A sugar chain exosome P-Tau-217, a marker for Alzheimer's disease detection, and its application

Abstract

The present invention provides a sugar chain exosome P-Tau-217, a detection marker for Alzheimer's disease, and a detection kit, a detection system and a method based on the above marker, which can quickly and effectively separate, enrich and detect the sugar chain exosome P-Tau-217 content in a blood sample, thereby realizing rapid, automated, intelligent, high-throughput, high-sensitivity and specific detection of the sugar chain exosome P-Tau-217 in the blood sample; the collected serum / plasma samples do not need to be processed in any way, and are matched with a fully automatic chemiluminescence immunoassay analyzer equipped with a sugar chain exosome separation and enrichment module and a detection module for detection, with an overall separation and enrichment time of less than or equal to 35 min, and no other equipment needs to be added during the detection process; the detection results can be applied to the detection and diagnosis of AD-MC I and / or AD, disease course monitoring, efficacy evaluation, prognosis evaluation, etc., effectively solving the deficiency that the existing technology cannot be widely used in clinical applications.

Description

Technical Field

[0001] The present invention relates to an Alzheimer's disease detection marker sugar chain exosome P-Tau-217 and application thereof, belonging to the technical field of biological detection. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease characterized by progressive cognitive decline, and its cause is still unclear.

[0003] AD detection is still mainly carried out through auxiliary means such as clinical history, behavioral and psychological scale assessment, and PET imaging examination. Although clinical history and behavioral and psychological scale assessment are simple and easy to perform, they are greatly affected by subjective factors and can only be used as auxiliary means. PET imaging examination can be used as an important examination method, but its equipment is expensive, the operation level is high, and the detection price is high. It is only used as an important diagnosis method in large clinical hospitals and cannot be carried out on a large scale. AD biological markers Aβ and tau protein derived from cerebrospinal fluid have been shown to have excellent detection performance, but cerebrospinal fluid samples require lumbar puncture, which is very harmful to the human body, has poor patient compliance, and is difficult to obtain samples, so it is not suitable for clinical implementation.

[0004] Existing studies have shown that abnormal phosphorylation of Tau protein is closely related to the onset of AD. Tau protein is a phosphorylated protein. Under normal circumstances, it is mainly involved in the assembly and stability maintenance of the cytoskeleton. The phosphorylation modification of Tau protein is strictly regulated. Once it is out of regulation, it may lead to the formation of neurofibrillary tangles (NFTs). In the brains of AD patients, Tau protein is abnormally phosphorylated to form NFTs, leading to impaired neuronal function and death. NFTs are an important pathology in AD patients. P-Tau-217 is a phosphorylated form of Tau protein, which is closely related to the pathogenesis of AD, but the content of P-Tau-217 in blood samples is extremely low, and current detection technologies are not widely used in clinical applications. Exosomes contain abundant bioactive molecules, such as proteins, nucleic acids and lipids, which can reflect the state and function of cells. In recent years, many studies have found that exosomes play an important role in the pathogenesis of AD. Exosomes and the bioactive molecule P-Tau-217 they contain are considered to be a potential biomarker for AD. The commonly used ultracentrifugation, differential centrifugation, ultrafiltration, size exclusion chromatography, precipitation and other methods for separation and enrichment of exosomes require special equipment or the addition of polymers, and are complex, time-consuming, and low in purity, which may affect the activity of exosomes or reduce the solubility of exosomes. The immunoaffinity method uses specific antibodies to bind to exosome surface antigens and separate exosomes through immune capture. Although this method is highly targeted, the sample processing time is long and complex, and it is easily affected by antibody specificity and affinity, as shown in Patent 20221. 1721260.9 stated: The exosome immune capture step is extremely complicated. The plasma pretreatment step requires three centrifugations including 15,000g centrifugation and a sample and enzyme incubation process of not less than 1 hour. The plasma sample after pretreatment is first separated and extracted by a total exosome column, and then the total exosome solution is used to capture the neural-specific exosome antibody, which takes more than 1.5 hours. The exosomes after antibody capture must be lysed at a constant temperature before subsequent inspection. The sample processing and exosome antibody capture separation time exceeds 3 hours. During the detection, the capture antibody and the detection antibody need to be dialyzed for more than 1 hour before use, and then the detection time is greater than 20 minutes. Therefore, the separation, enrichment and detection time of the entire exosome Tau217 exceed 4 hours and the result discrimination range is small; therefore, the above methods cannot be widely used in clinical practice. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an Alzheimer's disease detection marker sugar chain exosome P-Tau-217 and its application, which can realize rapid, automated, intelligent, high-throughput, high-sensitivity and specific detection of AD patient samples, and can be applied to the detection, diagnosis, disease course monitoring, efficacy evaluation, prognosis evaluation, etc. of AD mild cognitive impairment (AD-MCI) and / or AD.

[0007] In order to achieve the above objectives, the present invention provides the following technical solutions based on the aforementioned research results of this team (patent number: ZL202010060055.7).

[0008] (II) Technical solution

[0009] First, the present invention provides an Alzheimer's disease detection marker, characterized in that the detection marker is P-Tau-217 derived from sugar chain exosomes.

[0010] Preferably, the P-Tau-217 derived from the sugar chain exosomes is P-Tau-217 contained in any one of the glycosylated exosomes, namely, N-glycosylated exosomes, O-glycosylated exosomes and fucosylated exosomes.

[0011] More preferably, the P-Tau-217 derived from the sugar chain exosomes is P-Tau-217 contained in N-glycosylated exosomes, and the N-glycosylated exosomes can be separated and enriched using any one or more lectins selected from ConA, PVL, SBA, PAII, WGA, and AAL.

[0012] More preferably, the exosome-derived P-Tau-217 is exosome-derived P-Tau-217 in a plasma or serum sample.

[0013] Preferably, the P-Tau-217 marker derived from sugar chain exosomes is a detection marker for AD mild cognitive impairment (AD-MCI) detection or diagnosis, and / or AD detection or diagnosis, and / or AD disease course monitoring, and / or AD efficacy evaluation, and / or AD prognosis evaluation.

[0014] Secondly, another aspect of the present invention also provides a detection application of an Alzheimer's disease detection marker, characterized in that the detection application of the detection marker is a detection application of P-Tau-217 derived from sugar chain exosomes.

[0015] Preferably, the detection application of P-Tau-217 derived from sugar chain exosomes is the detection application of P-Tau-217 contained in any one of glycosylated exosomes derived from N-glycosylated exosomes, O-glycosylated exosomes, and fucosylated exosomes.

[0016] More preferably, the detection application of P-Tau-217 derived from sugar chain exosomes is the detection application of P-Tau-217 of N-glycosylated exosomes, and the N-glycosylated exosomes can be separated and enriched by any one or more lectins of ConA, PVL, SBA, PAII, WGA, AAL and directly used for detection.

[0017] More preferably, the detection application of the sugar chain exosome-derived P-Tau-217 is the detection application of the sugar chain exosome-derived P-Tau-217 in plasma or serum samples.

[0018] Preferably, the detection application of P-Tau-217 derived from sugar chain exosomes is for detection or diagnosis of AD mild cognitive impairment (AD-MCI), and / or AD detection or diagnosis, and / or AD disease course monitoring, and / or AD efficacy evaluation, and / or AD prognosis evaluation and other detection applications.

[0019] Third, on the other hand, the present invention also provides an AD detection reagent for the aforementioned sugar chain exosome P-Tau-217 marker, characterized in that the AD detection reagent comprises a sugar chain exosome separation and enrichment reagent for AD detection and a P-Tau-217 marker detection reagent.

[0020] Preferably, the sugar chain exosome separation and enrichment reagent for AD detection includes sugar chain exosome separation magnetic beads for AD detection, a washing solution, and an eluent.

[0021] More preferably, the sugar chain exosome separation magnetic beads for AD detection are lectin-magnetic carrier coupling complexes.

[0022] More preferably, the sugar chain exosome separation and enrichment reagent for AD detection can be used to separate and enrich any one of N-glycosylated exosomes, O-glycosylated exosomes, and fucosylated exosomes for AD detection.

[0023] More preferably, the above-mentioned sugar chain exosome separation and enrichment reagent for AD detection is a sugar chain exosome separation and enrichment reagent for separating and enriching N-glycosylated exosomes, and the N-glycosylated exosome separation and enrichment reagent includes any one or more lectins of ConA, PVL, SBA, PAII, WGA, and AAL.

[0024] Preferably, the P-Tau-217 marker detection reagent comprises a detection antibody and a detection magnetic bead, wherein the detection antibodies are P-Tau-217 antibody and Tau antibody, and the Tau antibody, P-Tau-217 antibody and P-Tau-217 in the sample form a stable double antibody sandwich immune complex.

[0025] Fourthly, another aspect of the present invention further provides an AD detection kit for the aforementioned sugar chain exosome P-Tau-217 marker, wherein the kit comprises the aforementioned detection reagent.

[0026] Preferably, the detection kit further comprises a calibrator, a detection cleaning solution, and a luminescent substrate solution.

[0027] Fifth, another aspect of the present invention further provides an AD detection system and application of a sugar chain exosome P-Tau-217 marker, wherein the detection system comprises a sugar chain exosome separation and enrichment module and a detection module.

[0028] Preferably, the sugar chain exosome separation and enrichment module is pre-loaded with a sugar chain exosome separation and enrichment reagent to separate and enrich sugar chain exosomes for AD detection.

[0029] Preferably, the detection module detects the P-Tau-217 marker contained in the separated and enriched sugar chain exosomes through a pre-packaged P-Tau-217 marker detection reagent and outputs a detection signal.

[0030] More preferably, the separation and enrichment time of the detection system and application is less than or equal to 35 minutes.

[0031] More preferably, the detection signal output by the detection module is directly used for early screening of AD, and / or AD detection or diagnosis, and / or AD disease course monitoring, and / or AD efficacy evaluation, and / or AD prognosis evaluation and other detection and evaluation applications.

[0032] Preferably, the AD detection application of the sugar chain exosome P-Tau-217 marker includes the detection application of a fully automatic chemiluminescence immunoassay analyzer equipped with the above-mentioned sugar chain exosome separation and enrichment module and detection module.

[0033] In addition, the present invention also provides the use of a combination of a sugar chain exosome separation and enrichment reagent and a P-Tau-217 detection reagent in the preparation of an Alzheimer's disease detection reagent or a detection kit.

[0034] For the above application, preferably, the sugar chain exosome separation and enrichment reagent includes sugar chain exosome separation magnetic beads, a washing solution, and an eluent.

[0035] More preferably, the sugar chain exosome separation magnetic beads are lectin-magnetic carrier coupling complexes.

[0036] Preferably, the sugar chain exosome separation and enrichment reagent can be used to separate and enrich any one of N-glycosylated exosomes, O-glycosylated exosomes, and fucosylated exosomes for AD detection.

[0037] More preferably, the sugar chain exosome separation and enrichment reagent is a sugar chain exosome separation and enrichment reagent for separating and enriching N-glycosylated exosomes.

[0038] More preferably, the N-glycosylated exosome separation and enrichment reagent includes any one or more lectins selected from the group consisting of ConA, PVL, SBA, PAII, WGA, and AAL.

[0039] Preferably, the P-Tau-217 detection reagent comprises a detection antibody and a detection magnetic bead, and the detection antibody comprises a P-Tau-217 antibody and a Tau antibody.

[0040] (III) Beneficial effects

[0041] The technical effects of the present invention include: the present invention provides a sugar chain exosome P-Tau-217, a detection marker for Alzheimer's disease, and a detection kit, a detection system and a method based on the above marker, which can quickly and effectively separate, enrich and detect the sugar chain exosome P-Tau-217 content in a blood sample, thereby realizing rapid, automated, intelligent, high-throughput, high-sensitivity and specific detection of sugar chain exosome P-Tau-217 in a blood sample; the collected serum / plasma samples do not need to be processed in any way, and are matched with a fully automatic chemiluminescence immunoassay equipped with a sugar chain exosome separation and enrichment module and a detection module for detection, the overall separation and enrichment time is less than or equal to 35 minutes, and no other equipment is required in the detection process; the detection results of AD samples, AD-MCI samples and HC control samples all show very significant gradient differences, and the detection values ​​show a significant upward trend with the progression of AD, and the detection results can be applied to the detection and diagnosis of AD mild cognitive impairment (AD-MCI) and / or AD, disease course monitoring, efficacy evaluation, prognosis evaluation, etc., effectively solving the deficiency that the existing technology cannot be widely used in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 :The S / CO values ​​of AD samples, AD-MCI samples and HC control samples have significant gradient difference analysis diagram

[0043] Figure 2 :ROC curve of the test results of AD samples and HC control samples

[0044] Figure 3 :ROC curve of the test results of AD-MCI samples and HC control samples

[0045] Figure 4 : ROC curve of the test results of AD patient samples (AD and AD-MCI samples combined) and HC control samples

[0046] Figure 5: Trend consistency analysis of serum and plasma test results of AD samples, AD-MCI samples and HC control samples

[0047] Figure 6 : Regression curve analysis of serum and plasma test results of AD samples, AD-MCI samples and HC control samples

[0048] Figure 7 : Comparison of detection of P-Tau-217 in AD samples, AD-MCI samples and HC control samples after separation and enrichment of sugar chain exosomes and direct detection of P-Tau-217 in plasma samples without separation and enrichment of sugar chain exosomes

[0049] Figure 8 : Comparison of the results of direct detection of plasma samples among AD samples, AD-MCI samples and HC control samples. DETAILED DESCRIPTION

[0050] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0051] Implementation Case 1

[0052] This embodiment is a detection kit for sugar chain exosome P-Tau-217, a marker for Alzheimer's disease detection. The kit components include a sugar chain exosome separation and enrichment reagent and a P-Tau-217 marker detection reagent, wherein:

[0053] The reagent for separation and enrichment of sugar chain exosomes is prepared with reference to the aforementioned research results of this team (patent number: ZL202010060055.7), including sugar chain exosome separation magnetic beads, washing solution, and elution solution; the sugar chain exosome separation magnetic beads include a lectin-magnetic carrier coupling complex formed by coupling a lectin and a magnetic carrier, and a Tris buffer solution; the magnetic carrier includes magnetic beads with a macromolecular structure on the surface. In this implementation case and subsequent implementation cases, agarose magnetic beads are used for illustration; the lectin-magnetic carrier coupling complex is mainly used for separation and enrichment of sugar chain exosomes, including N-glycosylated exosomes Any one of glycosylated exosomes, O-glycosylated exosomes, and fucosylated exosomes is used for separation and enrichment of glycosylated exosomes for AD detection, and different types of lectins can be used to separate different glycosylated exosomes; to better illustrate the present invention, this embodiment and subsequent embodiments are all described by separating and enriching N-glycosylated exosomes; N-glycosylated exosome separation and enrichment reagents include any one or more lectins among ConA, PVL, SBA, PAII, WGA, and AAL, and the lectin WGA is used in the embodiment and subsequent embodiments to separate and enrich N-glycosylated exosomes for illustration.

[0054] The sugar chain exosome washing solution is a washing buffer or purified water without metal salt ions, including Tris buffer; the sugar chain exosome elution solution is a sugar-containing buffer without metal salt ions, including Tris buffer and / or boric acid buffer, etc.

[0055] The P-Tau-217 marker detection reagent includes detection antibodies and detection magnetic beads. The above-mentioned detection antibodies are P-Tau-217 antibodies and Tau antibodies. The P-Tau-217 antibodies are coated with magnetic beads to form coated antibodies, and the Tau antibodies are combined with ALP enzymes to form labeled antibodies. During the detection reaction using the kit, Tau antibodies and P-Tau-217 antibodies can form a stable double antibody sandwich immune complex with P-Tau-217 in the sample, and further form a double antibody sandwich immune complex of coated antibody-P-Tau-217 in the sample-labeled antibody for automatic detection of P-Tau-217 by a fully automatic chemiluminescence immunoassay instrument and output of detection signals / data. Similarly, the Tau antibody is coupled to magnetic beads to form a coated antibody, and the P-Tau-217 antibody is combined with the ALP enzyme to form a labeled antibody, which can also achieve the same effect. To better illustrate the present invention, this embodiment and subsequent embodiments are described by using the P-Tau-217 antibody and magnetic beads to form a coated antibody, and the Tau antibody is combined with the ALP enzyme to form a labeled antibody.

[0056] The detection kit also includes a calibrator, a calibration curve card, a detection cleaning solution, and a luminescent substrate solution; the above-mentioned calibrators include calibrators 1 and calibrators 2, and the calibrators are composed of high and low concentrations, including high-concentration P-Tau-217 antigen, low-concentration P-Tau-217 antigen, BSA buffer solution, and trehalose. The specific content of the calibrator is independently assigned based on the production inspection data of each batch of kits, which is used for fitting the standard curve during the detection process. The detection cleaning solution includes Tris buffer, and the detection substrate solution includes 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoacyl)-phenyl-1,2-dioxetane disodium salt solution.

[0057] Under the same detection principle as above, the above-mentioned ALP enzymatic luminescence system can be replaced with commonly used acridinium ester luminescence system, terpyridine ruthenium electrochemical luminescence system and other commonly used luminescence systems to achieve the same effect as above. In order to better illustrate the present invention, this implementation case and subsequent implementation cases are all described using the ALP enzymatic luminescence system.

[0058] The main biological raw and auxiliary materials in this implementation case are all commercial reagents, and the core key biologically active raw materials and magnetic carriers are derived from Kaijing Gene.

[0059] In this embodiment, the sugar chain exosome separation and enrichment reagent and the P-Tau-217 marker detection reagent are pre-packed into reagent boats or reagent strips.

[0060] In this embodiment, the Alzheimer's disease detection marker sugar chain exosome P-Tau-217 detection kit is used to detect the content of sugar chain exosome P-Tau-217 in a blood sample; sugar chain exosome P-Tau-217 is a biological marker for AD detection, and the sugar chain exosome P-Tau-217 content can be used for AD mild cognitive impairment (AD-MCI) detection or diagnosis, and / or AD detection or diagnosis, and / or AD disease course monitoring, and / or AD efficacy evaluation, and / or AD prognosis evaluation, etc.

[0061] Implementation Case 2

[0062] This implementation case is a method for separating and enriching sugar chain exosomes in blood samples and detecting P-Tau-217 from sugar chain exosomes using an Alzheimer's disease detection marker sugar chain exosome P-Tau-217 detection kit, and the specific steps include:

[0063] The Alzheimer's disease detection marker sugar chain exosome P-Tau-217 detection kit used in this implementation case is the kit prepared in Implementation Case 1, and the supporting detection equipment is a fully automatic chemiluminescence immunoassay with a sugar chain exosome separation and enrichment module and a detection module, namely, the C series fully automatic chemiluminescence immunoassay and MQ60 series fully automatic chemiluminescence immunoassay of Regeneron Biotechnology, the company where this research team is located.

[0064] Take no less than 100 μL of fresh serum or plasma sample, or serum or plasma sample stored at -20°C or below and repeatedly frozen and thawed no more than 3 times, thaw it to room temperature for use; balance the detection kit to room temperature for use.

[0065] Calibration procedure: Before using a new batch of kits for serum or plasma sample testing, first enter the standard calibration curve card information. The calibration curve card includes reagent batch information, master curve, calibration target value information and other key information of the kit; the specific steps are: open the instrument software, select to enter the calibration curve addition interface, use the scanner to scan the barcodes on the calibration curve card in turn, and after scanning, click Add, the instrument automatically recognizes and enters the information. Secondly, after entering the calibration curve card information, execute the calibration procedure, use the calibrator 1 and calibrator 2 in the kit for calibration, and the calibration curve formed after calibration is the working curve; the specific steps are: put the detection reagent into the reagent position of the instrument, draw no less than 100μL of fully mixed calibrator 1 and calibrator 2 and add them to the corresponding sample wells, select the calibrator in the sample type of the instrument software interface, click Start, the instrument automatically runs the calibration process, and after the operation, the instrument automatically calibrates and calculates the calibration curve based on the signal values ​​(RLU values) of calibrator 1 and calibrator 2.

[0066] Sample detection: Take no less than 100 μL of serum or plasma sample and add it to the sample position, place the pre-packaged reagent boat or reagent strip into the corresponding reagent position, click Start, and automatically run the detection program, which includes taking 100 μL of sample and mixing it with 200 μL of lectin-magnetic carrier coupling complex, incubating at 37°C for no more than 10 minutes, aspirating no less than 300 μL of separation washing solution to wash three times, aspirating 200 μL of separation elution solution to elute the separated sugar chain exosomes, and evenly mixing the eluted sugar chain exosomes with 50-100 μL of coated antibody and 50-100 μL of labeled antibody, incubating at 37°C for no more than 10 minutes, washing three times with no less than 300 μL, adding 150 μL of luminescent substrate solution, and the fully automatic chemiluminescence immunoassay analyzer automatically detects the luminescent signal and automatically calculates and outputs the signal value of the analyte in the sample according to the calibration curve, or the S / CO value (signal value / Cutoff value set by the standard curve) or concentration value; the above overall detection time is less than 35 minutes.

[0067] In this implementation case, the S / CO value automatically reported by the fully automatic chemiluminescent immunoassay analyzer is used for judgment, and S / CO=1.0 is used as the critical value for positive judgment; when the S / CO value is ≥1.0, the result is judged as positive; when the S / CO value is <1.0, the result is judged as negative.

[0068] Implementation Case 3

[0069] This implementation case is a study on the clinical applicability of the Alzheimer's disease detection marker sugar chain exosome P-Tau-217 detection reagent

[0070] 214 plasma samples of AD patients were collected from clinical institutions, including 65 samples of mild cognitive impairment (AD-MCI) and 149 samples of AD patients, all of which had clear clinical diagnosis results (scale evaluation and / or PET-CT imaging examination); 167 HC control plasma samples, including 146 plasma samples of healthy people and 21 samples of patients with Parkinson's and other diseases and dementia. The reagents and detection methods of implementation cases 1 and 2 were used to detect the above 381 samples, and the sample detection results S / CO were analyzed.

[0071] The analysis results are shown in Table 1 below; Table 1 shows that the S / CO values ​​of the test results of AD samples, AD-MCI samples and HC control samples showed significant gradient differences, the AD sample test results were significantly higher than the AD-MCI sample test results, and the AD-MCI sample test results were significantly higher than the HC control sample test results ( Figure 1 ); The positive detection rate of 149 AD patient samples was 96.64%, and the area under the ROC curve was 0.991 ( Figure 2); The positive detection rate of 65 AD-MCI patient samples was 78.46%, and the area under the ROC curve was 0.975 ( Figure 3 ); The overall detection rate of 214 AD patient samples (AD and AD-MCI samples combined) was 91.12%, and the area under the ROC curve was 0.986 ( Figure 4 ); healthy person samples; 167 HC control samples (including healthy person samples and samples of patients with Parkinson's disease and other dementia diseases) had a negative detection rate of 100%.

[0072] Table 1

[0073] .

[0074] The above test results of the present implementation case all show that the detection results of the Alzheimer's disease detection marker sugar chain exosome P-Tau-217 detection reagent of the present invention for AD samples, AD-MCI samples, and HC control samples all show very significant gradient differences, and the detection value shows a significant upward trend with the progression of AD, indicating that the present invention has high application value and potential for disease monitoring; the high detection sensitivity and high detection specificity of the present invention show high sensitivity on both AD samples and AD-MCI samples, showing excellent AD clinical diagnostic value and AD-MCI early diagnosis and screening value; secondly, in the detection process of the present invention, the plasma sample is not subjected to any special treatment and is directly tested on the machine, and the fully automatic chemiluminescence immunoassay analyzer equipped with a sugar chain exosome separation and enrichment module and a detection module is matched to realize automatic detection, and fully automatic detection of sample input and result output is realized, the detection time is less than or equal to 35 minutes, and the detection throughput exceeds 150 tests / h. The present invention truly realizes the rapid, automated, intelligent, high-throughput, high-sensitivity and specific detection of sugar chain exosome P-Tau-217 in blood samples. The detection results can be applied to the detection, diagnosis, disease course monitoring, efficacy evaluation, prognosis evaluation, etc. of AD-MCI and / or AD, and has the potential and value for wide clinical application.

[0075] Implementation Case 4

[0076] This implementation case compares and analyzes the test results of serum and plasma samples from the same source. 38 homologous serum and plasma samples from AD patients, 35 homologous serum and plasma samples from AD-MCI patients, and 42 homologous serum and plasma samples from HC controls were collected. The reagents and detection methods of implementation cases 1 and 2 were used to test the above serum and plasma samples, and the test results were analyzed.

[0077] By comparing the test results of homologous serum and plasma samples, the test results of serum and plasma samples of AD samples, AD-MCI samples and HC control samples were highly consistent, with no significant differences ( Figure 5); and the test results of serum and plasma samples of the above AD samples, AD-MCI samples and HC control samples were analyzed by regression curve analysis. The results showed that R 2 =0.9784( Figure 6 ); The results of this implementation case show that the test results of the present invention for serum and plasma samples from the same source have no significant difference and are highly consistent.

[0078] Implementation Case 5

[0079] This implementation case verifies and analyzes the source of sugar chain exosome P-Tau-217 in the sample. In addition, 29 AD-MCI plasma samples, 52 AD plasma samples, HC control, and 57 plasma samples were collected, and each of the above samples was divided into 2 groups; Group 1 performed sugar chain exosome separation and enrichment and P-Tau-217 detection according to the steps in Implementation Case 3, and Group 2 did not perform sugar chain exosome separation and enrichment in the sample, and directly used the detection module steps in Implementation Case 3 to detect P-Tau-217 in plasma samples.

[0080] At the same time, the P-Tau-217 test results in the homologous samples of group 1 and group 2 were analyzed, and the results showed that ( Figure 7 and Figure 8 ), the S / CO of the test results of group 1 was significantly higher than that of group 2, and there were significant gradient differences between the test results of AD-MCI, AD, and HC controls in group 1; there were no significant gradient differences between the test results of AD-MCI, AD, and HC controls in group 2, and the S / CO values ​​were all less than 1, indicating that the specificity of the test process was poor, indicating that the direct content of P-Tau-217 in plasma samples was very low, and conventional chemiluminescence immunoassays could not be directly used for detection.

[0081] According to the test results of this embodiment and combined with the analysis of existing literature, P-Tau-217 exists in large quantities in cerebrospinal fluid samples of AD patients, but due to the influence of the blood-brain barrier, P-Tau-217 cannot directly pass through the blood-brain barrier into the blood sample in large quantities, resulting in a very low direct content of P-Tau-217 in the blood sample, and conventional detection methods cannot achieve effective detection results; P-Tau-217 is mainly carried into the blood through abnormally glycosylated neurogenic exosomes and stably exists in the exosomes; Based on the above analysis, by isolating and enriching sugar chain exosomes in blood samples of AD patients, and detecting P-Tau-217 rich in sugar chain exosomes, it has significant clinical detection and diagnostic value for the detection or diagnosis of AD mild cognitive impairment (AD-MCI), and / or AD detection or diagnosis, and / or AD course monitoring, and / or AD efficacy evaluation, and / or AD prognosis evaluation.

[0082] Finally, it should be noted that the above implementation cases are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above implementation cases, those skilled in the art should understand that they can still modify the technical solutions recorded in the above implementation cases, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the implementation cases of the present invention.

Claims

1. An AD detection reagent based on the detection of P-Tau-217 protein derived from sugar chain exosomes, characterized in that: The AD detection reagent comprises a sugar chain exosome separation and enrichment reagent and a P-Tau-217 protein detection reagent, wherein the sugar chain exosome separation and enrichment reagent is a sugar chain exosome separation and enrichment reagent for separating and enriching N-glycosylated exosomes.

2. The AD detection reagent according to claim 1, characterized in that The sugar chain exosome separation and enrichment reagent comprises sugar chain exosome separation magnetic beads, a washing solution and an eluent.

3. The AD detection reagent according to claim 2, characterized in that The sugar chain exosome separation magnetic beads are lectin-magnetic carrier coupling complexes.

4. The AD detection reagent according to claim 1, characterized in that The sugar chain exosome separation and enrichment reagent for separating and enriching N-glycosylated exosomes includes any one or more lectins selected from ConA, PVL, SBA, PA II, WGA, and AAL.

5. The AD detection reagent according to claim 1, characterized in that The P-Tau-217 protein detection reagent comprises a detection antibody and a detection magnetic bead, wherein the detection antibody comprises a P-Tau-217 antibody and a Tau antibody.

6. An AD detection kit based on the detection of P-Tau-217 protein derived from sugar chain exosomes, characterized in that: The kit comprises the AD detection reagent according to any one of claims 1 to 5.

7. The AD detection kit according to claim 6, characterized in that The AD detection kit also includes a calibrator, a detection cleaning solution and a luminescent substrate solution.

8. An AD detection system based on detection of P-Tau-217 protein derived from sugar chain exosomes, characterized in that: The AD detection system includes a sugar chain exosome separation and enrichment module and a P-Tau-217 protein detection module, wherein the sugar chain exosome separation and enrichment module is used to separate and enrich N-glycosylated exosomes.

9. The AD detection system according to claim 8, characterized in that: The sugar chain exosome separation and enrichment module separates and enriches sugar chain exosomes through a pre-installed sugar chain exosome separation and enrichment reagent; wherein the sugar chain exosome separation and enrichment reagent is a sugar chain exosome separation and enrichment reagent for separating and enriching N-glycosylated exosomes, and / or the separated and enriched sugar chain exosomes are N-glycosylated exosomes.

10. The AD detection system according to claim 8, characterized in that: The P-Tau-217 protein detection module detects the P-Tau-217 protein contained in the N-glycosylated exosomes separated and enriched by the sugar chain exosome separation and enrichment module and outputs the detection signal through the pre-installed P-Tau-217 protein detection reagent.

11. The AD detection system according to claim 10, characterized in that: The detection signal output by the P-Tau-217 protein detection module is directly used for early screening of AD, AD detection or diagnosis, AD course monitoring, AD efficacy evaluation and / or AD prognosis evaluation.

12. The AD detection system according to any one of claims 8 to 11, characterized in that: The AD detection system is a fully automatic chemiluminescence immunoassay analyzer equipped with the sugar chain exosome separation and enrichment module and the P-Tau-217 protein detection module.

13. Use of a combination of a sugar chain exosome separation and enrichment reagent and a P-Tau-217 protein detection reagent in the preparation of an Alzheimer's disease detection reagent or detection kit, wherein: The sugar chain exosome separation and enrichment reagent is a sugar chain exosome separation and enrichment reagent used for separating and enriching N-glycosylated exosomes.

14. The use according to claim 13, characterized in that The sugar chain exosome separation and enrichment reagent comprises sugar chain exosome separation magnetic beads, a washing solution and an eluent.

15. The use according to claim 14, characterized in that: The sugar chain exosome separation magnetic beads are lectin-magnetic carrier coupling complexes.

16. The use according to claim 13, characterized in that: The sugar chain exosome separation and enrichment reagent for separating and enriching N-glycosylated exosomes includes any one or more lectins selected from ConA, PVL, SBA, PA II, WGA, and AAL.

17. The use according to claim 13, characterized in that: The P-Tau-217 protein detection reagent comprises a detection antibody and a detection magnetic bead, wherein the detection antibody comprises a P-Tau-217 antibody and a Tau antibody.

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