattc aacagaataa 1200 tgctacaaag acatttcgag aattttgtgg acctgctgat cctgaaattg cccggcattt 1260 acgccctgga actctcagag caatctttgg taaaactaag atccagaatg ctgttcactg 1320 tactgatctg ccagaggatg gcctattaga ggttcaatac ttcttcaaga tcttggataa 1380 ttagtggtgt ggaaagtaaa gaagtcacag gttgggacat ttagacaaga gtgaatcaca 1440 cacgaggaat gtgttcattc ttttattgtc cgttgtttta acctgactga atacaagatc 1500 aacaagagca ctgtactcct ggcaattatt acatatgtta gaacatggat tttgcactgt 1560 agacaacatt taacaccagt ctatggggta ctgcattgct ttttataaag ttcaaaataa 1620 agatttattt tcaaacaaga aaaaaaaaaa aaaaaa 1656 <210> 6 <211> 1540 <212> DNA <213> Homo sapiens <400> 6 ggcattcaga gagtagatgc cagtcctggg aaaggcaggg gaggagagga gagccacggc 60 tgacgcttgg ggacagaagg aggagcctga ggaggagaca ggacagagcg tctggagagg 120 caggaggaca ccgagttccc cgtgttggcc tccaggtcct gtgcttgcgg agccgtccgg 180 cggctgggat cgagccccga caatgggcaa cgcgcaggag cggccgtcag agactatcga 240 ccgcgagcgg aaacgcctgg tcgagacgct gcaggcggac tcgggactgc tgttggacgc 300 gctgctggcg cggggcgtgc tcaccgggcc agagtacgag gcattggatg cactgcctga 360 tgccgagcgc agggtgcgcc gcctactgct gctggtgcag ggcaagggcg aggccgcctg 420 ccaggagctg ctacgctgtg cccagcgtac cgcgggcgcg ccggaccccg cttgggactg 480 gcagcacgtg ggtccgggct accgggaccg cagctatgac cctccatgcc caggccactg 540 gacgccggag gcacccggct cggggaccac atgccccggg ttgcccagag cttcagaccc 600 tgacgaggcc gggggccctg agggctccga ggcggtgcaa tccgggaccc cggaggagcc 660 agagccagag ctggaagctg aggcctctaa agaggctgaa ccggagccgg agccagagcc 720 agagctggaa cccgaggctg aagcagaacc agagccggaa ctggagccag aaccggaccc 780 agagcccgag cccgacttcg aggaaaggga cgagtccgaa gattcctgaa ggccagagct 840 ctgacaggcg gtgccccgcc catgctggat aggacctggg atgctgctgg agctgaatcg 900 gatgccacca aggctcggtc cagcccagta ccgctggaag tgaataaact ccggagggtc 960 ggacgggacc tgggctctct ccacgattct ggctgtttgc ccaggaactt agggtgggta 1020 cctctgagtc ccagggacct gggcaggccc aagcccacca cgagcatcat ccagtcctca 1080 gccctaatct gcccttagga gtccaggctg caccctggag atcccaaacc tagcccccta 1140 gtgggacaag gacctgaccc tcctgcccgc atacacaacc catttcccct ggtgagccac 1200 ttggcagcat atgtaggtac cagctcaacc ccacgcaagt tcctgagctg aacatggagc 1260 aaggggaggg tgacttctct ccacataggg agggcttaga gctcacagcc ttgggaagtg 1320 agactagaag aggggagcag aaagggacct tgagtagaca aaggccacac acatcattgt 1380 cattactgtt ttaattgtct ggcttctctc tggactggga gctcagtgag gattctgacc 1440 agtgacttac acaaaaggcg ctctatacat attataatat attcgcttac taaatgaata 1500 aggactttcc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1540

Claims (16)

Wherein the at least one protein is selected from the group consisting of ATCAY (Ataxia, Cerebellar, Cayman Type), NME7 (NME / NM23 Family Member 7) and NOL3 (Nucleolar Protein 3).
ATCAY (Ataxia, Cerebellar, Cayman Type) Protein-containing dementia diagnostic kit.
The conjugate according to any one of claims 1 to 3, which is conjugated with a color-developing marker by reacting with an antibody specific to an autoantibody specifically binding to the protein; And a color development substrate solution washing solution or an enzyme reaction stop solution for a color reaction with the label.
3. The diagnostic kit of claim 1 or 2, wherein the protein is bound to a solid substrate.
[5] The method of claim 4, wherein the solid substrate is any one selected from the group consisting of a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl or polystyrene resin, and a glass slide glass .
The method of claim 1 or 2, wherein the coloring substrate is selected from the group consisting of TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino- bis (3- ethylbenzothiazoline- ] And OPD (o-phenylenediamine).
1) adding a sample derived from a subject to a fixed body coated with any one or more proteins selected from the group consisting of ATCAY, NME7 and NOL3 to perform an antigen-antibody reaction;
2) detecting the antigen-antibody reactant produced in step 1) using a chromogenic substrate solution; And
3) A method for detecting an autoantibody in order to provide information on the diagnosis of a mild cognitive impairment comprising the step of judging an increased test subject as compared with a normal autoantibody amount to a person having a mild cognitive impairment or a possibility of taking a mild cognitive impairment .
1) adding a sample derived from a subject to a solid body coated with an ATCAY protein to perform an antigen-antibody reaction;
2) detecting the antigen-antibody reactant produced in step 1) using a chromogenic substrate solution; And
3) determining whether the detected autoantibody amount is greater than that of the normal autoantibody, and determining that the autoantibody amount is greater than the normal autoantibody level.
9. The method according to claim 7 or 8, wherein the sample derived from the subject is selected from the group consisting of human serum, plasma and blood.
9. The method according to claim 7 or 8, wherein the fixture is selected from the group consisting of a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl or polystyrene resin, and a glass slide glass Lt; / RTI &gt;
The method according to claim 7 or 8, wherein the antigen-antibody reaction is selected from the group consisting of enzyme immunoassay, radioactive immunoassay, sandwich assay, Western blotting, immunoprecipitation, immunohistochemistry, fluorescent immunoassay, enzyme- &Lt; / RTI &gt; antibody aggregation method.
A sample pad on which the biological sample is absorbed;
A conjugate pad binding to any one or more selected from the group consisting of ATCAY (Ataxia, Cerebellar, Cayman Type), NME7 (NME / NM23 Family Member 7) and NOL3 (Nucleolar Protein 3);
A reaction membrane on which a test line and a control line containing each antibody of the protein are treated;
An absorption pad on which a sample of the remaining amount is absorbed; And
A strip for the diagnosis of mild cognitive impairment comprising a support.
A sample pad on which the biological sample is absorbed;
A conjugate pad coupled with ATCAY (Ataxia, Cerebellar, Cayman Type);
A reaction membrane on which a test line and a control line containing each antibody of the protein are treated;
An absorption pad on which a sample of the remaining amount is absorbed; And
A diagnostic strip for dementia comprising a support.
The diagnostic strip according to claim 12 or 13, wherein the biological sample is blood.
The diagnostic strip according to claim 12 or 13, wherein the reaction membrane is nitrocellulose, cellulose, poly ethylene terephthalate (PVDF), polyethersufone (PES), glass fiber or nylon.
14. A diagnostic strip according to claim 12 or 13, characterized in that the absorbent pad is a cellulose, an anonymous or hydrophilic porous polymer.

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