SERF2-containing protein antigen combinations and their applications

Abstract

This invention belongs to the field of biological detection, specifically relating to a protein antigen combination containing SERF2 and its application. The technical solution adopted is: an antigen combination for differentiating Alzheimer's disease from frontotemporal dementia and Lewy body dementia, and for detecting autoantibodies, wherein the antigen combination includes at least a protein fragment of SERF2. This invention provides a novel protein antigen composition that can not only effectively identify AD patients, but also effectively differentiate between AD, FTD, and DLB, achieving accurate identification of AD, and has significant application and research value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a combination of SERF2 protein antigens for detecting autoantibodies, used to differentiate Alzheimer's disease from frontotemporal dementia and Lewy body dementia. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized primarily by memory and cognitive impairment. It primarily affects the elderly and its progression is slow and irreversible. AD is the most common type of dementia, accounting for 60-70% of all dementia cases. Currently, the diagnosis of AD mainly relies on a combination of MMSE scores, imaging studies such as MRI, CT, and PET scans, and clinical symptoms.

[0003] Autoantibodies are antibodies produced by an individual's immune system against its own protein antigens. Normally, the immune system produces antibodies in response to exogenous proteins or substances in the body, but sometimes it also recognizes one or more endogenous components, leading to the production of autoantibodies. Extensive evidence confirms that various autoantibodies in serum are involved in neurological diseases and syndromes. We have disclosed a series of antigenic proteins (and / or fragments) and combinations thereof that can be used for the detection of autoantibodies in AD patients in Chinese Patent CN 110850104 B. These combinations have shown good clinical efficacy in diagnosing Alzheimer's disease (AD), but they do not yet address the differential diagnosis of AD from related dementias such as Lewy body dementia (DLB) and frontotemporal dementia (FTD).

[0004] Lewy body dementia (DLB) is one of the most common neurodegenerative diseases; its main pathological feature is the widespread distribution of Lewy bodies (LB) in the cerebral cortex and brainstem. Statistics show that the prevalence of DLB ​​in people over 65 years of age is 3.6%–6.6%, accounting for 10%–20% of dementia patients. DLB's clinical symptoms and pathological manifestations are very similar to those of Alzheimer's disease (AD), and LB is found in the brains of more than 40% of AD patients, demonstrating a high degree of overlap between the two diseases. Furthermore, both DLB and AD present with progressive cognitive impairment, and their severity is difficult to distinguish; the levels of Tau protein and β-amyloid protein in the cerebrospinal fluid also show similar changes in both. Therefore, differentiating between these two diseases clinically is extremely difficult. Currently, clinical differentiation is mainly achieved through imaging examinations. One method is cranial MRI, which reveals subtle differences in the areas of brain atrophy; another is cerebral blood flow SPECT / PET scans, which differentiate AD and DLB based on imaging changes. However, existing methods require a high level of experience from physicians.

[0005] Frontotemporal dementia (FTD) is an insidious, progressive neurodegenerative disease characterized by frontotemporal lobe atrophy, accounting for approximately 5%–15% of all dementia types. Clinically, both FTD and AD patients exhibit varying degrees of cognitive, behavioral, and language impairments, and the MMSE scores of FTD patients are not significantly different from those of AD patients. Currently, CT and MRI are generally used to differentiate between FTD and AD; AD shows widespread brain atrophy, while frontotemporal dementia shows atrophy of the frontal and / or temporal lobes; however, further histopathological examination is still necessary.

[0006] In current research, it is not possible to differentiate the above-mentioned diseases using biomarkers. Paterson et al. (Alzheimer's Research & Therapy (2018) 10:32) detected the levels and proportions of 10 proteins, including Tau protein, different fragments of β-amyloid (Aβ) and its precursor protein (APP), NFL, and YKL-40, in the cerebrospinal fluid of various dementia patients. The results showed that these biomarkers could not effectively differentiate between Alzheimer's disease (AD) and Dementia Leukemia (DLB). Among them, Aβ42 / 40 had some reference value in differentiating between AD and behavioral variant frontotemporal dementia (bvFTD). However, the drawback is that the use of cerebrospinal fluid samples poses certain risks to patients and is usually rejected by them.

[0007] Meanwhile, comparing autopsy results, even in specialized dementia treatment centers, the clinical diagnosis of Alzheimer's disease (AD) still has a misdiagnosis rate of 25-30%. AD is a progressive neurodegenerative disease, with symptoms gradually worsening as the disease progresses. It has a long course and progresses slowly in the early stages. Therefore, the earlier the diagnosis and intervention, the easier it is to control the disease, and the more the patient benefits. However, the accuracy of MRI and PET scans is lower in the early stages of the disease because pathological changes are not yet obvious. Furthermore, in the early stages, the clinical symptoms of AD are very similar to those of other dementias such as Freezing Toxic Disease (FTD) and Dementia Leukemia-Induced Bleeding (DLB), further increasing the difficulty of clinical diagnosis.

[0008] Therefore, developing a diagnostic method that can effectively differentiate AD from other forms of dementia such as FTD and DLB is of great practical significance. Summary of the Invention

[0009] The purpose of this invention is to provide a protein antigen combination and its application that can effectively distinguish AD from other forms of dementia such as FTD and DLB, and detect autoantibodies.

[0010] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is: an antigen combination, wherein the antigen combination includes at least SERF2 protein and / or SNAP25 protein.

[0011] Preferably, the amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 7, and the amino acid sequence of the SNAP25 protein is shown in SEQ ID NO: 8.

[0012] Preferably, it includes both SERF2 protein and SNAP25 protein.

[0013] Preferably, it also includes any one or more of the following protein fragments: MAPT protein fragment, RAGE protein fragment, ASXL1 protein fragment, JMJD2D protein fragment, P21 protein fragment, and DNAJC8 protein fragment.

[0014] Preferably, the amino acid sequence of the MAPT protein fragment is shown in SEQ ID NO: 1, the amino acid sequence of the RAGE protein fragment is shown in SEQ ID NO: 4, the amino acid sequence of the ASXL1 protein fragment is shown in SEQ ID NO: 5, the amino acid sequence of the JMJD2D protein fragment is shown in SEQ ID NO: 6, the amino acid sequence of the P21 protein fragment is shown in SEQ ID NO: 2, and the amino acid sequence of the DNAJC8 protein fragment is shown in SEQ ID NO: 3.

[0015] Accordingly, the antigen combination is used in the preparation of products for detecting / identifying Alzheimer's disease.

[0016] Accordingly, the antigen combination is used in the preparation of products that differentiate between Alzheimer's disease, frontotemporal dementia, and Lewy body dementia.

[0017] Accordingly, a kit for detecting Alzheimer's disease was prepared using the antigen combination.

[0018] Accordingly, the application of SERF2 protein and / or SNAP25 protein and / or MAPT protein fragment and / or RAGE protein fragment and / or ASXL1 protein fragment and / or JMJD2D protein fragment and / or P21 protein fragment and / or DNAJC8 protein fragment in the preparation of products for detecting / identifying Alzheimer's disease, wherein the amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 7; the amino acid sequence of the SNAP25 protein is shown in SEQ ID NO: 8; the amino acid sequence of the MAPT protein fragment is shown in SEQ ID NO: 1; the amino acid sequence of the RAGE protein fragment is shown in SEQ ID NO: 4; the amino acid sequence of the ASXL1 protein fragment is shown in SEQ ID NO: 5; the amino acid sequence of the JMJD2D protein fragment is shown in SEQ ID NO: 6; the amino acid sequence of the P21 protein fragment is shown in SEQ ID NO: 2; and the amino acid sequence of the DNAJC8 protein fragment is shown in SEQ ID NO: 3.

[0019] Accordingly, the application of SERF2 protein and / or SNAP25 protein and / or MAPT protein fragment and / or RAGE protein fragment and / or ASXL1 protein fragment and / or JMJD2D protein fragment and / or P21 protein fragment and / or DNAJC8 protein fragment in the preparation of products that differentiate between Alzheimer's disease, frontotemporal dementia, and Lewy body dementia, wherein the amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 7; the amino acid sequence of the SNAP25 protein is shown in SEQ ID NO: 8; the amino acid sequence of the MAPT protein fragment is shown in SEQ ID NO: 1; the amino acid sequence of the RAGE protein fragment is shown in SEQ ID NO: 4; the amino acid sequence of the ASXL1 protein fragment is shown in SEQ ID NO: 5; the amino acid sequence of the JMJD2D protein fragment is shown in SEQ ID NO: 6; the amino acid sequence of the P21 protein fragment is shown in SEQ ID NO: 2; and the amino acid sequence of the DNAJC8 protein fragment is shown in SEQ ID NO: 3.

[0020] The present invention has the following beneficial effects: The present invention provides a novel protein antigen composition that can not only effectively identify AD patients, but also effectively distinguish AD, FTD and DLB, achieving accurate identification of AD, and has important application value and research value. Detailed Implementation

[0021] The present application will be further explained below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods; the materials and reagents used are all commercially available; the data obtained are all average values ​​obtained after at least 3 repetitions, and each repetition yields valid data.

[0022] Example 1: Construction, expression, and purification of recombinant vectors for antigens

[0023] 1. Antigen Selection. Twenty-three antigen proteins highly associated with Alzheimer's disease were selected for construction, expression, and purification. Their respective database IDs are shown in Table 1.

[0024] Table 1. Database ID mapping table for the proteins to be tested.

[0025] protein name Database ID protein name Database ID MAPT NP_058519.3 ICAM1 NP_000192.2 ADARB1 NP_001103.1 CHGA NP_001266.1 P21 NP_000380.1 SNAP25 NP_001309832.1 DNAJC8 NP_055095.2 VSNL1 NP_001353732.1 RAGE NP_001127.1 CHI3L1 NP_001267.2 ASXL1 NP_056153.2 FABP3 NP_001307925.1 JMJD2D NP_060509.2 AD7c-NTP AAC08737.1 ApoE4 NP_001289617.1 RALGPS2 NP_689876.2 H2BC5 NP_066407.1 CTSH NP_004381.2 TOMM20 NP_055580.1 DOC2A NP_003577.2 PDPN NP_006465.3 ICA1L NP_001275551.1 SERF2 NP_001018118.1 \ \

[0026] 2. Construction and Expression of Recombinant Antigen Vectors. Using a human cDNA library (purchased from Invitrogen) or fully synthesized DNA as templates, primers were designed, and the full-length gene of the protein was cloned into the pET28 plasmid using molecular cloning techniques such as PCR, enzyme digestion, and ligation. Simultaneously, HIS and c-myc tags were added to the N-terminus of the protein to form a fusion protein. The resulting recombinant expression vector was identified by DNA sequencing to confirm the presence of the correct protein gene fragment. It should be noted that the added tags are only for facilitating protein identification and extraction and do not decisively affect the protein's function as an antigen. When using this vector, it is acceptable to omit the tags or add other tags as needed.

[0027] The recombinant plasmid containing the protein gene fragments listed in Table 1 was transformed into *E. coli* BL21(DE3) competent cells. Clones were picked and inoculated into LB medium and cultured in a shaker at 37°C. When the bacterial density reached OD200... 600 When the temperature reached approximately 0.8, the temperature was lowered to 16°C, and 0.1 mM isopropyl thio-β-D-galactoside (IPTG) was added to each LB medium to induce expression overnight, thus obtaining bacterial cells.

[0028] 3. Antigen Purification. Collect the induced bacterial cells by centrifugation and wash twice with PBS. Resuspend and disperse the cells with lysis buffer (5-10 mL per gram of cells), place on ice, and sonicate to disrupt the cells (200 W, 5 seconds of disruption, 5 seconds of rest). After disruption, centrifuge at 13000 rpm, 10°C for 20 minutes. Collect the supernatant and purify it using Ni column affinity chromatography and molecular sieve chromatography. Analyze the purified protein using SDS-PAGE electrophoresis to confirm its molecular weight and purity. Determine the concentration using the Bradford method and store at -80°C for later use. The purified protein is then obtained.

[0029] Example 2: Screening for candidate antigens from the proteins to be tested

[0030] 1. The solutions and reagents used in this embodiment are as follows:

[0031] (1) The coating buffer is PBS buffer, pH=7.4. The preparation method is as follows: accurately weigh 3.58g Na2HPO4·12H2O, 0.23g KH2PO4·2H2O, 0.2g KCl, and 8.0g NaCl, dissolve them in water, and make up to 1L with distilled water.

[0032] (2) Blocking buffer / sample diluent / antibody diluent: Dissolve 10g BSA (bovine serum albumin) in coating buffer and bring the volume up to 1L with water.

[0033] (3) Washing solution: Prepare fresh before use. Before use, add 0.5% Tween 20 (V / V) to the coating buffer, pH=7.4.

[0034] (4) TMB colorimetric reagent, purchased from KPL Company.

[0035] (5) Termination solution: 1M hydrochloric acid.

[0036] 2. Solid-phase coating of the test protein. Dilute the purified test protein obtained in Example 1 to 5 μg / ml with coating buffer, add 50 μl to each well of a 96-well plate, and coat overnight at 4°C. The next day, discard the solution, spin dry, and wash three times with washing buffer, 200 μl per well each time. Then add 200 μl of blocking buffer to each well, incubate at room temperature for 1 h, discard the blocking buffer, spin dry, and wash three more times with washing buffer, 200 μl per well each time, and spin dry again; obtain the solid-phase coated antigen in the 96-well plate.

[0037] 3. Add the test sample. Dilute the human serum sample 100-fold with the sample diluent, and add it to the 96-well plate containing the test protein. Add 50 μl of the diluted test sample to each well. Then place the 96-well plate on a microplate shaker and incubate at room temperature for 1 hour. Shake dry, wash three times with washing buffer, 200 μl per well each time, and shake dry again.

[0038] 4. Add enzyme-labeled secondary antibody. Dilute 1.0 mg / ml horseradish peroxidase-labeled recombinant goat anti-human immunoglobulin G antibody (purchased from Jackson ImmunoResearch Inc.) 20,000 times with antibody dilution buffer, and add 50 μl to each well of the 96-well plate treated in step 3. Then place the 96-well plate on a microplate shaker and incubate at room temperature for 0.5 h. Shake the plate dry, wash three times with washing buffer, 200 μl per well each time, and shake dry again.

[0039] 5. Colorimetric reaction and optical density reading. In the 96-well plate after step 4, add 50 μl of TMB colorimetric reagent to each well, shake for 15 s, react at room temperature in the dark for 15 min, and then add 50 μl of stop solution; then use a microplate reader to read the absorbance value at a wavelength of 450 nm to obtain the detection signal (S) of each sample.

[0040] 6. Sensitivity and Specificity Analysis. 180 positive samples (serum from patients diagnosed with Alzheimer's disease) and 180 negative samples (serum from healthy subjects) were collected. The detection signal (S) of each sample was measured using the method described above (absorbance at 450 nm wavelength). Using the negative samples as negative reference samples, the mean (M) and standard deviation (SD) of the detection signals (S) of all negative reference samples were calculated, with M+3SD as the cut-off value. Samples with a detection signal (S) ≥ the cut-off value (S ≥ M+3SD) were defined as positive; samples with a detection signal (S) < the cut-off value (S < M+3SD) were defined as negative.

[0041] Specificity and sensitivity were calculated based on positive and negative sample results. Specificity refers to the proportion of healthy subject samples correctly identified as negative, calculated as the number of correctly identified negative samples divided by the total number of negative samples. Sensitivity refers to the proportion of Alzheimer's patient samples correctly identified as positive, calculated as the number of correctly identified positive samples divided by the total number of positive samples. The sensitivity and specificity were calculated when using each tested protein as an antigen for sample testing, and the results are shown in Table 2.

[0042] Table 2 shows the sensitivity and specificity of each tested protein / protein fragment as an antigen.

[0043]

[0044]

[0045] 7. From the above-mentioned proteins or fragments, proteins with a sensitivity ≥15% and a specificity ≥85% were selected as candidate antigens. The screening results are shown in Table 3.

[0046] Table 3. Candidate antigens selected from the tested proteins.

[0047] Test antigen Amino acid initiation and termination positions Sensitivity Specificity Corresponding amino acid sequence MAPT 502-758 40.56％ 90.00％ SEQ ID NO: 1 P21 2-164 37.78％ 90.56％ SEQ ID NO: 2 DNAJC8 114-253 35.00％ 88.89％ SEQ ID NO: 3 RAGE 23-54 34.44％ 89.44％ SEQ ID NO: 4 ASXL1 1-84 33.89％ 90.00％ SEQ ID NO: 5 JMJD2D 1-160 33.33％ 89.44％ SEQ ID NO: 6 SERF2 (whole protein) 1-59 25.56％ 95.56％ SEQ ID NO: 7 SNAP25 (whole protein) 1-206 28.89％ 95.56％ SEQ ID NO: 8

[0048] Example 3: Demonstration of the effect of candidate antigen detection on DLB and FTD patient samples

[0049] Forty-three DLB-positive samples (serum from patients diagnosed with DLB), 63 FTD-positive samples (serum from patients diagnosed with FTD), and 232 negative samples (serum from healthy subjects) were collected. Following the method in Example 2, the detection signal (S) of each sample was measured using the candidate antigens (protein fragments) listed in Table 3. Using the negative samples as negative reference samples, the mean (M) and standard deviation (SD) of the detection signals (S) of all negative reference samples were calculated, with M+3SD as the cut-off value. Samples with a detection signal (S) ≥ the cut-off value (S ≥ M+3SD) were defined as positive; samples with a detection signal (S) < the cut-off value (S < M+3SD) were defined as negative.

[0050] Specificity and sensitivity were calculated based on positive and negative sample results. Specificity refers to the proportion of healthy subject samples correctly identified as negative, calculated as the number of correctly identified negative samples divided by the total number of negative samples. DLB sample sensitivity refers to the proportion of DLB ​​patient samples correctly identified as positive, calculated as the number of correctly identified positive DLB samples divided by the total number of positive samples. FTD sample sensitivity refers to the proportion of correctly identified positive FTD patient samples, calculated as the number of correctly identified positive FTD samples divided by the total number of positive samples. The sensitivity and specificity were calculated for each tested protein as an antigen, and the results are shown in Table 4.

[0051] Table 4 shows the test results of DLB ​​and FTD patient samples.

[0052] Test antigen Amino acid initiation and termination positions DLB Sensitivity FTD Sensitivity Specificity MAPT 502-758 4.65％ 19.05％ 90.09％ P21 2-164 0％ 1.59％ 90.52％ DNAJC8 114-253 0％ 4.76％ 89.66％ RAGE 23-54 9.30％ 1.59％ 90.52％ ASXL1 1-84 16.28％ 4.76％ 90.95％ JMJD2D 1-160 4.65％ 7.94％ 90.09％ SERF2 1-59 4.65％ 4.76％ 95.69％ SNAP25 1-206 4.65％ 6.35％ 95.26％

[0053] Example 4: Demonstration of the effectiveness of candidate antigen combination in detecting AD, FTD and DLB

[0054] 1. Select proteins or protein fragments from the candidate protein antigens in Table 3 to form different antigen combinations containing SERF2 and SNAP25. The specific combination methods are shown in Table 5.

[0055] Table 5. Comparison of different antigen combinations

[0056]

[0057]

[0058]

[0059] 2. Serum samples were collected from 94, 63, and 44 patients with AD, FTD, and DLB, respectively, and serum samples were collected from 197 healthy subjects. Following the detection method in Example 2, the sensitivity and specificity of each sample were tested using the antigen combinations listed in Table 5.

[0060] In antigen combinations, the definitions of positive and negative for individual antigens are as described in Examples 2 and 3. The overall sensitivity and specificity of the antigen combination are defined as follows:

[0061] For an antigen combination, a serum sample is considered positive if it receives a positive detection signal when tested with any one of the antigens in the combination; otherwise, it is considered negative. Based on this definition of positive and negative, the positive and negative results obtained from detecting an antigen combination are used to calculate the sensitivity of that antigen combination in patient samples.

[0062] Sensitivity refers to the proportion of AD (or FTD, or DLB) patient samples that are correctly identified as positive, which is the number of positive AD (or FTD, or DLB) patient samples divided by the total number of AD (or FTD, or DLB) patient samples. Specificity refers to the proportion of healthy subject samples that are correctly identified as negative, which is the number of negative healthy subject samples divided by the total number of healthy subject samples.

[0063] The sensitivity and specificity results obtained from the detection of each antigen combination are shown in Table 6.

[0064] Table 6 Comparison of Antigen Combination Detection Results for Each Group

[0065]

[0066]

[0067]

[0068] As shown in Table 6, the specificity of each combination detection is very good, all above 81%, with some combinations reaching 85% or even over 90%. The detection sensitivity for Alzheimer's disease (AD) patients is several times higher than that for frontotemporal dementia (FTD) and Lewy body dementia (DLB). For example, combination 13 has a sensitivity of 72.34% for AD, but only 7.94% for FTD and a very low sensitivity of only 4.55% for DLB, demonstrating excellent differential diagnostic value. In future applications, one or more candidate antigens or antigen combinations provided by this invention can be selected as needed to prepare kits for the diagnosis of AD and / or to differentiate between FTD and DLB.

[0069] Example 5: Demonstration of the effect of candidate antigen combinations and corresponding full-length protein combinations in detecting AD, FTD, and DLB.

[0070] 1. Select four different antigen combinations from Table 5 of Example 4. The proteins or protein fragments are all from Table 3. Based on the full-length proteins corresponding to each protein fragment in the antigen combinations of groups 1 to 4, set up corresponding full-length protein combinations, namely control groups 1 to 4, as shown in Table 7. If the protein in Table 3 is already a full-length protein, then no adjustment is made to that protein.

[0071] Table 7 Comparison of Antigen Combinations

[0072]

[0073]

[0074] 2. Referring to the detection method in Example 2, the antigen combinations in Table 7 were used to detect the sensitivity and AD specificity of each sample in Example 4 for AD, FTD, and DLB. The results are shown in Table 8.

[0075] Table 8 Comparison of Antigen Combination Detection Results for Each Group

[0076] combination AD sensitivity FTD Sensitivity DLB Sensitivity Specificity Group 1 63.83％ 9.52％ 15.91％ 89.85％ Group 2 72.34％ 7.94％ 4.55％ 89.85％ Group 3 80.85％ 14.29％ 11.36％ 87.82％ Group 4 95.74％ 26.98％ 36.36％ 81.22％ Control group 1 57.45％ 17.46％ 22.73％ 83.25％ Control group 2 70.21％ 25.40％ 27.27％ 80.20％ Control group 3 78.72％ 30.16％ 27.27％ 74.62％ Control group 4 89.36％ 52.38％ 52.27％ 56.85％

[0077] As can be seen from the results in Table 8, compared with the combination that uses the full-length protein, the combination containing fragments showed better specificity, all above 81%. At the same time, the combination containing fragments not only maintained high detection sensitivity for AD, but also had lower detection sensitivity for FTD and DLB, and had better differential diagnostic significance.

[0078] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. An antigen combination, characterized in that: The antigen combination includes SERF2 protein and SNAP25 protein; the amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 7, and the amino acid sequence of the SNAP25 protein is shown in SEQ ID NO: 8; It also includes any one or more of the following protein fragments: MAPT, RAGE, ASXL1, JMJD2D, P21, and DNAJC8; the amino acid sequence of the MAPT protein fragment is shown in SEQ ID NO: 1, the amino acid sequence of the RAGE protein fragment is shown in SEQ ID NO: 4, the amino acid sequence of the ASXL1 protein fragment is shown in SEQ ID NO: 5, the amino acid sequence of the JMJD2D protein fragment is shown in SEQ ID NO: 6, the amino acid sequence of the P21 protein fragment is shown in SEQ ID NO: 2, and the amino acid sequence of the DNAJC8 protein fragment is shown in SEQ ID NO:

3.

2. The use of the antigen combination of claim 1 in the preparation of products for detecting / identifying Alzheimer's disease.

3. The use of the antigen combination of claim 1 in the preparation of products that differentiate between Alzheimer's disease, frontotemporal dementia, and Lewy body dementia.

4. A kit for detecting Alzheimer's disease prepared using the antigen combination of claim 1.

5. The application of SERF2 protein, SNAP25 protein, MAPT protein fragment, RAGE protein fragment, ASXL1 protein fragment, JMJD2D protein fragment, P21 protein fragment, and DNAJC8 protein fragment in the preparation of products for detecting / identifying Alzheimer's disease, characterized by: The amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 7; the amino acid sequence of the SNAP25 protein is shown in SEQ ID NO: 8; the amino acid sequence of the MAPT protein fragment is shown in SEQ ID NO: 1; the amino acid sequence of the RAGE protein fragment is shown in SEQ ID NO: 4; the amino acid sequence of the ASXL1 protein fragment is shown in SEQ ID NO: 5; the amino acid sequence of the JMJD2D protein fragment is shown in SEQ ID NO: 6; the amino acid sequence of the P21 protein fragment is shown in SEQ ID NO: 2; and the amino acid sequence of the DNAJC8 protein fragment is shown in SEQ ID NO:

3.

6. The application of SERF2 protein, SNAP25 protein, MAPT protein fragment, RAGE protein fragment, ASXL1 protein fragment, JMJD2D protein fragment, P21 protein fragment, and DNAJC8 protein fragment in the preparation of products that differentiate between Alzheimer's disease, frontotemporal dementia, and Lewy body dementia, characterized by: The amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 7; the amino acid sequence of the SNAP25 protein is shown in SEQ ID NO: 8; the amino acid sequence of the MAPT protein fragment is shown in SEQ ID NO: 1; the amino acid sequence of the RAGE protein fragment is shown in SEQ ID NO: 4; the amino acid sequence of the ASXL1 protein fragment is shown in SEQ ID NO: 5; the amino acid sequence of the JMJD2D protein fragment is shown in SEQ ID NO: 6; the amino acid sequence of the P21 protein fragment is shown in SEQ ID NO: 2; and the amino acid sequence of the DNAJC8 protein fragment is shown in SEQ ID NO: 3.

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