A novel biomarker for diagnosing Alzheimer's disease discovered from blood-derived exosomes and a method for diagnosing Alzheimer's disease using the same

Abstract

The present invention relates to a novel exosome-derived biomarker for diagnosing Alzheimer's disease, a composition for diagnosing Alzheimer's disease containing the biomarker, a kit for diagnosing Alzheimer's disease containing the composition, and a method for providing information for diagnosing Alzheimer's disease using the biomarker, composition, or kit.The use of the biomarker for diagnosing Alzheimer's disease provided by the present invention not only increases the diagnostic rate of Alzheimer's disease, but also improves accuracy, sensitivity, and specificity through detailed diagnosis according to the progression stage of Alzheimer's disease (normal, MCI, and AD), and can therefore be widely used for the effective diagnosis and further treatment of Alzheimer's disease.

Description

[Technical field]

[0001] The present invention relates to a novel biomarker for diagnosing Alzheimer's disease discovered from blood-derived exosomes and a method for diagnosing Alzheimer's disease using the same. More specifically, the present invention relates to a novel exosome-derived biomarker for diagnosing Alzheimer's disease, a composition for diagnosing Alzheimer's disease comprising the biomarker, a kit for diagnosing Alzheimer's disease comprising the composition, and a method for providing information for diagnosing Alzheimer's disease using the biomarker, composition, or kit. [Background technology]

[0002] Alzheimer's disease, a degenerative brain disease that accounts for 60-70% of dementia cases, is the most common disease among the elderly, with 10% of people aged 65-74, 19% of people aged 75-84, and 47% of people aged 85 or older developing the disease. The incidence rate is increasing year by year, making it a major social problem.

[0003] Conventional products only provide information about the proteins identified in Alzheimer's disease through enzyme-linked immunosorbent assay (ELISA) kits for amyloid β peptide, total tau, and phosphorylated tau proteins, which have been identified in conventional Alzheimer's disease research, and are not currently of much use in diagnosis.

[0004] The market share and awareness of leading products in Alzheimer's disease diagnostic kits is small, and the accuracy of diagnosing Alzheimer's disease using a single biomarker is low, so there is a need to form a diagnostic biomarker panel for Alzheimer's disease to improve the diagnosis rate.

[0005] On the other hand, exosome-derived extracts have a clearer correlation between diseases and biomarkers than general plasma extracts and can be purified to a high degree of purity, so research to find biomarkers from exosome-derived extracts is currently being actively conducted. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent No. 10-1274765 [Patent Document 2] Korean Patent No. 10-0784437 [Patent Document 3] Korean Patent No. 10-1547643 Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have discovered a new, more reliable biomarker for diagnosing Alzheimer's disease from blood-derived exosomes, and have developed a method for using it to provide information necessary for diagnosing Alzheimer's disease. [Means for solving the problem]

[0008] The main object of the present invention is to provide a novel biomarker for diagnosing Alzheimer's disease discovered from blood-derived exosomes. Another object of the present invention is to provide a method for providing information necessary for diagnosing Alzheimer's disease using the biomarker, composition, or kit.

[0009] A further object of the present invention is to provide a composition for diagnosing Alzheimer's disease, comprising a preparation for measuring the expression level of the biomarker. A further object of the present invention is to provide a kit for diagnosing Alzheimer's disease, which comprises the composition.

[0010] A further object of the present invention is to provide a method for providing information for identifying the progression stage of Alzheimer's disease using the biomarker, composition or kit. A further object of the present invention is to provide use of the biomarker for diagnosing Alzheimer's disease for the preparation of a composition for diagnosing Alzheimer's disease.

[0011] A further object of the present invention is to provide use of the composition for diagnosing Alzheimer's disease for diagnosing Alzheimer's disease. Effect of the Invention

[0012] Use of the biomarkers for diagnosing Alzheimer's disease provided by the present invention not only increases the rate of diagnosis of Alzheimer's disease, but also improves accuracy, sensitivity and specificity through detailed diagnosis according to the progression stage of Alzheimer's disease (normal, MCI, and AD), and can therefore be widely used for the effective diagnosis of Alzheimer's disease and further for the treatment of Alzheimer's disease. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram showing a method for providing information for identifying the progression stage of Alzheimer's disease provided by the present invention. [Figure 2a] FIG. 1 shows the results of primary and secondary proteomic analysis of exosome samples, divided into markers that increased or decreased. [Figure 2b] FIG. 1 shows the results of primary and secondary proteomic analysis of exosome samples, divided into markers that increased or decreased. [Diagram 3] FIG. 1 shows six biomarkers (MYH9, TSP1, TERA, APOM, APOC3, and APOA4) whose increase and decrease patterns significantly overlap in the results of the primary and secondary proteomics analyses. [Figure 4a] This pattern was set based on candidates showing a tendency for the expression difference to be lowest in the normal group, followed by mild cognitive impairment and Alzheimer's disease, in that order. [Figure 4b] This is a pattern set based on candidates that tend to be specifically elevated in mild cognitive impairment. [Figure 4c] This is a pattern set as a candidate that shows a tendency to be specifically elevated in Alzheimer's disease. [Figure 4d]This pattern was established based on the candidate showing the highest expression in the normal group. [Diagram 5] FIG. 1 is a schematic diagram summarizing the process of selecting transitions for a target peptide. [Figure 6] FIG. 1 is a schematic diagram summarizing the transition optimization process for a target SIS peptide. [Figure 7] This is a graph showing the results of MRM analysis using the final SIS peptide after optimization. [Figure 8a] 1 is a graph showing the results of confirming and verifying differential expression of transitions by AuDIT analysis of AD-specific samples. [Figure 8b] 13 is a graph showing the results of confirming and verifying transition expression differences by AuDIT analysis of MCI-specific samples. [Figure 9] 1 is a graph showing the results of individual sample analysis of the training set. [Figure 10] FIG. 1 shows ROC curves for biomarker candidates 1 and 5 from individual sample analysis. [Figure 11] 1 is a graph showing the results of an analysis of the expression levels of three biomarkers (ITGB3, APOA4, and CRP) at each stage of progression of Alzheimer's disease. [Figure 12] FIG. 1 shows the results of paired testing of eight clone antibodies by sandwich ELISA. [Figure 13] 1 is a graph showing the results of paired testing of eight clone antibodies by sandwich ELISA. [Figure 14a] FIG. 1 is a schematic diagram showing a process for measuring antigen-antibody affinity. [Figure 14b] FIG. 1 shows the results of measuring the affinity of Antibody 1 to an antigen. [Figure 14c] FIG. 1 shows the results of measuring the affinity of antibody 3 to an antigen. [Figure 14d] FIG. 1 shows the results of measuring the affinity of antibody 6 to an antigen. [Figure 15]FIG. 1 is a schematic diagram showing the development and performance testing process of an Alzheimer's disease diagnostic kit. [Figure 16] FIG. 1 is a schematic diagram showing a combination of biomarkers included in an Alzheimer's disease diagnostic kit. [Figure 17] FIG. 1 is a schematic diagram showing the process of assaying after immobilization of a biomarker in the preparation of a kit, taking as an example Tau, a marker associated with Alzheimer's disease. [Figure 18] FIG. 1 is a schematic diagram showing the immobilization order of three biomarkers. [Figure 19] 13 is a set of photographs showing the process of establishing the shape conditions of spots based on the concentration difference of the immobilized antibody. [Figure 20a] 1 shows photographs showing the results of an experiment screening for APOA4 immobilization conditions. [Figure 20b] FIG. 1 shows the results of testing low-, medium-, and high-titer samples under conditions that are believed to be most suitable for the APOA4 immobilization composition. [Figure 21] Photographs showing the results of an ITGB3 screening test. [Figure 22] FIG. 1 shows photographs and a graph showing the results of an immobilization test carried out on a CRP marker. [Diagram 23] FIG. 1 shows specific immobilization compositions for APOA4, CRP, and ITGB3. [Figure 24] 13 is a photograph showing the results of testing conditions for stabilizing antibodies immobilized after spotting of three types of markers. [Diagram 25] This is a photograph showing the results of a blocking test performed on antibodies immobilized after spotting of three types of markers. [Figure 26] This is a photograph showing the results of a dry test performed on fixed antibodies after spotting of three types of markers. [Figure 27] 13 is a photograph showing the results of testing conditions of a specimen dilution solution. [Figure 28] 13 is a photograph showing the results of testing conjugate dilution conditions. [Figure 29a]FIG. 1 shows photographs and a diagram showing the results of Western blot analysis of ITGB3. [Figure 29b] FIG. 1 shows photographs and a diagram showing the results of Western blot analysis of APOA4. [Figure 29c] Photographs showing the results of Western blot analysis of CRP. [Diagram 30] Photographs showing the results of analyzing actual samples using the final prepared kit. [Diagram 31] 1 is a graph showing the results of a correlation analysis of the ITGB3 marker. [Diagram 32] 1 is a graph showing the results of a correlation analysis of the APOA4 marker. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] To achieve the above object, one embodiment of the present invention provides a biomarker for diagnosing Alzheimer's disease, comprising a protein selected from the group consisting of APOA4, ITGB3, and a combination thereof.

[0015] By using the SG Cap technology, which is the core technology of the applicant, to develop a multiple biomarker diagnostic kit that can simultaneously detect not a single biomarker but a conventional marker and a newly discovered marker, it is expected that the accuracy, sensitivity, and specificity of diagnosis, which were insufficient in conventional single biomarker kits, can be improved. The details of the core technology are described in Patent Documents 1, 2, and 3.

[0016] In order to develop a method for objectively evaluating and diagnosing the onset of Alzheimer's disease, the present inventors conducted various studies to find a protein marker whose expression level changes depending on the onset of Alzheimer's disease among proteins in serum. As a result, it was confirmed that the expression levels of APOA4 and ITGB3 change depending on the onset of Alzheimer's disease, and they were found to be markers.

[0017] "Diagnosis" in this invention includes determining a subject's susceptibility to a particular disease or disorder, determining whether a subject currently has a particular disease or disorder, determining the prognosis of a subject having a particular disease or disorder, or therametrics (e.g., monitoring a subject's condition to provide information regarding the effectiveness of a treatment).

[0018] In the present invention, Alzheimer's disease diagnosis is interpreted as meaning the act of objectively determining whether or not a patient of interest has developed Alzheimer's disease, or, if a patient has developed Alzheimer's disease, the act of objectively determining the level of progression of the disease.

[0019] In the present invention, "APOA4 (Apolipoprotein A-IV)" refers to a type of plasma protein expressed by the APOA4 gene, and is also called apoA-IV, apoAIV, or apoA4. A protein consisting of 396 amino acids is expressed from the gene, and then APOA4, a glycoprotein consisting of 376 amino acids, is expressed through a maturation process. It is known that in most mammals, it is expressed in the intestine and secreted into the circulatory system. The specific amino acid sequence of APOA4 or the nucleotide sequence information of the gene encoding it is reported in databases such as NCBI. For example, it is reported in GenBank Accession Nos: NP_031494, NM_007468, NM_000482, etc.

[0020] In the present invention, "ITGB3 (Integrin β3)" refers to a peptide constituting integrin, a type of cell surface protein, and integrin is known to be involved in cell adhesion and cell surface-mediated signal transduction. The specific amino acid sequence of ITGB3 or the base sequence information of the gene encoding it is reported in databases such as NCBI. For example, it is reported in GenBank Accession Nos: NM_000212, NM_016780, NP_000203, NP_058060, etc.

[0021] The biomarkers for diagnosing Alzheimer's disease provided in the present invention may further include CRP. In the present invention, "CRP (C-reactive protein)" refers to a cyclic pentamer protein found in human plasma, which is synthesized in the liver. Since the concentration of CRP in blood increases during an inflammatory state, it is frequently used as a means of testing whether the human body is in an inflammatory state. The specific amino acid sequence of the CRP or the base sequence information of the gene encoding it is reported in databases such as NCBI. For example, it is reported in GenBank Accession Nos: NM_007768, NP_031794, etc.

[0022] Another embodiment of the invention provides a method of using said biomarkers, compositions or kits to provide information necessary for the diagnosis of Alzheimer's disease. Specifically, the method of the present invention for providing information necessary for determining whether or not a patient has Alzheimer's disease includes the steps of (a) quantitatively analyzing the expression level of a marker protein selected from the group consisting of APOA4, ITGB3, and a combination thereof in a serum sample from an individual, excluding humans, suspected of having Alzheimer's disease, and (b) correlating the level of the quantitatively analyzed marker protein with the determination of whether or not a patient has Alzheimer's disease.

[0023] In the present invention, an "individual" may be any animal, such as a mouse, livestock, mammal, including human, farmed fish, etc., that has developed or is at risk of developing Alzheimer's disease.

[0024] In the present invention, the step of quantitatively analyzing the expression level of a protein may be performed by any method known to those skilled in the art, including, but not limited to, PCR, ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, nucleic acid sequence-based amplification (NASBA), etc. Here, the amino acid sequence of the marker protein for diagnosing Alzheimer's disease according to the present invention or the nucleotide sequence of the gene encoding the same is publicly available in databases such as NCBI, and therefore, those skilled in the art can use an appropriate means required to measure the expression level of the protein.

[0025] In addition, since the quantitative analysis level of each of the above proteins varies depending on the condition of the patient, it is not easy to use only the fragmentary quantitative analysis level of the above proteins to determine whether or not Alzheimer's disease has developed. Therefore, by combining and analyzing the quantitative analysis levels of each of the above proteins, it can be used to determine whether or not Alzheimer's disease has developed.

[0026] As an example of a method for combining and analyzing the quantitative analysis results of each of the above proteins, a method is used in which the quantitative analysis levels of each protein measured from a serum sample are used alone or in combination to determine the presence or absence of Alzheimer's disease.

[0027] Another example of a method for combining and analyzing the quantitative analysis results for the protein is a conventional statistical analysis method, which is not particularly limited to, for example, linear or nonlinear regression analysis, linear or nonlinear classification analysis, ANOVA, neural network analysis, genetic analysis, support vector machine analysis, hierarchical analysis or cluster analysis, decision tree algorithm or kernel principal component analysis, Markov blanket, recursive feature elimination or entropy-based recursive feature elimination analysis, forward floating search or backward floating search analysis, which may be used alone or in combination.

[0028] The combination of the quantitative analysis results may also be performed using computer algorithms that perform these statistical methods automatically. The method of the present invention for providing information necessary for determining the presence or absence of Alzheimer's disease may further comprise a step of quantitatively analyzing the expression level of CRP protein in the step (a).

[0029] Yet another embodiment of the present invention provides a composition for diagnosing Alzheimer's disease comprising a formulation for measuring the expression level of said biomarkers. In the present invention, the "preparation for measuring expression level" refers to a preparation that specifically binds to the protein or the mRNA encoding it to enable recognition, or amplifies miRNA. Specific examples include, but are not limited to, an antibody that specifically binds to the protein, and a primer or probe that specifically binds to the miRNA. Those skilled in the art can select an appropriate preparation according to the purpose of the invention.

[0030] The preparation is directly or indirectly labeled to measure the expression level of the protein or mRNA. Specifically, the label may be, but is not limited to, a ligand, a bead, a radioactive nuclide, an enzyme, a substrate, a cofactor, an inhibitor, a fluorescent substance, a chemiluminescent substance, a magnetic particle, a hapten, a dye, etc. Specific examples of the ligand include biotin, avidin, streptavidin, etc., the enzyme includes luciferase, peroxidase, β-galactosidase, etc., and the fluorescent substance includes, but is not limited to, fluorescein, coumarin, rhodamine, phycoerythrin, sulforhodamine acid chloride (Texas red), etc. Almost any known label may be used as these detectable labels, and a person skilled in the art can select an appropriate label according to the purpose of the invention.

[0031] In the present invention, the term "antibody" refers to a proteinaceous molecule that specifically binds to an antigenic site of a protein or peptide molecule. Such an antibody can be prepared by cloning each gene into an expression vector by a conventional method, obtaining a protein encoded by the marker gene, and then using the obtained protein by a conventional method. The form of the antibody is not particularly limited, and the antibody of the present invention includes polyclonal antibodies, monoclonal antibodies, and any part thereof that has antigen-binding ability, and includes not only all immunoglobulin antibodies but also special antibodies such as humanized antibodies. The antibody also includes not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. The functional fragment of the antibody molecule refers to a fragment that has at least an antigen-binding function, such as Fab, F(ab'), F(ab')2, Fv, etc.

[0032] In the present invention, the term "primer" refers to a short base sequence having a short free 3' hydroxyl group, capable of forming a base pair with a complementary template and functioning as a starting point for copying the template. In the present invention, the primer used for amplifying the miRNA is a single-stranded oligonucleotide that acts as a starting point for template-directed DNA synthesis under appropriate conditions (e.g., four different nucleotide triphosphates and a polymerization agent such as DNA, RNA polymerase, or reverse transcriptase) in an appropriate buffer and at an appropriate temperature, but the appropriate length of the primer varies depending on the purpose of use. The primer sequence does not need to be completely complementary to the polynucleotide of the miRNA of the gene or its complementary polynucleotide, but only needs to be sufficiently complementary to be hybridized.

[0033] In the present invention, the term "probe" refers to a labeled nucleic acid fragment or peptide that specifically binds to miRNA. Specific examples of the probe include an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, an oligonucleotide peptide probe, and a polypeptide probe.

[0034] Yet another embodiment of the present invention provides a kit for diagnosing Alzheimer's disease, comprising a quantification device for measuring the expression level of the biomarker. The quantitative determination device included in the diagnostic kit of the present invention may be one that measures the expression level of the marker protein. Specific examples include an RT-PCR kit and an ELISA kit, but are not limited thereto as long as they can measure the expression level of miRNA or protein.

[0035] Here, the RT-PCR kit may be a kit containing essential elements required for performing RT-PCR. For example, the RT-PCR kit may contain, in addition to each primer specific to the gene, a test tube or other suitable container, a reaction buffer (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC water (DEPC-water), sterile water, etc. Also, the kit may contain a primer pair specific to a gene used as a quantitative control group.

[0036] Yet another embodiment of the invention provides a method of using said biomarkers, compositions or kits to provide information for staging the progression of Alzheimer's disease. Specifically, the method of the present invention for providing information for identifying the progression stage of Alzheimer's disease includes the steps of (a) quantitatively analyzing the expression level of a marker protein selected from the group consisting of APOA4, ITGB3, and a combination thereof in a serum sample of an individual, excluding humans, suspected of developing Alzheimer's disease, and (b) correlating the level of the quantitatively analyzed marker protein with a determination of the presence or absence of Alzheimer's disease.

[0037] In the above method, "individuals," "quantitative analysis of protein expression levels," "combination of quantitative analysis results," and the like are as described above. The method for providing information for identifying the progression stage of Alzheimer's disease provided by the present invention may further comprise a step of quantitatively analyzing the expression level of CRP protein in step (a).

[0038] In the method for providing information for identifying the progression stage of Alzheimer's disease provided by the present invention, the progression stages of Alzheimer's disease increase in severity in the order of normal (HC)-mild cognitive impairment (MCI)-Alzheimer's disease (AD), and these stages may be classified and diagnosed. For example, APOA4 is a MCI-specific marker and can be used to distinguish and diagnose HC, AD, and MCI, and ITGB3 can be used to distinguish and diagnose HC and AD (Figure 1).

[0039] FIG. 1 is a schematic diagram showing a method for providing information for identifying the progression stage of Alzheimer's disease provided by the present invention. Yet another embodiment of the present invention provides a use of the biomarker for diagnosing Alzheimer's disease for the preparation of a composition or kit for diagnosing Alzheimer's disease.

[0040] Yet another embodiment of the present invention provides a use of the Alzheimer's disease diagnostic composition or kit for diagnosing Alzheimer's disease. EXAMPLES

[0041] The present invention will be described in detail below with reference to examples. However, these examples are merely illustrative of the present invention and are not intended to limit the present invention. EXAMPLES

[0042] Biomarker selection To search for candidates for predictive biomarkers for Alzheimer's disease, exosomes were extracted from the blood of normal subjects, mild cognitive impairment patients, and Alzheimer's disease patients, and the same samples were subjected to primary and secondary analyses using proteomics. Next, as a result of the primary and secondary analyses, 50 biomarkers with large protein increases and decreases between the normal subjects, mild cognitive impairment patients, and Alzheimer's disease patients were selected for each stage. Finally, biomarkers with significant overlap in the primary and secondary increase and decrease types were organized (Figures 2a, 2b, and 3).

[0043] Figures 2a and 2b show the results of the primary and secondary proteomic analyses of exosome samples divided into markers that increased or decreased, and Figure 3 shows six biomarkers (MYH9, TSP1, TERA, APOM, APOC3, and APOA4) whose increase and decrease types significantly overlap in the results of the primary and secondary proteomic analyses. EXAMPLES

[0044] Verification of the association of the biomarkers selected in Example 1 with Alzheimer's disease In order to develop a biomarker for the early diagnosis of Alzheimer's disease obtained in Example 1, exosomes were extracted from plasma samples of Alzheimer's disease (AD), mild cognitive impairment (MCI), and normal subjects obtained from Switzerland (Universite de Geneva, Neurix), and a study was conducted to develop and verify a biomarker for predicting Alzheimer's disease and mild cognitive impairment.

[0045] Example 2-1: Organizing the expression of selected protein candidates into patterns before biomarker validation For the selected protein candidates, a total of four expression patterns were constructed by comparing the expression differences between Alzheimer's disease and mild cognitive impairment patients (Figures 4a to 4d).

[0046] Figure 4a is a pattern set with candidates showing the lowest expression difference in the normal group, followed by those showing a tendency for the expression difference to increase in mild cognitive impairment and Alzheimer's disease, in that order; Figure 4b is a pattern set with candidates showing a tendency for the expression difference to be specifically higher in mild cognitive impairment, Figure 4c is a pattern set with candidates showing a tendency for the expression difference to be specifically higher in Alzheimer's disease, and Figure 4d is a pattern set with candidates showing the highest expression in the normal group.

[0047] Example 2-2: Selecting representative target peptides and transitions for MRM validation of biomarker candidates For MRM validation of the selected Alzheimer's disease and mild cognitive impairment disease predictive biomarker candidate group, target peptides that specifically represent each protein were selected. The peptides were selected to contain approximately 6 to 20 amino acids in consideration of detection efficiency, and peptides containing post-translational modifications of proteins or sequences that are not fragmented by trypsin were excluded. Transitions, which are combinations of precursor ions (Q1) and precursor ions (Q3) that show quantitative values ​​during MRM analysis, were also set in the range of 50 to 1350 m / z to match the Agilent equipment used in the laboratory, and at least three transitions were selected per peptide (Figure 5).

[0048] FIG. 5 is a schematic diagram summarizing the process of selecting transitions for a target peptide. Example 2-3: SIS peptide transition optimization process At least three target peptides were selected for each target protein by referring to a web-based library (SRMAtlas, National Cancer Institute of Cancer Clinical Proteomics, PeptideAtlas). In addition, SIS peptides with the same sequence as the target peptides but labeled with isotopes (13C and 15N) at the carbon and nitrogen of lysine and arginine were preferentially synthesized and prepared. The SIS peptides were then analyzed and transition optimization was performed (Figure 6).

[0049] FIG. 6 is a schematic diagram summarizing the transition optimization process for the target SIS peptide. The transitions optimized by SIS peptides were spiked into pooled samples divided into control, mild cognitive impairment, and Alzheimer's disease groups, and MRM analysis was performed simultaneously. Simultaneous analysis of SIS peptides and pooled samples allows for standardization by grasping accurate elution times and absolute quantification. The detected transitions were re-verified as reliable transitions for quantification by applying the analytical reproducibility (coefficient of variation, CV<20%) and statistical criterion value (p-value<0.05) of the AuDIT (Automated Detection of Inaccurate and Imprecise Transition) program (Figure 7).

[0050] FIG. 7 is a graph showing the results of MRM analysis using the final SIS peptide after optimization. As a result of the AuDIT analysis, 1572 transitions corresponding to 215 peptides were selected as a final candidate set for validation in individual samples.

[0051] Example 2-4: Select the final biomarker to be used in kit development and verify the final biomarker with patient samples To increase the reliability of the validation of individual samples, all samples were divided into two independent cohorts, a training set and a test set, for analysis. Based on the results of the individual sample analysis of the training set, the results of the analysis of Alzheimer's disease, mild cognitive impairment, and normal groups using polynomial regression analysis and C statistics were used as the basis for biomarker selection, and the final biomarkers were selected taking into account various other factors as follows:

[0052] Here, as a result of analyzing some individual samples, it was confirmed that some candidate groups showed significant expression differences between each disease group and the normal group (FIGS. 8a and 8b). Figure 8a is a graph showing the results of confirming and verifying the difference in transition expression by AuDIT analysis of AD-specific samples, and Figure 8b is a graph showing the results of confirming and verifying the difference in transition expression by AuDIT analysis of MCI-specific samples.

[0053] In addition, the AUC value was confirmed using the Receiver Operating Characteristic (ROC) curve to verify the biomarker candidates, and a group of biomarker candidates with an AUC value of 0.7 or more, which is the evaluation criterion, was selected. After that, the candidates verified using the training set were re-verified as Alzheimer's disease-specific biomarkers by individual sample analysis of the test set (Figure 9).

[0054] FIG. 9 is a graph showing the results of an individual sample analysis of the training set. Based on the results of analyzing individual samples, MCI-specific biomarker candidate 5 (APOA) was selected as the biomarker to be used in the diagnostic kit. Even APOA biomarkers belonging to the same group showed different MS / MS results, and it was confirmed that APOA4 in particular was specific to MCI (Figure 10).

[0055] FIG. 10 shows ROC curves for biomarker candidates 1 and 5 based on individual sample analysis. Since the ROC value of the AD-specific biomarker candidate 1 (ITGB3) was 0.71, we decided to use it together with APOA4 in the diagnostic kit. The expression level of the AD-specific marker increases gradually as the disease worsens in the normal group, MCI group, and AD group, and we determined that it is useful for diagnosing Alzheimer's disease and selected it.

[0056] Finally, the CRP biomarker was also immobilized in the same well. Although CRP was slightly below the ROC value, which is the internal standard for selecting biomarkers, considering that the biomarker is involved in inflammatory responses and is a marker related to Alzheimer's disease in the literature, we decided to investigate the significant association between Alzheimer's disease and the biomarker in question once more data is accumulated in the future.

[0057] Example 2-5: Results of exosome proteomic analysis In searching for Alzheimer's disease biomarkers, exosomes extracted from the blood of patients at the normal, mild cognitive impairment (MCI), and Alzheimer's disease (AD) stages (16 HC, 9 MCI, 17 AD) were pooled with those at the same disease stage to prepare three pooled samples for each disease stage, and proteomic analysis of the three biomarkers (ITGB3, APOA4, and CRP) confirmed as described above was performed (Figure 11).

[0058] FIG. 11 is a graph showing the results of analyzing the expression levels of three biomarkers (ITGB3, APOA4, and CRP) at each stage of progression of Alzheimer's disease. As shown in FIG. 11, the expression levels of the three biomarkers vary at each stage of Alzheimer's disease, and it is therefore possible to diagnose the stage of Alzheimer's disease based on the expression patterns of the three biomarkers. EXAMPLES

[0059] Development of eight antibodies against validated biomarkers and selection of antibody pairs by sandwich ELISA We first produced an antibody to APOA4, a biomarker specific to MCI, for use in diagnosis. We did not produce antibodies to the other two biomarkers, ITGB3 and CRP, but instead used commercially available antibodies for development.

[0060] Example 3-1: Development of eight antibodies against validated biomarkers APOA4 antigen was injected into mice to induce an immune reaction, and hybridomas were obtained from them to produce a total of five clone antibodies, and three clone antibodies were similarly produced using goats. The eight clone antibodies described above were arbitrarily named antibody 1 to antibody 8 for convenience, and then reacted with APOA4 antigen to perform a pair test by ELISA.

[0061] A pair test was performed by sandwich ELISA for the eight antibodies prepared as described above, and each of the eight antibodies prepared was used as a capture antibody and a detection antibody (Figures 12 and 13).

[0062] FIG. 12 shows the results of a paired test of eight clone antibodies by sandwich ELISA, and FIG. 13 is a graph showing the results. As can be seen from Figures 12 and 13, two pairs that are considered suitable for use in diagnostic kits were selected: (Capture-Antibody 1, Detection-Antibody 6), (Capture-Antibody 3, Detection-Antibody 6). Example 3-2: Affinity measurement using sandwich ELISA and selection of antibody pairs The affinity of the antigens with Antibody 1, Antibody 3, and Antibody 6, selected from the results of the pair test, was measured (Figure 14a). The antigens used in the affinity measurement were the same as those used to produce the antibodies. Antigen-antibody affinity was measured for each of the three types of antibodies (Antibody 1-Antibody 6, Antibody 3-Antibody 6). The measurement method basically used direct ELISA, and affinity was measured by an experiment inducing competitive binding between the antigen and antibody. The amount of detection antibody used when conducting the competitive binding experiment between the antigen and antibody was determined using a Klotz plot graph.

[0063] The determined amount of detection antibody was reacted with serially diluted antigen, then dispensed into wells coated with the antigen, and fluorescence was measured at 450 nm. A curve was generated from the measured OD values ​​using Sigma plot software, and the dissociation constant Kd value, which indicates the antigen-antibody affinity, was measured.

[0064] FIG. 14a is a schematic diagram showing the process of measuring antigen-antibody affinity. Example 3-2-1: Measurement of affinity between antibody 1 and antigen An affinity test between Antibody 1 and the antigen was performed by self-evaluation (Figure 14b).

[0065] FIG. 14b shows the results of measuring the affinity of Antibody 1 to the antigen. As shown in Figure 14b, the dissociation constant Kd value calculated using Sigma plot software was 11.38 nM, which was confirmed to be approximately 10-fold lower than the evaluation criterion of affinity of 1 nM or more. Therefore, it was found that Antibody 1 was not suitable for use in kit development.

[0066] Example 3-2-2: Measurement of affinity between antibody 3 and antigen An affinity test between antibody 3 and the antigen was performed by self-evaluation (Figure 14c). FIG. 14c shows the results of measuring the affinity of antibody 3 with the antigen.

[0067] As shown in FIG. 14c, the dissociation constant Kd value calculated using Sigma plot software was 0.2035 nM, which was confirmed to be approximately 5-fold higher than the evaluation criterion of affinity of 1 nM or higher, indicating that Antibody 3 is suitable for use in kit development.

[0068] Example 3-2-3: Measurement of affinity between antibody 6 and antigen An affinity test between antibody 6 and the antigen was performed by self-evaluation (Figure 14d). FIG. 14d shows the results of measuring the affinity of antibody 6 with the antigen.

[0069] As shown in Figure 14d, the dissociation constant Kd value calculated using Sigma plot software was 0.2755 nM, which was confirmed to be approximately four times higher than the evaluation criterion of an affinity of 1 nM or more, indicating that antibody 6 is suitable for use in kit development.

[0070] Based on the affinity measurement results, a diagnostic kit was developed using Antibody 3 and Antibody 6 as the capture antibody and detection antibody, respectively. EXAMPLES

[0071] Development of an Alzheimer's disease diagnostic kit Example 4-1: Overview of diagnostic kit The steps from kit production to performance testing can be roughly divided into three steps: the antibody immobilization (spotting) process, the immobilized antibody stabilization process, and the assay reaction process. Since conditions such as pH and antibody concentration at each step affect the performance of the kit, it can be said that the key to kit development is to explore these conditions and select the optimal conditions (Figure 15).

[0072] FIG. 15 is a schematic diagram showing the development and performance testing process of an Alzheimer's disease diagnostic kit. Considering the advantage of the applicant's technology in enabling multiplexed diagnosis, it was determined that preparing an Alzheimer's disease diagnostic kit using two or more biomarkers would be more advantageous in terms of reliability and accuracy of results than preparing an Alzheimer's disease diagnostic kit using a single noble biomarker.

[0073] The final goal of developing a diagnostic kit was multiplexed diagnosis using three different biomarkers, with the three biomarkers consisting of APOA4, which is specific to MCI, ITGB3, which is specific to AD, and CRP, which is expected to be significant in additional clinical trials (Figure 16).

[0074] FIG. 16 is a schematic diagram showing a combination of biomarkers contained in an Alzheimer's disease diagnostic kit. Example 4-2: Immobilization of biomarkers Antibody 3, which showed the highest affinity in the results of the affinity analysis measured in Example 3-2, was used to carry out immobilization using the sol-gel method, which is a core technology of PCL Corporation.

[0075] As the immobilization process, the capture antibody was coated (spotted) on the bottom of the plate well, and then the antigen and detection antibody were reacted by sandwich ELISA, and then the signal value was measured by scanning at 532 nm (FIG. 17).

[0076] FIG. 17 is a schematic diagram showing the process of assaying after immobilization of a biomarker in the preparation of a kit, taking as an example Tau, a marker associated with Alzheimer's disease. In the assay before using real samples, the assay was performed with the commercially available antigen used for antibody production. This was in order to more accurately validate the assay using a large amount of real samples from actual patients later once the sol-gel immobilization process was established.

[0077] First, we explored the immobilization composition of the APOA4 marker, which is an MCI-specific biomarker. Once the composition of the APOA4 marker was established, we then immobilized it together with ITGB3, and finally immobilized all three markers, including CRP, to complete the kit production (Figure 18).

[0078] FIG. 18 is a schematic diagram showing the immobilization order of three biomarkers. In particular, for APOA4, the antibody we had prepared was used to prepare the kit, and for the other markers, ITGB3 and CRP, we used commercially available antibodies rather than newly prepared antibodies for fixation and reaction. However, we also performed screening tests to select suitable antibodies against these two markers, and used those.

[0079] When the applicant mixed the capture antibody with the sol-gel and spotted it according to a protocol that he had previously determined in relation to the sol-gel composition, he observed a phenomenon in which the spots shrunk. Based on his empirical inference that this phenomenon was mainly caused by the very low concentration of the capture antibody, he concentrated the capture antibody from the conventional concentration of 0.5 mg / ml to 1 mg / ml and spotted it again with the same sol-gel composition, thereby improving the shrinkage of the spots (Figure 19).

[0080] FIG. 19 is a set of photographs showing the process of establishing the shape conditions of spots based on the concentration difference of the immobilized antibody. Based on the results in FIG. 19, the capture antibody concentration was set at 1 mg / ml in subsequent experiments using various compositions of antibody and sol-gel immobilization to reflect these results.

[0081] For the APOA4 marker, we carried out capture antibody and sol-gel immobilization experiments with various compositions, and found a composition (CBS-3) that seemed to have a somewhat higher signal value (FIGS. 20a and 20b).

[0082] FIG. 20a is a photograph showing the results of an experiment to explore APOA4 immobilization conditions, and FIG. 20b is a diagram showing the results of testing low-titer, medium-titer, and high-titer samples to determine the conditions that are thought to be most suitable for the APOA4 immobilization composition.

[0083] Example 4-3: ITGB3 immobilization test For the ITGB3 marker, a screening test was performed with 10 to 12 immobilization compositions according to the protocol established by the applicant, and compositions that were deemed significant were selected. As a result, a significant signal value was obtained in the CBS composition to which nothing was added, so this was selected as the immobilization condition (Figure 21).

[0084] FIG. 21 is a photograph showing the results of an ITGB3 screening test. Example 4-4: CRP immobilization test In the CRP marker, similar to the ITGB3 marker screening, spotting was performed using the immobilization composition established according to the applicant's protocol, and screening tests were performed using only the composition. As a result, significant signal values ​​were obtained in two compositions, R-1 and R-1-1, in which the ratio of Sol-Gel was very low. However, in R-1-1, a very viscous substance called Sol3 was added to the raw material of the composition, which is thought to be difficult in the process or in the quality control when mass-producing the kit later, so the R-1 composition was finally selected (Figure 22).

[0085] FIG. 22 shows photographs and a graph showing the results of an immobilization test carried out on the CRP marker. Thus, the immobilization composition for APOA4, CRP, and ITGB3 was established (FIG. 23).

[0086] FIG. 23 shows specific immobilization compositions for APOA4, CRP, and ITGB3. Thereafter, an aging test, a blocking test and a drying test of the immobilized antibody, which are processes for stabilizing the immobilized antibody, were carried out.

[0087] Example 4-4-1: Aging test of immobilized antibody APOA4, ITGB3, and CRP were all spotted, and then the conditions of the aging step, which is a step to support the stabilization of the spots, were tested (Figure 24). In summary, the aging condition test was performed in two locations: a production room with a high humidity of about 60% and a desiccator with a low humidity of about 9%, and for two periods of time: 2 hours and overnight.

[0088] FIG. 24 is a photograph showing the results of testing conditions for stabilizing the immobilized antibodies after spotting of three types of markers. As can be seen from Figure 24, it was confirmed that the signal value increased when aging was performed in a desiccator, and the shape of the spots was more stable after 2 hours than after overnight. Since the stability of the spots improved when the aging location was a desiccator at room temperature and the aging time was 2 hours, these were determined as the aging conditions.

[0089] Example 4-4-2: Blocking test of immobilized antibodies After determining the aging conditions, a blocking buffer test was performed, which is a process aimed at removing non-specific reactions. Three types of blocking buffers were tested: the buffer used in the applicant's conventional protocol (BSA 1%, goat serum 0.1%, Tween® 20 1 g, sodium azide 2 g), and SG01 and ST01, which are stabilizers from StabilBlock (Figure 25).

[0090] FIG. 25 is a photograph showing the results of a blocking test carried out on antibodies immobilized after spotting of three types of markers. As shown in FIG. 25, it was confirmed that blocking with ST01 showed the best signal in terms of signal value and spot stability.

[0091] Example 4-4-3: Dry condition test After the blocking process, two humidity and time conditions were tested for drying the wells. Humidity was set to 60% in the production room and 9% in a desiccator, and time was set to 2 hours and overnight (Figure 26).

[0092] FIG. 26 is a photograph showing the results of a drying test performed on antibodies immobilized after spotting of three types of markers. As shown in Figure 26, the difference in signal values ​​under each drying condition was not large. However, taking into consideration that the wells did not dry completely under any of the conditions other than overnight in a desiccator, the final drying condition was determined to be overnight in a desiccator.

[0093] Example 4-5: Final assay standardization test of diagnostic kit The standards for the assay process of the diagnostic kit were determined based on the protocol established by the applicant, and the optimum assay conditions were selected by changing the conditions of the specimen dilution solution, the conjugate dilution solution, etc. based on the standards.

[0094] Example 4-5-1: Sample dilution solution condition test During the assay, a condition test was conducted on the diluent to be dispensed together with the specimen (Figure 27). FIG. 27 is a photograph showing the results of testing the conditions of the specimen dilution solution.

[0095] As shown in Figure 27, the ratio of specimen to specimen diluent was set at 1:4, and a total volume of 100 uL was dispensed and reacted. The most improved results were obtained when the specimen diluent contained goat serum.

[0096] Example 4-5-2: Conjugate dilution solution condition test Conditions were tested in which dilutions of the conjugate were reacted with the specimen and then aliquoted into a second reaction (FIG. 28).

[0097] FIG. 28 is a photograph showing the results of testing the conditions of the conjugate dilution solution. As shown in FIG. 28, the results confirmed that the signal values ​​for all markers increased in the reaction using a buffer containing BSA.

[0098] Example 4-5-3: Final assay standardization information for diagnostic kits The final assay standardization information of the diagnostic kit finally confirmed from the results of the above examples is as follows:

[0099] i. Aging information 1. Room temperature desiccator for 2 hours ii.Blocking Information 1. Room temperature for 1 hour iii. Drying information 1. Desiccator at room temperature overnight iv. Driving information 1. Equipment used: Shaker 2. Assay Protocol

[0100] [Table 1]

[0101] v. Analysis information 1. Imaging device: Sensovation device Examples 4-6: Preparation of final kit and actual sample testing Based on the above immobilization conditions, three markers were immobilized in one well, and then the performance of the prepared kit was tested using actual HC, MCI, and AD samples. Samples extracted from 380 patients were pooled by disease stage, with 3-4 samples each. Finally, a total of 69 samples were set, with 23 samples for each of the HC, MCI, and AD groups.

[0102] Example 4-6-1: Western blot analysis results of exosomes To evaluate the biomarkers APOA4, CRP, and ITGB3, a large number of exosome samples (23 per stage, total of 69) were examined by Western blot analysis, where the order of samples was randomly set, rather than collecting each patient according to their disease stage, to account for the difference in Western blot gels (Figures 29a, 29b, and 29c).

[0103] FIG. 29a is a photograph and a diagram showing the results of Western blot analysis for ITGB3, FIG. 29b is a photograph and a diagram showing the results of Western blot analysis for APOA4, and FIG. 29c is a photograph showing the results of Western blot analysis for CRP.

[0104] As shown in Figures 29a, 29b and 29c, for ITGB3 and APOA4, the WB results correspond to the proteomics results, but for CRP, WB gave results that were difficult to analyze, and although the results are attached, they were excluded from the correlation analysis in the final conclusion.

[0105] Example 4-6-2: Actual sample analysis results after final kit production The results were obtained by reacting 69 plasma samples from the same patients as in the Western blot analysis experiment with the kit prepared (FIG. 30).

[0106] FIG. 30 is a photograph showing the results of analyzing an actual sample using the final kit. Example 4-6-3: Correlation between actual sample analysis results and WB analysis results The correlation between the data obtained in Example 4-6-2 and the Western blot intensity was compared and analyzed (FIGS. 31 and 32).

[0107] First, FIG. 31 is a graph showing the results of a correlation analysis targeting the ITGB3 marker. As shown in Figure 31, for the ITGB3 marker, the proteomics analysis results, Western blot analysis results, and the analysis results of the prepared kit all showed a trend in which the protein expression level increased as the disease progressed from HC to MCI to AD, and the correlation coefficient R2 was also confirmed to be 80% or more.

[0108] Next, FIG. 32 is a graph showing the results of a correlation analysis targeting the APOA4 marker. As shown in FIG. 32, it was confirmed that the expression level of the APOA4 marker was extremely high at the MCI stage, but tended to decrease again at the AD stage.

[0109] Because the goal of the diagnostic kit is to diagnose Alzheimer's disease early and because it is clear that a patient has Alzheimer's disease once the diagnostic kit has reached the AD stage, it is important to make a rapid diagnosis at the MCI stage.

[0110] It was confirmed that the APOA4 marker showed a correlation coefficient R2 of approximately 88% between the diagnostic kit prepared using MCI stage samples and the results of Western blot analysis. Finally, although it is difficult to determine the quantitative value of the CRP marker by Western blot analysis, it is considered preferable to use it as an auxiliary indicator marker, since it is believed that the expression level of CRP decreases at the MCI stage based on the results of proteomics analysis and literature information.

[0111] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A marker composition for diagnosing Alzheimer's disease, comprising APOA4 and ITGB3 as marker proteins, wherein APOA4 is an MCI (mild cognitive impairment) specific marker protein.

2. The composition of claim 1 , wherein the marker protein further comprises CRP.

3. A marker composition for distinguishing between Alzheimer's disease and mild cognitive impairment, the composition comprising APOA4 and ITGB3 as marker proteins, and APOA4 being an MCI (mild cognitive impairment) specific marker protein.

4. 1. A method for distinguishing between Alzheimer's disease and mild cognitive impairment, comprising: (a) quantitatively analyzing the expression levels of marker proteins APOA4 and ITGB3 in serum samples from individuals, other than humans, suspected of developing Alzheimer's disease; (b) correlating the levels of the quantified marker proteins with a distinction between Alzheimer's disease and mild cognitive impairment; the step of ascertaining a stage of progression of Alzheimer's disease; The method, wherein the progression stage is any of normal, MCI (mild cognitive impairment), and AD (Alzheimer's disease).

5. A kit for diagnosing the onset of Alzheimer's disease, the kit including a quantitative device for measuring expression levels of APOA4 and ITGB3, the kit being for confirming the progression stage of Alzheimer's disease, the progression stage being either normal, MCI (mild cognitive impairment), or AD (Alzheimer's disease).

6. 1. A method for providing information for staging Alzheimer's disease, comprising: (a) quantitatively analyzing the expression levels of marker proteins APOA4 and ITGB3 in serum samples from individuals, other than humans, suspected of developing Alzheimer's disease; (b) correlating the quantitatively analyzed levels of marker proteins with the presence or absence of Alzheimer's disease; the step of ascertaining a stage of progression of Alzheimer's disease; The method, wherein the progression stage is any of normal, MCI (mild cognitive impairment), and AD (Alzheimer's disease).

7. The method according to claim 6 , wherein the step of associating is performed by combining the quantitative analysis results of each marker protein.

8. 8. The method of claim 7, wherein the combination of the quantitative analysis results is performed using an analysis method selected from the group consisting of linear or nonlinear regression analysis, linear or nonlinear classification analysis, ANOVA, neural network analysis, genetic analysis, support vector machine analysis, hierarchical analysis or cluster analysis, decision tree algorithm or kernel principal component analysis, Markov blanket, recursive feature elimination or entropy-based recursive feature reduction analysis, forward floating search or backward floating search analysis, and combinations thereof.

9. The method of claim 7 , wherein the combining of the quantitative analyses is performed using a computer algorithm.

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