CN118150843A - Alzheimer disease detection marker sugar chain exosome P-Tau-217 and application thereof - Google Patents
Alzheimer disease detection marker sugar chain exosome P-Tau-217 and application thereof Download PDFInfo
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Abstract
The invention provides an Alzheimer disease detection marker sugar chain exosome P-Tau-217, and a detection kit, a detection system and a detection method based on the marker, which can rapidly and effectively separate, enrich and detect the sugar chain exosome P-Tau-217 content in a blood sample, and realize rapid, automatic, intelligent, high-flux, high-sensitivity and specific detection of the sugar chain exosome P-Tau-217 in the blood sample; the collected serum/plasma sample does not need any treatment, is matched with a full-automatic chemiluminescence immunoassay analyzer with a sugar chain exosome separation and enrichment module and a detection module for detection, the total separation and enrichment time is less than or equal to 35mi, and no other equipment is needed in the detection process; the detection result can be applied to detection and diagnosis of AD-MC I and/or AD, disease course monitoring, curative effect evaluation, prognosis evaluation and the like, and the defect that the prior art cannot be widely applied to clinic is effectively overcome.
Description
Technical Field
The invention relates to an Alzheimer disease detection marker sugar chain exosome P-Tau-217 and application thereof, belonging to the technical field of biological detection.
Background
Alzheimer's disease (A l zhe imer's d i sease, AD) is a common neurodegenerative disease, which is mainly manifested by progressive decline of cognitive functions, the etiology of which is not completely understood.
The detection of AD is mainly carried out by auxiliary means such as clinical history, behavioral psychology evaluation, PET image examination and the like at present; clinical history and behavioral mind scale evaluation are simple and easy, but are greatly affected by subjective factors, such as: the experience, the supervisor judgment, the environmental factors and the like of the evaluation personnel, the psychological factors, the living environment and the like of the subject can only be used as auxiliary means. The PET image inspection can be used as an important inspection means, but the equipment is expensive, the operation level is high, the detection price is high, and the PET image inspection can not be developed on a large scale only as an important diagnosis confirming means in large clinical hospitals. AD biological markers Abeta and tau protein from cerebrospinal fluid have proved to have excellent detection performance, but cerebrospinal fluid samples need lumbar puncture, so that the patients have large damage to human bodies, poor patient compliance and large sample acquisition difficulty, and are not suitable for clinical development.
Previous studies have suggested that abnormal phosphorylation of Tau protein is closely related to the pathogenesis of AD; tau protein is a phosphorylation-modified protein that is normally involved mainly in cytoskeletal assembly and stability maintenance, and is tightly regulated, once deregulated, possibly leading to the formation of neurofibrillary tangles (Neurof i br I L L ARY TANG L ES, NFTs); in the brain of AD patients, tau proteins are abnormally phosphorylated, forming NFTs, which are important pathologies in AD patients, leading to impaired neuronal function and death. P-Tau-217 is a phosphorylation modification form of Tau protein, is closely related to pathogenesis of AD, but the content of P-Tau-217 in blood samples is extremely low, and the detection technology at present has no wide application in clinical application. The exosomes contain abundant bioactive molecules, such as proteins, nucleic acids, lipids and the like, which can reflect the state and the function of cells; in recent years, a plurality of researches find that exosomes play an important role in the pathogenesis of AD, and exosomes and bioactive molecules P-Tau-217 contained in the exosomes are considered as potential biomarkers of AD, and the problems that special equipment or polymers are required to be added, the operation is complex, the time is long, the purity is low, the exosomes activity is influenced or the exosome solubility is reduced due to the fact that the common ultracentrifugation method, differential centrifugation method, ultrafiltration method, size exclusion chromatography method, precipitation method and the like are used for separating and enriching the exosomes; the immunoaffinity method utilizes specific antibodies to combine with exosome surface antigens, and exosome is separated through immunocapture, and the method has strong pertinence, but long and complex sample treatment time and is easy to be influenced by antibody specificity, affinity and the like, as described in patent 202211721260.9: the exosome immune capturing step is extremely complex, the plasma pretreatment step comprises 3 times of centrifugation including 15000g centrifugation and incubation treatment process of samples and enzymes not less than 1h, the plasma samples after pretreatment are firstly subjected to total exosome column separation and extraction, then the antibody capturing of the nerve source specific exosome is carried out through total exosome solution, the duration is longer than 1.5h, the exosome after the antibody capturing can be subjected to subsequent examination after the constant-temperature pyrolysis, the sample treatment and exosome antibody capturing and separating time is longer than 3h, the capturing antibody and the detecting antibody are required to be subjected to dialysis treatment for more than 1h respectively before the use, then the detection is carried out, and the detection time is longer than 20min, so that the separation, enrichment and detection time of the whole exosome Tau217 are longer than 4h and the result discrimination interval is small; therefore, none of the above methods is widely used in clinical applications.
Disclosure of Invention
First, the technical problem to be solved
In view of the above-mentioned shortcomings and disadvantages of the prior art, the present invention provides a sugar chain exosome P-Tau-217 as a marker for detecting Alzheimer's disease and its application, which can realize rapid, automatic, intelligent, high-throughput, high-sensitivity and specific detection of AD patient samples, and can be applied to detection, diagnosis/disease course monitoring, efficacy evaluation, prognosis evaluation, etc. of AD mild cognitive impairment (AD-MC I) and/or AD.
In order to achieve the purpose, the invention provides the following technical scheme based on the research result (patent number: ZL 202010060055.7) of the team.
(II) technical scheme
Firstly, the invention provides an Alzheimer disease detection marker, which is characterized in that the detection marker is P-Tau-217 derived from sugar chain exosomes.
Preferably, the P-Tau-217 derived from the sugar chain exosome is P-Tau-217 contained in any one of N-glycosylated exosome, O-glycosylated exosome and fucosylated exosome.
More preferably, the P-Tau-217 derived from the sugar chain exosome is P-Tau-217 contained in the N-glycosylated exosome, and the N-glycosylated exosome can be separated and enriched by using one or more lectins selected from ConA, PVL, SBA, PA I, WGA and AAL.
More preferably, the sugar chain exosome-derived P-Tau-217 is a sugar chain exosome-derived P-Tau-217 in a blood plasma or serum sample.
Preferably, the P-Tau-217 marker derived from sugar chain exosomes is a detection marker for detection or diagnosis of AD mild cognitive impairment (AD-MC I), and/or detection or diagnosis of AD, and/or monitoring of the course of AD, and/or evaluation of AD efficacy, and/or prognosis of AD, etc.
Secondly, the invention also provides detection application of the Alzheimer disease detection marker, which is characterized in that the detection application of the detection marker is detection application of P-Tau-217 derived from sugar chain exosomes.
Preferably, the detection application of the P-Tau-217 derived from the sugar chain exosome is the detection application of the P-Tau-217 contained in any one of the glycosylated exosome derived from the N-glycosylated exosome, the O-glycosylated exosome and the fucosylated exosome.
More preferably, the detection application of the P-Tau-217 derived from the sugar chain exosome is the detection application of the P-Tau-217 for detecting the N-glycosylated exosome, and the N-glycosylated exosome can be separated and enriched by any one or more than two lectins of ConA, PVL, SBA, PA I, WGA and AAL and is directly subjected to detection application.
More preferably, the detection application of the sugar chain exosome-derived P-Tau-217 is a detection application of the sugar chain exosome-derived P-Tau-217 in a blood plasma or serum sample.
Preferably, the detection application of the P-Tau-217 derived from the sugar chain exosome is used for detection or diagnosis of AD mild cognitive impairment (AD-MC I), and/or detection or diagnosis of AD, and/or monitoring of the disease course of AD, and/or evaluation of the curative effect of AD, and/or evaluation of the prognosis of AD, and the like.
In a third aspect, the present invention also provides an AD detection reagent for the aforementioned P-Tau-217 marker of sugar chain exosomes, 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.
Preferably, the sugar chain exosome separation and enrichment reagent for AD detection comprises sugar chain exosome separation magnetic beads for AD detection, a cleaning solution and an eluent.
More preferably, the sugar chain exosome separation magnetic beads for AD detection are lectin-magnetic carrier coupled complexes.
More preferably, the sugar chain exosome separation and enrichment reagent for AD detection can be used for separating and enriching any one of N-glycosylated exosome, O-glycosylated exosome and fucosylated exosome for AD detection.
More preferably, the 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 comprises any one or more lectins of ConA, PVL, SBA, PA ii, WGA and AAL.
Preferably, the P-Tau-217 marker detection reagent comprises a detection antibody and a detection magnetic bead, wherein the detection antibody is a P-Tau-217 antibody and a Tau antibody, and the Tau antibody and the P-Tau-217 antibody form a stable double-antibody sandwich immune complex with the P-Tau-217 in the sample.
Fourth, the invention also provides an AD detection kit of the sugar chain exosome P-Tau-217 marker, which comprises the detection reagent.
Preferably, the detection kit further comprises a calibrator, a detection cleaning solution and a luminescent substrate solution.
Fifth, the invention also provides an AD detection system of the sugar chain exosome P-Tau-217 marker and application thereof, wherein the detection system comprises a sugar chain exosome separation and enrichment module and a detection module.
Preferably, the sugar chain exosome separation and enrichment module performs separation and enrichment of sugar chain exosomes for AD detection by pre-loading a sugar chain exosome separation and enrichment reagent.
Preferably, the detection module detects and outputs detection signals of the P-Tau-217 marker contained in the separated and enriched sugar chain exosomes through a pre-packaged P-Tau-217 marker detection reagent.
More preferably, the separation and enrichment time of the detection system and the application is less than or equal to 35min.
More preferably, the detection signal output by the detection module is directly used for detection and evaluation applications such as AD early screening, AD detection or diagnosis, AD disease course monitoring, AD curative effect evaluation, AD prognosis evaluation and the like.
Preferably, the AD detection application of the sugar chain exosome P-Tau-217 marker comprises detection application of a full-automatic chemiluminescence immunoassay analyzer with the sugar chain exosome separation and enrichment module and the detection module.
In addition, the invention also provides application of the combination of the sugar chain exosome separation and enrichment reagent and the P-Tau-217 detection reagent in preparing the Alzheimer disease detection reagent or the detection kit.
For the above application, preferably, the sugar chain exosome separation and enrichment reagent includes sugar chain exosome separation magnetic beads, a washing liquid, and an eluent.
More preferably, the sugar chain exosome separation magnetic beads are lectin-magnetic carrier conjugate complexes.
Preferably, the sugar chain exosome separation and enrichment reagent can be used for separating and enriching any one of N-glycosylated exosomes, O-glycosylated exosomes and fucosylated exosomes for AD detection.
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.
More preferably, the N-glycosylated exosome separation enrichment reagent comprises any one or more lectins of ConA, PVL, SBA, PA I, WGA, AAL.
Preferably, the P-Tau-217 detection reagent comprises a detection antibody and detection magnetic beads, wherein the detection antibody comprises a P-Tau-217 antibody and a Tau antibody.
(III) beneficial effects
The technical effects of the invention include: the invention provides an Alzheimer disease detection marker sugar chain exosome P-Tau-217, and a detection kit, a detection system and a detection method based on the marker, which can rapidly and effectively separate, enrich and detect the sugar chain exosome P-Tau-217 content in a blood sample, and realize rapid, automatic, intelligent, high-flux, high-sensitivity and specific detection of the sugar chain exosome P-Tau-217 in the blood sample; the collected serum/plasma sample does not need any treatment, is matched with a full-automatic chemiluminescence immunoassay analyzer with a sugar chain exosome separation and enrichment module and a detection module for detection, the total separation and enrichment time is less than or equal to 35min, and no other equipment is needed in the detection process; the detection results of the AD sample, the AD-MC I sample and the HC control sample show very remarkable gradient difference, the detection value shows a remarkable rising trend along with the progress of the AD disease course, and the detection results can be applied to detection and diagnosis of AD mild cognitive impairment (AD-MC I) and/or AD, disease course monitoring, curative effect evaluation, prognosis evaluation and the like, so that the defect that the prior art cannot be widely applied to clinic is effectively overcome.
Drawings
Fig. 1: S/CO values of detection results of AD sample, AD-MC I sample and HC control sample have significant gradient difference analysis chart
Fig. 2: ROC curve graph of detection results of AD sample and HC control sample
Fig. 3: ROC curve graph of detection results of AD-MC I sample and HC control sample
Fig. 4: ROC profile of overall AD patient sample (sum of AD and AD-MC I samples) versus HC control sample test results
Fig. 5: serum and plasma sample detection result trend consistency analysis chart of AD sample, AD-MC I sample and HC control sample
Fig. 6: regression curve analysis chart is carried out on detection results of serum and plasma samples of AD sample, AD-MC I sample and HC control sample
Fig. 7: P-Tau-217 detection after separation and enrichment of sugar chain exosomes in AD sample, AD-MC I sample and HC control sample and direct detection of P-Tau-217 in plasma sample without sugar chain exosome separation and enrichment
Fig. 8: results for direct detection of plasma samples from AD samples, AD-MC I samples and HC control samples.
Detailed Description
The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.
Example 1
The embodiment is a kit for detecting a sugar chain exosome P-Tau-217 of an Alzheimer disease detection marker, and the kit comprises a sugar chain exosome separation and enrichment reagent and a P-Tau-217 marker detection reagent, wherein:
the sugar chain exosome separation and enrichment reagent is carried out according to the previous research result (patent number: ZL 202010060055.7) of the team and comprises sugar chain exosome separation magnetic beads, cleaning liquid and eluent; the sugar chain exosome separation magnetic beads comprise lectin-magnetic carrier coupling compound formed by coupling lectin and magnetic carrier, tris buffer solution; the magnetic carrier comprises magnetic beads with macromolecular structures coated on the surfaces, and agarose magnetic beads are adopted for illustration in the embodiment and the subsequent embodiment; the lectin-magnetic carrier coupling compound is mainly used for separating and enriching sugar chain exosomes, including separating and enriching any one of N-glycosylated exosomes, O-glycosylated exosomes and fucosylated exosomes for AD detection, and different glycosylated exosomes can be separated by adopting different types of lectins; to better illustrate the present invention, the present embodiment and the subsequent embodiments are described using isolation and enrichment of N-glycosylated exosomes; the N-glycosylated exosome separation and enrichment reagent comprises one or more lectins of ConA, PVL, SBA, PA I, WGA and AAL, and the N-glycosylated exosome separation and enrichment by using the lectin WGA in the embodiment and the subsequent embodiment is described.
The sugar chain exosome cleaning solution is a cleaning buffer solution or purified water without metal salt ions, and comprises Tris buffer solution; the sugar chain exosome eluent is a buffer solution containing sugar and free of metal salt ions, and comprises Tris buffer solution, boric acid buffer solution and/or the like.
The P-Tau-217 marker detection reagent comprises a detection antibody and detection magnetic beads, wherein the detection antibody is a P-Tau-217 antibody and a Tau antibody, the P-Tau-217 antibody and the magnetic beads are coated to form a coated antibody, and the Tau antibody and ALP enzyme are combined to form a labeled antibody; in the detection reaction process by using the kit, the Tau antibody and the P-Tau-217 antibody can form a stable double-antibody sandwich immune complex with the P-Tau-217 in the sample, and the double-antibody sandwich immune complex coated with the P-Tau-217-labeled antibody in the antibody-sample is further formed for the automatic detection of the P-Tau-217 of the full-automatic chemiluminescence immunoassay analyzer and the output of detection signals/data. Similarly, the Tau antibody and the magnetic beads are coupled to form a coated antibody, the P-Tau-217 antibody and the ALP enzyme are combined to form a labeled antibody, and the P-Tau-217 antibody and the magnetic beads are coated to form a coated antibody, and the Tau antibody and the ALP enzyme are combined to form a labeled antibody for better explaining the invention.
The detection kit further comprises a calibrator, a calibration curve card, a detection cleaning solution and a luminescent substrate solution; the calibrator comprises a calibrator 1 and a calibrator 2, wherein the calibrator comprises a high concentration P-Tau-217 antigen and a low concentration P-Tau-217 antigen, a BSA buffer solution and trehalose. The specific content of the calibrator is independently assigned according to the production test data of each batch of kits and is used for fitting standard curves in the detection process. The detection cleaning solution comprises Tris buffer solution, and the detection substrate solution comprises 3- (2-spiral adamantane) -4-methoxy-4- (3-phosphorus oxy acyl) -phenyl-1, 2-dioxy cyclohexane disodium salt solution.
Under the condition that the detection principle is the same, the ALP enzymatic luminescence system is replaced by a common acridinium ester luminescence system, and common luminescence systems such as a terpyridyl ruthenium electrochemical luminescence system and the like can achieve the same effects.
In the embodiment, main biological raw materials and auxiliary materials are all commercial reagents, and core key bioactive raw materials and magnetic carriers are derived from a prospect gene.
In this embodiment, the sugar chain exosome separation and enrichment reagent and the P-Tau-217 marker detection reagent are pre-packaged in a reagent boat or reagent strip.
In the embodiment, the Alzheimer's disease detection marker sugar chain exosome P-Tau-217 detection kit is used for detecting the sugar chain exosome P-Tau-217 content in a blood sample; sugar chain exosome P-Tau-217 is a biological marker for AD detection, and the content of the sugar chain exosome P-Tau-217 can be used for AD mild cognitive impairment (AD-MC I) detection or diagnosis, and/or AD disease course monitoring, and/or AD curative effect evaluation, and/or AD prognosis evaluation, and the like.
Example 2
The embodiment is a method for separating and enriching sugar chain exosomes in a blood sample by using an Alzheimer disease detection marker sugar chain exosome P-Tau-217 detection kit, which specifically comprises the following steps:
The Alzheimer disease detection marker sugar chain exosome P-Tau-217 detection kit used in the embodiment is the kit prepared in embodiment 1, and the matched detection equipment is a full-automatic chemiluminescence immunoassay instrument with a sugar chain exosome separation enrichment module and a detection module, namely, a full-automatic chemiluminescence immunoassay instrument of the thermal scene organism C series and a full-automatic chemiluminescence immunoassay instrument of the MQ60 series of enterprises of the research team.
Taking fresh serum or plasma sample not less than 100 mu L, or repeatedly freezing and thawing the serum or plasma sample not more than 3 times at-20deg.C for later use; the detection kit is balanced to room temperature for standby.
Calibration procedure: before a new batch of reagent boxes are adopted to detect serum or plasma samples, firstly, standard input calibration curve card information is input, and the calibration curve card comprises reagent batch number information, main curves, reagent box key information such as calibrator target value information and the like; the method comprises the following specific steps: opening instrument software, selecting to enter a calibration curve adding interface, sequentially scanning bar codes on a calibration curve card by using a scanning gun, clicking for adding after scanning is completed, and automatically identifying and inputting information by the instrument. Secondly, after the calibration curve card information is recorded, executing a calibration program, and calibrating by using a calibrator 1 and a calibrator 2 in the kit, wherein a calibration curve formed after calibration is a working curve; the method comprises the following specific steps: placing the detection reagent into the reagent position of the instrument, sucking not less than 100 mu L of the calibrator 1 and the calibrator 2 which are fully and uniformly mixed, adding the calibrator into corresponding sample holes, selecting the calibrator at the sample type of the instrument software interface, clicking to start, automatically operating the calibration flow of the instrument, and automatically calibrating and calculating to obtain a calibration curve according to the signal values (RLU values) of the calibrator 1 and the calibrator 2 after the operation is finished.
Sample detection: adding not less than 100 mu L of serum or plasma sample into a sample position, placing a pre-packaged reagent boat or reagent strip into a corresponding reagent position, clicking to start, automatically running a detection program, wherein the detection program comprises the steps of taking 100 mu L of sample and 200 mu L of lectin-magnetic carrier coupling compound, incubating for not more than 10min at 37 ℃, absorbing not less than 300 mu L of separation cleaning liquid, cleaning for three times, absorbing 200 mu L of separation eluent, eluting separated sugar chain exosomes, uniformly mixing the eluted sugar chain exosomes with 50-100 mu L of coating antibody and 50-100 mu L of labeled antibody, incubating for not more than 10min at 37 ℃, cleaning for three times with not less than 300 mu L of luminescent substrate liquid, adding 150 mu L of luminescent substrate liquid, automatically detecting luminescent signals by a full-automatic chemiluminescence immunoassay analyzer, and automatically calculating and outputting signal values, S/CO values (signal values/standard curve-set Cutoff values) or concentration values of detected objects in the sample according to a calibration curve; the whole detection time is less than 35min.
In the embodiment, the automatic analysis light immunoassay analyzer automatically reports the S/CO value to judge, and the S/CO=1.0 is used as a positive judgment critical value; when the S/CO value is more than or equal to 1.0, the result is judged to be positive; when the S/CO value is less than 1.0, the result is judged as negative.
Example 3
The embodiment is the research on the clinical applicability of the detection reagent of the Alzheimer's disease detection marker sugar chain exosome P-Tau-217
214 Samples of plasma from AD patients were collected from a clinical institution, 65 samples of mild cognitive impairment (AD-MC I), 149 samples of AD patients, all with clear clinical diagnostic results (scale and/or PET-CT image examination); 167 HC control plasma samples, 146 healthy human plasma samples, 21 Parkinson and other dementia patients. The sample 381 is detected by using the reagents and the detection method of the embodiment cases 1 and 2, and the sample detection result S/CO is analyzed.
The analysis results are shown in the following Table 1; table 1 shows: the S/CO values of the detection results of the AD sample, the AD-MC I sample and the HC control sample show significant gradient difference, the detection result of the AD sample is significantly higher than the detection result of the AD-MC I sample, and the detection result of the AD-MC I sample is significantly higher than the detection result of the HC control sample (figure 1); the positive detection rate of 149 AD patient samples is 96.64%, and the area under the ROC curve is 0.991 (figure 2); the positive detection rate of the samples of 65 AD-MC I patients is 78.46%, and the area under the ROC curve is 0.975 (figure 3); the overall detection rate of 214 AD patient samples (sum of AD and AD-MC I samples) was 91.12%, and the area under the ROC curve was 0.986 (FIG. 4); a healthy human sample; the negative detection rate of 167 HC control samples (including healthy human samples and samples of patients suffering from Parkinson and other diseases and dementia) is 100 percent.
TABLE 1
The detection results of the embodiment show that the detection reagent of the Alzheimer disease detection marker sugar chain exosome P-Tau-217 shows very remarkable gradient difference on detection results of an AD sample, an AD-MC I sample and an HC control sample, and the detection values show obvious rising trend along with the progress of the AD disease course, so that the detection reagent has very high application value and potential for disease course monitoring; the high detection sensitivity and the high detection specificity of the invention show very high sensitivity on both AD samples and AD-MC I samples, and show excellent AD clinical diagnosis value and AD-MC I early diagnosis and screening value; and secondly, in the detection process, the plasma sample is directly subjected to on-machine detection, and the full-automatic detection is realized by matching with a full-automatic chemiluminescence immunoassay analyzer with a sugar chain exosome separation and enrichment module and a detection module, so that the full-automatic detection of the sample inlet and outlet results is realized, the detection time is less than or equal to 35min, and the detection flux exceeds 150 tests/h. The invention truly realizes the rapid, automatic, intelligent, high-flux, high-sensitivity and specific detection of the sugar chain exosome P-Tau-217 in the blood sample, and the detection result can be applied to the detection, diagnosis, disease course monitoring, curative effect evaluation, prognosis evaluation and the like of AD-MC I and/or AD, and has the potential and value of wide clinical application.
Example 4
The embodiment carries out comparison analysis on the detection results of serum and plasma samples from the same source. 38 cases of homologous serum plasma samples of AD patients, 35 cases of homologous serum plasma samples of AD-MC I patients and 42 cases of homologous serum plasma samples of HC control are collected. The reagents of embodiments 1 and 2 and the detection method thereof are used for respectively detecting the serum and the plasma samples, and the detection results are analyzed.
By comparing the detection results of homologous serum and plasma samples, the detection results of the serum and plasma samples of the AD sample, the AD-MC I sample and the HC control sample have high consistency and no obvious difference (figure 5); regression curve analysis is carried out on the detection results of serum and plasma samples of the AD sample, the AD-MC I sample and the HC control sample, and the result shows that R 2 = 0.9784 (figure 6); the results of the embodiment show that the invention has high consistency for the detection results of serum and plasma samples from the same source without obvious difference.
Example 5
In the embodiment, the source of the sugar chain exosome P-Tau-217 in the sample is verified and analyzed. In addition, 29 cases of AD-MC I plasma samples, 52 cases of AD plasma samples and 57 cases of HC control plasma samples are collected, and each collected sample is divided into 2 groups; group 1 carries out sugar chain exosome separation and enrichment and P-Tau-217 detection according to the steps in embodiment 3, group 2 does not carry out sugar chain exosome separation and enrichment in the sample, and the detection module step in embodiment 3 is directly adopted to carry out P-Tau-217 detection in the plasma sample.
Meanwhile, the detection results of P-Tau-217 in the homologous samples of the 1 st group and the 2 nd group are analyzed, and the results show (shown in fig. 7 and 8), wherein the detection results S/CO of the 1 st group are obviously higher than the detection results S/CO of the 2 nd group, and the detection results of the 1 st group AD-MC I, AD and HC contrast have obvious gradient differences; the detection results of the group 2 AD-MC I, AD and HC controls have no obvious gradient difference, the S/CO value is less than 1, the specificity is poor in the detection process, and the fact that the direct content of P-Tau-217 in a plasma sample is very low is indicated that the conventional chemiluminescent immunology detection cannot be directly used for detection.
According to the detection result of the embodiment and the combination of the existing literature analysis, the P-Tau-217 exists in a large amount in the cerebrospinal fluid sample of the AD patient, but the P-Tau-217 cannot directly enter the blood sample in a large amount through the blood brain barrier due to the influence of the blood brain barrier, so that the direct content of the P-Tau-217 in the blood sample is very low, and the conventional detection method cannot achieve the effective detection effect; P-Tau-217 is carried into the blood mainly through abnormally glycosylated neurogenic exosomes and stably exists in the exosomes; in conclusion, the analysis is carried out, and the separation and enrichment of sugar chain exosomes in blood samples of AD patients and the detection of P-Tau-217 enriched in the sugar chain exosomes are of significant clinical detection and diagnosis value to AD mild cognitive impairment (AD-MC I) detection or diagnosis, and/or AD disease course monitoring, and/or AD curative effect evaluation, and/or AD prognosis evaluation, etc.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme recorded in each embodiment can be modified or part of technical features in the technical scheme can be replaced equivalently; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (33)
1. The Alzheimer's disease detection marker is characterized in that the detection marker is P-Tau-217 derived from sugar chain exosomes.
2. The detection marker according to claim 1, wherein the sugar chain exosome-derived P-Tau-217 is P-Tau-217 contained in any one of an N-glycosylated exosome, an O-glycosylated exosome and a fucosylated exosome.
3. The detection marker according to claim 1, wherein the sugar chain exosome-derived P-Tau-217 is an N-glycosylated exosome-contained P-Tau-217.
4. The detection marker according to claim 3, wherein the N-glycosylated exosomes are enriched by isolation with one or more lectins selected from ConA, PVL, SBA, PA ii, WGA, AAL.
5. The detection marker of claim 1, wherein the sugar chain exosome-derived P-Tau-217 is sugar chain exosome-derived P-Tau-217 in a plasma or serum sample.
6. The detection marker according to claim 1, wherein the P-Tau-217 marker derived from sugar chain exosomes is a detection marker for use in detection or diagnosis of AD mild cognitive impairment (AD-MCI), and/or detection or diagnosis of AD, and/or monitoring of the course of AD, and/or evaluation of the efficacy of AD, and/or evaluation of the prognosis of AD.
7. The detection application of the Alzheimer disease detection marker is characterized in that the detection application is of P-Tau-217 derived from sugar chain exosomes.
8. The detection application according to claim 7, wherein the sugar chain exosome-derived P-Tau-217 detection application is a detection application for detecting P-Tau-217 contained in any one of an N-glycosylated exosome, an O-glycosylated exosome and a fucosylated exosome.
9. The detection application according to claim 7, wherein the sugar chain exosome-derived P-Tau-217 detection application is a detection application for detecting N-glycosylated exosome P-Tau-217.
10. The detection application according to claim 9, wherein the N-glycosylated exosomes can be isolated and enriched with any one or more lectins of ConA, PVL, SBA, pai, WGA, AAL and directly used for detection.
11. The use of claim 7, wherein the use of P-Tau-217 derived from sugar chain exosomes is the use of P-Tau-217 derived from sugar chain exosomes in a blood plasma or serum sample.
12. The use according to claim 7, wherein the P-Tau-217 detection of carbohydrate exosomes is used for detection or diagnosis of mild cognitive impairment (AD-MCI), and/or detection or diagnosis of AD, and/or monitoring of the course of AD, and/or evaluation of the efficacy of AD, and/or evaluation of prognosis of AD.
13. An AD detection reagent based on sugar chain exosome P-Tau-217 marker detection, which is characterized by comprising a sugar chain exosome separation enrichment reagent for AD detection and a P-Tau-217 marker detection reagent.
14. The AD detection reagent according to claim 13, wherein the sugar chain exosome separation and enrichment reagent for AD detection comprises sugar chain exosome separation magnetic beads for AD detection, a washing liquid, and an eluent.
15. The AD detection reagent according to claim 14, wherein the sugar chain exosome separation magnetic beads for AD detection are lectin-magnetic carrier conjugate complexes.
16. The reagent for detecting AD according to claim 14, wherein the reagent for separating and enriching sugar chain exosomes is useful for separating and enriching any one of N-glycosylated exosomes, O-glycosylated exosomes, and fucosylated exosomes for use in AD detection.
17. The AD detection reagent according to claim 14, wherein the sugar chain exosome separation-enrichment reagent is a sugar chain exosome separation-enrichment reagent for separating and enriching an N-glycosylated exosome.
18. The AD detection reagent according to claim 17, wherein the N-glycosylated exosome separation enrichment reagent comprises one or more lectins selected from ConA, PVL, SBA, PA ii, WGA, and AAL.
19. The AD detection reagent according to claim 13, wherein the P-Tau-217 marker 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.
20. An AD detection kit based on sugar chain exosome P-Tau-217 marker detection, characterized in that the kit comprises the detection reagent of claim 13.
21. The AD detection kit of claim 20, further comprising a calibrator, a detection wash, and a luminescent substrate solution.
22. The AD detection system based on sugar chain exosome P-Tau-217 marker detection is characterized by comprising a sugar chain exosome separation and enrichment module and a detection module.
23. The AD detection system of claim 22, wherein the sugar chain exosome separation and enrichment module performs separation and enrichment of sugar chain exosomes for AD detection by pre-loading sugar chain exosome separation and enrichment reagents.
24. The AD detection system according to claim 22, wherein the detection module detects and outputs a detection signal for the P-Tau-217 marker contained in the sugar chain exosomes isolated and enriched by claim 22 by means of a pre-packaged P-Tau-217 marker detection reagent.
25. The AD detection system according to claim 22, wherein the detection signal output by the detection module is directly used for detection and evaluation applications such as early detection of AD, and/or detection or diagnosis of AD, and/or monitoring of AD disease course, and/or evaluation of AD efficacy, and/or evaluation of AD prognosis.
26. The AD detection system according to claim 22, wherein the AD detection application of the sugar chain exosome P-Tau-217 marker comprises detection application using a full-automatic chemiluminescence immunoassay analyzer having the sugar chain exosome separation enrichment module and the detection module.
27. The application 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 disease detection reagent or a detection kit.
28. The use according to claim 27, wherein the sugar chain exosome separation and enrichment reagent comprises sugar chain exosome separation magnetic beads, a washing liquid, and an eluent.
29. The use according to claim 28, wherein the sugar chain exosome separation magnetic beads are lectin-magnetic carrier conjugate complexes.
30. The use according to claim 27, wherein the sugar chain exosome separation and enrichment reagent is useful for separating and enriching any one of an N-glycosylated exosome, an O-glycosylated exosome, and a fucosylated exosome for AD detection.
31. The use according to claim 30, 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.
32. The use of claim 31, wherein the N-glycosylated exosome separation enrichment reagent comprises any one or more lectins of ConA, PVL, SBA, PA ii, WGA, AAL.
33. The use of claim 27, wherein the P-Tau-217 detection reagent comprises a detection antibody and a detection magnetic bead, the detection antibody comprising a P-Tau-217 antibody, a Tau antibody.
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