Protein antigen combination for alzheimer's disease detection and use

Abstract

A protein antigen for Alzheimer's disease detection comprises at least any two of DOC2A, LGALS1, KDM4D, and ADARB1 proteins at the same time, can be used for early detection or diagnosis of Alzheimer's disease, and is suitable for risk assessment and prediction of before the onset of Alzheimer's disease; moreover, the protein antigen can distinguish Alzheimer's disease from other types of dementia, and can be further prepared into a related reagent or kit according to requirements.

Description

A protein antigen combination for Alzheimer's disease detection and its application Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a protein antigen combination and application for early detection of Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is one of the most common dementia diseases, accounting for 60% to 80% of all dementia cases. According to the patient's condition, AD is usually divided into preclinical stage, mild cognitive impairment (MCI), mild (early), moderate (middle) and severe (late). The preclinical stage (including the subjective cognitive decline stage of AD) and MCI stage are defined as the very early stage of AD. In the preclinical and MCI stages, the patient's symptoms are often less obvious, but the biomarker indicators have become abnormal. If timely and accurate diagnosis can be achieved in the early stage when there are no (obvious) symptoms, patients can intervene in the disease 10 to 20 years in advance.

[0003] In 2023, the Alzheimer's Association International Conference (AAIC) announced the draft NIA-AA Revised Clinical Criteria for Alzheimer's Disease. The draft categorizes biomarkers into three main categories: blood-derived biomarkers (BBBM), cerebrospinal fluid (CSF) biomarkers, and gold standard imaging (PET / MRI) biomarkers.

[0004] Cerebrospinal fluid (CSF) biomarkers can be used for asymptomatic detection, showing changes earlier than PET imaging. However, these tests require an invasive lumbar puncture and have low clinical acceptance. Brain PET / MRI imaging can detect MCI (mild cognitive impairment) and asymptomatic AD, but changes occur later than CSF biomarkers. Furthermore, these tests are expensive, and the availability of testing equipment is low. Only a few hospitals in China can perform these tests. Furthermore, these tests require the injection of contrast agent, which is radioactive, and patient acceptance remains low.

[0005] Compared to biomarker tests such as cerebrospinal fluid (CSF) and brain PET / MRI imaging, blood-based biomarker testing offers advantages such as minimal invasiveness, low cost, ease of use, and widespread access. Compared to CSF ​​and PET imaging, blood-based biomarker testing is less expensive, faster, and simpler to perform. It requires only a venous blood draw, is radiation-free, and is non-invasive. It can be used by a wide range of patients and offers valuable guidance for the early detection and prevention of AD.

[0006] Using blood markers for early AD detection can shorten diagnosis and treatment time and is a crucial tool in the AD referral pathway. Specifically, a positive BBBM further aids in cognitive function assessment and confirms AD pathology (CSF examination and PET imaging); whereas a negative BBBM aids in AD pathology diagnosis, excluding other causes of cognitive decline and reducing unnecessary CSF and PET imaging.

[0007] Subjective cognitive decline (SCD) refers to a patient's subjective perception of a decline in memory or cognitive function compared to their previous normal state, while objective neuropsychological tests are within normal limits. SCD lies between normal cognitive aging and mild cognitive impairment (MCI). SCD can be caused by a variety of factors, including preclinical Alzheimer's disease, depression, anxiety, personality disorders, and sleep disorders.

[0008] SCD is quite common among the elderly. Population-based studies have shown that 50% to 80% of cognitively normal adults over 65 years old report self-perceived cognitive decline. However, SCD has received little clinical attention. Factors such as the form of cognitive decline, individual personality traits, cultural background, and statistical false positives make the classification and quantification of SCD extremely difficult. Because SCD is a subjective cognitive experience and lacks objective criteria for judgment, researchers have proposed more self-assessment scales for SCD. Currently, commonly used scales include the Subjective Cognitive Decline Questionnaire (SCD-Q) and the Measurement of Everyday Cognition (Ecog).

[0009] Many clinical epidemiological studies have shown that individuals with SCD have a higher risk of developing AD than those without SCD. However, not all SCD patients will develop AD or dementia.

[0010] Mild cognitive impairment (MCI) is a heterogeneous clinical syndrome characterized by mild cognitive impairment that does not significantly impact daily life. It represents a transitional stage between normal aging and mild dementia. MCI can be caused by neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and frontotemporal lobar degeneration, as well as other systemic, neurological, and psychiatric disorders. Targeted treatment based on the underlying cause of MCI may reverse or prevent further progression of cognitive impairment.

[0011] Therefore, if Alzheimer's disease can be screened out in the preclinical stage to the subjective cognitive decline (SCD) and mild cognitive impairment (MCI) stage, and Alzheimer's disease can be effectively distinguished from other types of dementia, it will be possible to intervene early and provide targeted treatment to prevent the disease from worsening.

[0012] Summary of the Invention

[0013] The purpose of the present invention is to provide a protein antigen combination and application for early detection of Alzheimer's disease.

[0014] To achieve the above object of the invention, the technical solution adopted by the present invention is: an antigen combination, which at least simultaneously includes: any two of the DOC2A protein, LGALS1 protein, KDM4D protein and ADARB1 protein.

[0015] Preferably, the amino acid sequence of the DOC2A protein is shown in SEQ ID NO: 1, and / or the amino acid sequence of the LGALS1 protein is shown in SEQ ID NO: 2, and / or the amino acid sequence of the KDM4D protein is shown in SEQ ID NO: 7, and / or the amino acid sequence of the ADARB1 protein is shown in SEQ ID NO: 9.

[0016] Preferably, the antigen combination further includes: SERF2 protein.

[0017] Preferably, the amino acid sequence of the SERF2 protein is shown in SEQ ID NO: 6.

[0018] Preferably, the antigen combination further comprises any one or more of: HIST1H2BD protein, ICAM1 protein, RIOK2 protein and DNAJC8 protein.

[0019] Preferably, the amino acid sequence of the HIST1H2BD protein is shown in SEQ ID NO: 3, and / or the amino acid sequence of the ICAM1 protein is shown in SEQ ID NO: 4, and / or the amino acid sequence of the RIOK2 protein is shown in SEQ ID NO: 5, and / or the amino acid sequence of the DNAJC8 protein is shown in SEQ ID NO: 8.

[0020] Accordingly, the antigen combination is used in the preparation of products for detecting / identifying Alzheimer's disease.

[0021] Preferably, the application in the preparation of products for detecting / identifying Alzheimer's disease in the middle stage and earlier stages has outstanding advantages, especially in the preparation of products for detecting / identifying Alzheimer's disease in the early stage and earlier stages.

[0022] Accordingly, a reagent or kit for detecting / identifying Alzheimer's disease is prepared using the antigen combination.

[0023] Accordingly, the use of LGALS1 protein and / or HIST1H2BD protein and / or RIOK2 protein in the preparation of products for detecting / identifying Alzheimer's disease, the amino acid sequence of the LGALS1 protein is shown in SEQ ID NO: 2, and / or the amino acid sequence of the HIST1H2BD protein is shown in SEQ ID NO: 3, and / or the amino acid sequence of the RIOK2 protein is shown in SEQ ID NO: 5.

[0024] Accordingly, products for detecting / identifying Alzheimer's disease, such as reagents or kits, prepared using the protein have outstanding advantages in preparing products for detecting / identifying Alzheimer's disease in the middle and earlier stages, especially in the early and earlier stages.

[0025] The present invention has the following beneficial effects:

[0026] The present invention provides several protein antigens that can be used to effectively differentiate Alzheimer's disease autoantibody detection from the preclinical stage to the subjective cognitive decline (SCD) and mild cognitive impairment (MCI) stages. The detection method is simple and convenient, does not involve radiation, and does not cause trauma that is difficult for the subjects to accept. The test results are highly consistent with those obtained using cerebrospinal fluid or "gold standard" testing. The protein antigens involved can be produced by methods such as artificial synthesis and genetic recombination.

[0027] The antigens or combinations thereof provided by the present invention can be used to test the presence or level of autoantibodies against the above-mentioned antigen combination in biological samples from subjects, thereby determining whether the subject is in the early stages of Alzheimer's disease, or whether he or she has a risk of developing Alzheimer's disease, and at the same time identifying whether the type of dementia suffered by the subject is Alzheimer's disease. Wherein, the biological sample can be serum, plasma, whole blood, saliva, oral mucosal swabs, urine, lymph, cerebrospinal fluid, etc. Depending on the specific circumstances, the biological sample can be pretreated by extraction, dilution, enrichment and other means, and the method of use is diverse, simple and easy to operate. When the antigens and combinations provided by the present invention are used for the above-mentioned tests, the presence or level of the autoantibodies is tested by causing the proteins or fragments thereof in the antigens and combinations to bind or interact with the corresponding autoantibodies that may be present.

[0028] The protein antigens or combinations thereof provided by the present invention can also be used to prepare early screening reagents for Alzheimer's disease-related autoantibodies or early screening diagnostic reagents for Alzheimer's disease. It should be understood that the protein antigen combination can also be used to prepare a kit for detecting early screening Alzheimer's disease-related autoantibodies. The kit can be prepared by referring to the methods and reagents for early screening of Alzheimer's disease-related autoantibodies using the protein antigen combination in the embodiments of the present invention, and can also be adjusted accordingly as needed. In addition to being used for early screening of Alzheimer's disease, the protein antigens or combinations thereof provided by the present invention also have excellent performance in identifying early and mid-stage AD.

[0029] In summary, the present invention provides several new protein antigens and their combinations, which can be used for early screening, detection or diagnosis of Alzheimer's disease, and are particularly suitable for risk assessment and prediction before the onset of Alzheimer's disease; at the same time, they can distinguish Alzheimer's disease from other types of dementia; and can be further prepared into related reagents or kits as needed. DETAILED DESCRIPTION

[0030] The present invention first discovered that DOC2A, LGALS1, HIST1H2BD, ICAM1, RIOK2, SERF2, DNAJC8, KDM4D (also known as JMJD2D), and ADARB1 proteins are closely associated with the very early diagnosis of Alzheimer's disease. In particular, LGALS1, HIST1H2BD, and RIOK2 proteins were first found to be associated with the diagnosis of Alzheimer's disease. The DOC2A protein sequence is shown in SEQ ID NO: 1. The LGALS1 protein sequence is shown in SEQ ID NO: 2. The HIST1H2BD protein sequence is shown in SEQ ID NO: 3. The ICAM1 protein sequence is shown in SEQ ID NO: 4. The RIOK2 protein sequence is shown in SEQ ID NO: 5. The SERF2 protein sequence is shown in SEQ ID NO: 6. The KDM4D protein sequence is shown in SEQ ID NO: 7. The DNAJC8 protein sequence is shown in SEQ ID NO: 8. The ADARB1 protein sequence is shown in SEQ ID NO: 9.

[0031] Based on this new discovery, the present invention provides the use of the above proteins in the preparation of products (such as kits) for detecting and diagnosing Alzheimer's disease. The above proteins can be used alone or in combination to form a protein composition.

[0032] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are the average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0033] Example 1: Construction, expression and purification of recombinant antigen vectors

[0034] The method for obtaining the antigen can include synthesizing DNA encoding the protein antigen, using the synthesized DNA as a template, designing primers, and cloning the gene fragment of the protein antigen or its fragment into an expression plasmid through molecular cloning methods such as PCR, enzyme digestion, and ligation. The protein is then expressed in E. coli, yeast, or cells, and purified by chromatography to obtain the target protein. Alternatively, tags such as Trx, GST, AVI, HIS, and c-myc can be added to the protein antigen or its fragment. Adding these tags facilitates purification or labeling of the protein antigen but does not fundamentally alter the binding properties of the antigen with its own antibodies.

[0035] DOC2A, LGALS1, HIST1H2BD, ICAM1, RIOK2, SERF2, DNAJC8, KDM4D and ADARB1 proteins were selected, and their respective database IDs are shown in Table 1.

[0036] Table 1 Comparison table of candidate protein database IDs

[0037] Using a human cDNA library (purchased from Invitrogen) or fully synthesized DNA as a template, primers were designed, and the gene fragment of the protein was cloned into the pET28 plasmid by molecular cloning methods such as PCR, enzyme digestion, and ligation. At the same time, tags such as HIS and FLAG 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 that it contained the correct protein gene fragment. It should be noted that the addition of the tag is only for the convenience of identifying and extracting the protein, and does not have a decisive effect on the function of the protein as an antigen. When used, no tag is added or other tags are added as needed or other marking / identification methods are adopted.

[0038] The recombinant plasmid containing the protein gene fragment was transformed into E. coli BL21 (DE3) competent cells, and clones were picked and inoculated into LB medium and cultured in a shaking incubator at 37°C. 600When the pH value was about 0.8, the temperature was lowered to 16°C, 0.1 mM isopropylthio-β-D-galactopyranoside (IPTG) was added to each LB culture medium, and the expression was induced overnight to obtain bacteria.

[0039] The induced expression bacteria were collected by centrifugation and rinsed twice with PBS. The bacteria were resuspended and dispersed with lysis buffer (5-10 mL of lysis buffer per gram of bacteria), placed in an ice bath, and ultrasonically disrupted (ultrasonic power 200W, disruption for 5 seconds, rest for 5 seconds). After disruption, the supernatant was collected and purified by two steps of Ni column affinity chromatography and molecular sieve chromatography. The protein was analyzed by SDS-PAGE electrophoresis to confirm its molecular weight and purity. The concentration was determined by the Bradford method and stored at -80°C until use. The purified candidate proteins were obtained.

[0040] Example 2: Demonstration of the effect of each candidate protein alone in detecting AD-derived SCD and AD-derived MCI samples 1. The solutions and reagents used in this example are as follows:

[0041] (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 add water to make up to 1L.

[0042] (2) Blocking solution / sample diluent / antibody diluent: Dissolve 10 g of BSA (bovine serum albumin) in 800 ml of coating buffer and adjust the volume to 1 L with coating buffer.

[0043] (3) Washing solution: Prepare immediately before use. Add 0.5% Tween 20 (V / V) to the coating buffer, pH = 7.4.

[0044] (4) TMB colorimetric reagent, purchased from KPL.

[0045] (5) Stop solution: 1M hydrochloric acid.

[0046] 2. Solid-phase coating of the protein to be tested. Dilute the purified candidate proteins 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 solution, 200 μL per well each time. Then, add 200 μL of blocking solution to each well, incubate at room temperature for 1 hour, discard the blocking solution, spin dry, and wash three times with washing solution, 200 μL per well each time, and spin dry again; obtain the solid-phase coated antigen in the 96-well plate.

[0047] 3. Add the test sample. Dilute the human serum to be tested 100-fold with sample diluent and add 50 μL of the diluted test sample to each well of the 96-well plate containing the test protein. Place the 96-well plate on a microplate shaker and incubate at room temperature for 1 hour. Spin dry, then wash three times with washing solution, adding 200 μL per well each time, and spin dry again.

[0048] 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-fold in antibody diluent and add 50 μL per well to the 96-well plate treated in step 3. Place the 96-well plate on a microplate shaker and incubate at room temperature for 0.5 h. Shake dry the plate and wash three times with 200 μL per well of washing buffer, then shake dry again.

[0049] 5. Color development and optical density reading. Add 50 μL of TMB colorimetric reagent to each well of the 96-well plate treated in step 4. Shake for 15 seconds. Incubate in the dark at room temperature for 15 minutes, then add 50 μL of stop solution. Read the absorbance at 450 nm using a microplate reader to obtain the detection signal (S) for each sample.

[0050] 6. Sensitivity and specificity analysis: 300 positive samples (defined as positive samples at enrollment) and 300 negative samples (sera from healthy subjects) were collected.

[0051] Positive inclusion criteria were defined as: subjective memory loss without other cognitive impairments, onset less than 5 years prior, age greater than 55 years, concern about cognitive decline, and perceived memory worse than age-matched individuals. Patients had no recent (within 7 days) or long-term (≥5 years) use of the following medications: immunomodulators, antidepressants or neuroleptics containing anticholinergic components, antiparkinsonian medications, or other investigational drugs; and no autoimmune diseases, primarily rheumatoid arthritis, hyperthyroidism, systemic lupus erythematosus, or primary biliary cirrhosis. Assessments included a Global Dementia Scale (GDs) score of 2–3, a Clinical Dementia Rating (CDR) score of 0.5 or less, a memory score less than 1.5 standard deviations of an age- and education-matched control group, a Mini-Mental State Examination (MMSE) score greater than 24, and a Mattis Dementia Rating (DRs) score greater than 123.

[0052] It should be noted that the current common Alzheimer's disease diagnosis is not completely accurate, and it is especially difficult to accurately diagnose patients with very early (potential) Alzheimer's disease. Therefore, the samples defined as positive here do not represent all true (very early) Alzheimer's disease patients. Among them are normal people who are misdiagnosed / misjudged, and people with cognitive decline, cognitive impairment and memory loss caused by other reasons other than Alzheimer's disease.

[0053] The inclusion criteria for the negative sample (healthy people) were that the following conditions were met simultaneously: (1) the subjects were conscious, with stable vital signs, no abnormal neurological symptoms or signs, and no neurological or psychiatric diseases; (2) there was no subjective memory decline or other history of neurological or psychiatric diseases, and no recent insomnia or chronic insomnia (≥3 times / week, lasting ≥3 months); (3) the Mini-Mental State Examination (MMSE) score was >26 points; (4) the Montreal Cognitive Assessment (MoCA) score was >26 points; (5) the Hamilton Depression Scale (HAMD) < 7 points; (6) Hamilton Anxiety Rating Scale-HAMA (14 items) < 7 points; (7) Clinical Dementia Rating Scale (CDR) = 0 points; (8) No recent (within 7 days) or long-term (≥ 5 years) use of the following medications, including: immunomodulators, antidepressants or neuroleptics containing anticholinergic components, anti-Parkinson drugs, or other clinical experimental drugs; (9) No autoimmune diseases, mainly including rheumatoid arthritis, hyperthyroidism, systemic lupus erythematosus, and primary biliary cirrhosis.

[0054] Measure the detection signal (S) of each sample according to the method in step 4 (absorbance at a wavelength of 450 nm). Using the negative sample as the negative reference sample, calculate the mean (M) and standard deviation (SD) of the detection signal (S) of all negative reference samples, and use M + 3SD as the cutoff value.

[0055] Samples with a detection signal (S) ≥ Cut Off value (S ≥ M + 3SD) were defined as positive; samples with a detection signal (S) < Cut Off value (S < M + 3SD) were defined as negative.

[0056] Specificity and sensitivity were calculated based on the positive and negative results. Specificity refers to the proportion of healthy subject samples correctly identified as negative (i.e., the number of negative samples identified as negative divided by the total number of negative samples). Sensitivity refers to the proportion of positive patient samples identified as positive (i.e., the number of positive samples identified as positive divided by the total number of positive samples). The sensitivity and specificity of each protein tested were calculated using the protein as an antigen.

[0057] Positive samples were also tested at the hospital for β-amyloid accumulation (CSF / PET), FDG-PET / fMRI, or tau-mediated neuronal injury (CSF). The hospital test results served as a control for calculating sensitivity. Furthermore, because tests for β-amyloid accumulation and other conditions involve radiation / trauma, normal subjects (negative samples) were not tested accordingly, and therefore no specificity data are available. The results are shown in Table 2.

[0058] Table 2 Comparison table of detection results of candidate proteins

[0059] The results showed that the existing commonly used convenient methods (self-assessment, scale assessment) have low accuracy in detecting very early Alzheimer's disease, and the samples defined as positive contain a large number of misdiagnosed patients.

[0060] Example 3: Demonstration of the effect of protein composition in combined detection of AD-derived SCD and AD-derived MCI samples 1. Detection was performed using the antigen composition using the "single indicator" method.

[0061] The "single indicator" method means: each antigen is tested separately, and one antigen corresponds to one data; when a sample to be tested is tested using a certain antigen combination, as long as any one of the antigens in the antigen combination obtains a positive test signal, the result of the antigen combination detecting the sample is considered positive, otherwise it is negative.

[0062] Different antigen combinations were formed using the candidate protein antigens from Example 2, as shown in Table 3. The specificity and sensitivity of the positive and negative samples from Example 1 were tested using each antigen combination, as described in Example 2. The test results are shown in Table 4. It should be noted that the inventors did not conduct only the combination tests in Table 3; they obtained the antigen combinations shown in Table 3 after extensive preliminary experiments.

[0063] Table 3 Comparison table of antigen combinations

[0064] Table 4 Comparison of test results of each antigen combination using the “single indicator” method

[0065] The results showed that when using a "single indicator" for judgment, the sensitivity was higher than that of the three commonly used existing methods, and the specificity was lower than that of each individual protein antigen test, indicating that the accuracy of the test may be further improved.

[0066] 2. Use the "dual indicator" method to detect using the antigen combination.

[0067] The "double indicator" method means that when a certain antigen combination is used to detect a certain sample to be tested, if the detection signals obtained by two or more antigens in the antigen combination are positive detection signals, then the test result of the sample is positive, otherwise it is negative. The rest of the operations are the same as the "single indicator" method. When multiple existing technologies are used for detection at the same time, the "double indicator" method is also used for judgment, that is, the test results are defined as positive only when the test results of the two detection methods are both positive, otherwise it is negative. For example: the combination of β-Amyloid Accumulation + FDG-PET / fMRI (CSF / PET) requires that the test results of β-Amyloid Accumulation and FDG-PET / fMRI (CSF / PET) are both positive, and the test result is positive. The results are shown in Table 5.

[0068] Table 5 Comparison of test results of each antigen combination using the "double index method"

[0069] The results showed that the detection results of the "dual index" method were closer to the detection results of β-Amyloid Accumulation and other methods, and the specificity was significantly higher, indicating that the detection accuracy of the "dual index" method was higher.

[0070] 3. Use individual protein antigens or a combination of antigens via a "single marker" or "dual marker" method to detect samples that are defined as positive for β-amyloid protein or other tests.

[0071] To further confirm the overlap between the positive results detected by individual antigens or combinations of antigens and the positive results detected by existing methods, each antigen combination was used to retest the samples that were positive for β-Amyloid Accumulation, FDG-PET / fMRI (CSF / PET), Tau-mediated neuronal injury (CSF), β-Amyloid Accumulation + FDG-PET / fMRI (CSF / PET), β-Amyloid Accumulation + Tau-mediated neuronal injury (CSF), and FDG-PET / fMRI + Tau-mediated neuronal injury (CSF) in Table 5. The results are shown in Table 6. The totals in Table 6 correspond to the number and samples detected positive by each existing method in Table 5.

[0072] Table 6 Comparison of test results for each antigen or antigen combination

[0073] The results showed that the test results of combinations 7, 8, and 9 were highly consistent with the existing detection methods that are generally recognized to have high accuracy, and all patients who were considered positive by the existing methods could be accurately detected.

[0074] Example 4: Demonstration of the effectiveness of protein combination combined detection in patients with early and mid-stage AD. 100 samples clinically diagnosed as positive for early-stage AD and 100 samples clinically diagnosed as positive for mid-stage AD were collected. The test signal (S) of each sample was measured using the candidate antigens and combinations listed in Table 3, following the method of Example 2. Samples with a test signal (S) ≥ the Cut Off value (S ≥ M + 3SD) were defined as positive; samples with a test signal (s) < the Cut Off value (S < M + 3SD) were defined as negative.

[0075] Specificity and sensitivity were calculated based on the positive and negative sample results. Specificity refers to the proportion of healthy subject samples correctly identified as negative, i.e., the number of negative samples identified as negative divided by the total number of negative samples. Sensitivity refers to the proportion of positive patient samples identified as positive, i.e., the number of positive samples identified as positive divided by the total number of positive samples. The sensitivity and specificity of sample testing using each test protein as an antigen were calculated. The results are shown in Table 7.

[0076] Table 7 Comparison of test results for each antigen or antigen combination

[0077] The results showed that the detection results of combinations 5, 6, 7, 8, and 9 had high specificity and good sensitivity in the early and middle stages of AD.

[0078] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. An antigen combination, characterized in that: The antigen combination includes at least any two of DOC2A protein, LGALS1 protein, KDM4D protein, and ADARB1 protein simultaneously.

2. The antigen combination according to claim 1, wherein: The amino acid sequence of the DOC2A protein is as shown in SEQ ID NO: 1, and / or the amino acid sequence of the LGALS1 protein is as shown in SEQ ID NO: 2, and / or the amino acid sequence of the KDM4D protein is as shown in SEQ ID NO: 7, and / or the amino acid sequence of the ADARB1 protein is as shown in SEQ ID NO:

9.

3. The antigen combination according to claim 1 or 2, characterized in that: The antigen combination further includes: SERF2 protein; the amino acid sequence of the SERF2 protein is as shown in SEQ ID NO:

6.

4. The antigen combination according to claim 3, wherein: The antigen combination further includes: any one or more of HIST1H2BD protein, ICAM1 protein, RIOK2 protein, and DNAJC8 protein.

5. The antigen combination according to claim 4, wherein: The amino acid sequence of the HIST1H2BD protein is as shown in SEQ ID NO: 3, and / or the amino acid sequence of the ICAM1 protein is as shown in SEQ ID NO: 4, and / or the amino acid sequence of the RIOK2 protein is as shown in SEQ ID NO: 5, and / or the amino acid sequence of the DNAJC8 protein is as shown in SEQ ID NO:

8.

6. Use of the antigen combination according to any one of claims 1 to 5 in the preparation of a product for detecting / identifying Alzheimer's disease.

7. The application according to claim 6, wherein: Use in the preparation of a product for detecting / identifying Alzheimer's disease in the middle stage and earlier stages.

8. A reagent or kit for detecting / identifying Alzheimer's disease prepared by using the antigen combination according to any one of claims 1 to 5.

9. Use of LGALS1 protein and / or HIST1H2BD protein and / or RIOK2 protein in the preparation of a product for detecting / identifying Alzheimer's disease, characterized in that: The amino acid sequence of the LGALS1 protein is as shown in SEQ ID NO: 2, and / or the amino acid sequence of the HIST1H2BD protein is as shown in SEQ ID NO: 3, and / or the amino acid sequence of the RIOK2 protein is as shown in SEQ ID NO:

5.

10. A product for detecting / identifying Alzheimer's disease prepared by using the protein according to claim 9.

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