Alzheimer's disease detection marker and application thereof

Abstract

The present application relates to a kind of Alzheimer's disease detection marker and its application, by detecting the p-Tau217 of exosome (plasma or urine source total exosome or nerve source specificity exosome), realize the noninvasive detection diagnosis of Alzheimer's disease.The kit of the present application has the advantages of noninvasive detection, high sensitivity, strong specificity, does not depend on large equipment, etc., solves the existing Alzheimer's disease detection kit sample acquisition invasiveness, low accuracy, large equipment dependence and poor precision etc.Problems, suitable for the needs of clinical large sample detection, can be used for early diagnosis of Alzheimer's disease, disease progression monitoring and efficacy evaluation etc..

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology and relates to an Alzheimer's disease detection biomarker and its application. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly. Its main pathological changes include diffuse atrophy of the cerebral cortex, neurofibrillary tangles, and the formation of a large number of senile plaques between nerve cells. Its main clinical symptoms are progressive cognitive impairment and memory decline.

[0003] Dementia is a human problem, and it is also a global problem.

[0004] Early diagnosis of Alzheimer's disease (AD) primarily relies on clinical history, imaging examinations, scale assessments, and CSF biomarker detection. Imaging examinations such as PET scans are not only expensive but also require sophisticated equipment, limiting their widespread availability in general hospitals. While psychological scale assessments are convenient, they are influenced by numerous factors, such as the subject's educational level, the assessor's subjective judgment, and environmental factors. CSF biomarkers like Aβ and tau have been validated for their high specificity and sensitivity, but their clinical application is limited by the invasiveness of lumbar puncture and the difficulty in obtaining samples. Plasma is more readily available than cerebrospinal fluid, facilitating clinical trials; however, the accuracy of studies on Aβ1-42 in plasma varies, possibly because Aβ1-42 in plasma originates from more than just the central nervous system. Detection of pTau181 or pTau217 in plasma relies on large single-molecule detection equipment due to their extremely low levels and lacks precision. Therefore, current detection technologies, due to their invasive sample acquisition, low accuracy, reliance on large equipment, and poor precision, are not suitable for widespread clinical application. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an Alzheimer's disease detection biomarker and its application, enabling non-invasive detection and diagnosis of Alzheimer's disease. It has advantages such as non-invasive detection, high sensitivity, strong specificity, and no reliance on large equipment. It solves the problems of existing Alzheimer's disease detection kits, such as invasive sample acquisition, low accuracy, reliance on large equipment, and poor precision. It is suitable for the needs of large-scale clinical sample testing and can be used for early diagnosis, disease progression monitoring, and efficacy evaluation of Alzheimer's disease.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. The application of p-Tau217 as a biomarker for Alzheimer's disease detection, characterized in that the p-Tau217 is derived from exosomes.

[0008] As one of the preferred technical solutions, the exosomes are total exosomes or neural-derived specific exosomes.

[0009] As a further preferred technical solution, the neural-derived specific exosomes are exosomes that express any one of the proteins CD171, GLAST, PLP1, and Ibal on the membrane surface.

[0010] As a further preferred technical solution, the exosomes are plasma exosomes or urinary exosomes.

[0011] As one of the preferred technical solutions, the Alzheimer's disease detection biomarkers are biomarkers for the diagnosis of Alzheimer's disease, and / or disease progression monitoring, and / or disease efficacy evaluation, and / or disease prognosis evaluation.

[0012] 2. A biomarker for Alzheimer's disease detection, characterized in that the biomarker is p-Tau217 derived from exosomes.

[0013] As one of the preferred technical solutions, the exosomes are total exosomes or neural-derived specific exosomes.

[0014] As a further preferred technical solution, the neural-derived specific exosomes are exosomes that express any one of the proteins CD171, GLAST, PLP1, and Ibal on the membrane surface.

[0015] As a further preferred technical solution, the exosomes are plasma exosomes or urinary exosomes.

[0016] 3. Application of the aforementioned biomarkers in the preparation of Alzheimer's disease detection agents.

[0017] 4. Detection reagents used to detect the aforementioned markers.

[0018] As one of the preferred technical solutions, the detection reagent includes p-Tau217 detection reagent and exosome enrichment reagent.

[0019] As a further preferred technical solution, the p-Tau217 detection reagent contains a detection antibody, which is a Tau antibody.

[0020] As a further preferred technical solution, the detection antibody also includes a p-Tau217 antibody.

[0021] As a further preferred technical solution, the detection antibody is labeled with acridine ester.

[0022] As one of the preferred technical solutions, the exosome enrichment reagent includes total exosome extraction reagent and / or neural-derived specific exosome capture reagent.

[0023] As a further preferred technical solution, the neural-derived specific exosome capture reagent comprises a solid-phase carrier labeled with one or more of the following: CD171 antibody, GLAST antibody, PLP1 antibody, and Ibal antibody.

[0024] As a further preferred technical solution, the detection reagent also includes a capture antibody, which is a biotin-labeled p-Tau217 antibody.

[0025] As one of the preferred technical solutions, the detection reagent further includes a pre-activation solution, an activation solution, and a washing solution.

[0026] 5. Application of the aforementioned detection reagents in the preparation of Alzheimer's disease detection reagents or kits.

[0027] 6. An Alzheimer's disease detection kit containing the aforementioned detection reagent.

[0028] 7. An Alzheimer's disease detection system, the system comprising: a sample processing module, a diagnostic module, and an information output module.

[0029] As one of the preferred technical solutions, the diagnostic module is used to collect exosome p-Tau217 detection signals.

[0030] As one of the preferred technical solutions, the sample processing module is used for exosome enrichment, namely total exosome extraction and / or neural-derived specific exosome capture, wherein the neural-derived specific exosomes are captured by a solid-phase carrier labeled with one or more of CD171 antibody, GLAST antibody, PLP1 antibody, and Ibal antibody.

[0031] As one of the preferred technical solutions, the information output module is used to output the results of Alzheimer's disease diagnosis, and / or disease progression monitoring, and / or disease efficacy evaluation, and / or disease prognosis evaluation.

[0032] The beneficial effects of this invention are as follows:

[0033] This invention provides an Alzheimer's disease detection kit that achieves non-invasive detection and diagnosis of Alzheimer's disease by detecting p-Tau217 derived from exosomes (total exosomes from plasma or urine, or nerve-derived specific exosomes). The kit offers advantages such as non-invasive detection, high sensitivity, strong specificity, and no reliance on large equipment. It addresses the problems of invasive sample acquisition, low accuracy, reliance on large equipment, and poor precision in existing Alzheimer's disease detection kits, making it suitable for the needs of large-scale clinical sample testing. It can be used for early diagnosis, disease progression monitoring, and efficacy evaluation of Alzheimer's disease.

[0034] The specific analysis is as follows:

[0035] (1) The present invention has the advantages of minimal trauma, high acceptability and low difficulty in obtaining human plasma or urine samples.

[0036] (2) In this invention, after capturing nerve-derived specific exosomes on magnetic beads, the exosomes are not eluted but directly lysed, thus avoiding the damage that polar elution reagents may cause to the integrity of exosome particles and exosome markers.

[0037] (3) The present invention detects plasma neurogenic exosome marker pTau217, specifically captures it with high tissue specificity and pTau217 has a very high disease association, so the detection results have higher accuracy; at the same time, the marker abundance is high, and it does not rely on large single-molecule equipment for detection. It is not only highly adaptable, but also has good precision and simple operation, making it suitable for large-scale detection of clinical samples.

[0038] (4) This invention can be used for early diagnosis of Alzheimer's disease, monitoring of disease progression and evaluation of treatment efficacy. Attached Figure Description

[0039] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0040] Figure 1 When p-Tau217 antibody and Tau antibody mixed antibody are used as detection antibodies, the sample detection results of the p-Tau217 detection system are shown.

[0041] Figure 2 The test results of the p-Tau217 detection system when Tau antibody is used as the detection antibody;

[0042] Figure 3 The results show the detection of p-Tau217 in total exosome-derived plasma samples.

[0043] Figure 4 The results show the detection of p-Tau217 in plasma samples;

[0044] Figure 5 The results show the detection of p-Tau217 in total exosome-derived urine samples.

[0045] Figure 6 The results show the detection of p-Tau217 in urine samples derived from nerve-derived exosomes.

[0046] Figure 7 The ROC curve of the kit for sample detection;

[0047] Figure 8 The kit provides detection results for different types of sample concentrations.

[0048] Figure 9 Clinical performance comparison of p-Tau217 from CD171-positive neural-derived exosomes;

[0049] Figure 10 Clinical performance comparison of p-Tau217 from neurogenic exosomes that are positive for GALST;

[0050] Figure 11 Clinical performance comparison of p-Tau217 in PLP1-positive neural-derived exosomes;

[0051] Figure 12 Clinical performance comparison of p-Tau217, a neurogenic exosome that is positive for Iba1. Detailed Implementation

[0052] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0053] Example 1

[0054] This embodiment describes an Alzheimer's disease detection kit, which includes the following components:

[0055] Total exosome extraction reagent: This reagent comprises three components: a CL-6B packing column, a column washing buffer (phosphate buffer), and an exosome extraction buffer (phosphate buffer). It is used to extract total exosomes from plasma samples.

[0056] Neural-derived specific exosome capture reagent: contains three components, namely biotinylated antibody, streptavidin magnetic beads, and magnetic bead washing solution.

[0057] Exosome lysis reagents consist of one component, the main component of which is a solution containing surfactants such as Triton X-100. Exosome lysis reagents are used for the lysis of total exosomes or tissue-specific exosomes.

[0058] The p-Tau217 detection reagent includes streptavidin-conjugated magnetic beads, a biotin-labeled capture antibody, a calibrator, an acridinium ester-labeled detection antibody, a pre-activation solution, an activation solution, and a washing solution. The capture antibody is a p-Tau217 antibody, and the detection antibody is a Tau antibody.

[0059] Example 2

[0060] This embodiment describes the method for using an Alzheimer's disease detection kit to extract total exosomes from plasma samples, capture and lyse neural-derived specific exosomes, and detect p-Tau217. Specific operational steps include:

[0061] Plasma pretreatment: Remove plasma from the -80℃ freezer, thaw 2 mL of each plasma sample rapidly, centrifuge at 800g for 10 min at room temperature, collect the supernatant, and then centrifuge at 15000g for 10 min. Add 0.6 mL of prothrombin-D and incubate at room temperature for 60 min. Add 1.4 mL of 0.1M phosphate buffer containing a 1X protease inhibitor complex (Shanghai Sangon Biotech, C600387). Centrifuge at 3000g for 20 min at 4℃. Collect the supernatant after centrifugation.

[0062] Total exosome extraction: The separation column was cleaned with column washing buffer. 4 mL of the buffer was loaded onto the column, allowing the solution to remain in the column. This process was repeated three times. The processed plasma sample was then loaded onto the separation column, and 2 mL of the flow-through was collected as total exosomes.

[0063] Neural-derived specific exosome capture: Streptavidin magnetic beads were washed three times with magnetic bead washing buffer and set aside. 2 μL of biotinylated CD171 antibody was added to 2 mL of total exosome solution and incubated at 4°C for 1 hour. Then, streptavidin magnetic beads were added and incubated at 4°C for 30 min. The magnetic beads were then washed three times with magnetic bead washing buffer.

[0064] Exosome lysis: 0.5 mL of exosome lysis buffer was added to the captured exosomes, and the mixture was incubated at 37 °C for 10 min and vortexed for 10 seconds to completely lyse the exosomes.

[0065] p-Tau217 detection: A fully automated chemiluminescence analyzer was used as the detection tool. The method was a double-antibody sandwich method, in which 50 μL of biotin-labeled capture antibody (capture antibody and biotin (Thermo Scientific, 21343) at a 1:20 molar ratio were reacted at 25℃ for 1 h, followed by dialysis to remove free biotin), 50 μL of sample (calibrator or exosome lysis buffer sample), and 50 μL of acridinium ester-labeled detection antibody (detection antibody and acridinium ester (MTW-CLIA-001) at a 1:20 molar ratio were reacted at 25℃ for 1 h, followed by dialysis to remove free acridinium ester) were added to the instrument. After reacting for 15 min, 20 μL of streptavidin magnetic beads were added and reacted for 5 min. Magnetic separation was performed and the sample was washed 3 times. 100 μL of chemiluminescent substrate pre-excitation solution (Abbott, 6E23-83) and 100 μL of chemiluminescent substrate excitation solution (Abbott, 6C55-83) were added sequentially to induce a luminescence reaction. The luminescence intensity was recorded, and the p-Tau217 content of the sample was calculated based on the calibration curve. The linear range of the calibration curve was 7.8125–500 pg / mL.

[0066] Example 3

[0067] Validation of the discriminative power of different paired antibody combinations in the p-Tau217 detection system: Thirty AD plasma samples and 30 healthy plasma samples were collected. Total exosomes were extracted, and neural-derived specific exosomes were captured and lysed according to the method described in Example 2. The detection method for p-Tau217 was the same as described in Example 2, except that the capture antibody was still p-Tau217 antibody, and the detection antibody was used in two ways. Method 1: A mixed antibody of p-Tau217 antibody and Tau antibody (preferred) was used as the detection antibody; Method 2: Only Tau antibody was used as the detection antibody (the method described in Example 2). The detection results of 60 samples are shown in Table 1. The ROC curve analysis of the mixed antibody combination of p-Tau217 antibody and Tau antibody in Method 1 showed an AUC of 0.996. Figure 1 The ROC of method two is 0.989, see... Figure 2 Both studies showed good differentiation between the AD group samples and the healthy group samples.

[0068] Table 1. Detection results of samples with different antibody combinations in the p-Tau217 detection system.

[0069]

[0070]

[0071] Example 4

[0072] Comparison of p-Tau217 discrimination among samples from different sources: Thirty paired plasma and urine samples from AD patients and 30 paired plasma and urine samples from healthy individuals were collected. Total exosome p-Tau217 in plasma, neurogenic exosome p-Tau217 in plasma, p-Tau217 in undiluted plasma, total exosome p-Tau217 in urine, and neurogenic exosome p-Tau217 in urine were detected, and the discrimination of p-Tau217 among samples from different sources was compared.

[0073] The detection method for total plasma exosomes p-Tau217 followed the sample pretreatment and total exosome extraction method described in Example 2. After extraction, the total exosomes were concentrated to 0.25 mL using a 100 kD ultrafiltration tube, and 0.25 mL of lysis buffer was added for lysis. The lysed exosomes were directly used as samples for detection as described in Example 2 using the p-Tau217 detection method. The detection results from 30 AD samples and 30 healthy samples are shown in Table 2. ROC curve analysis was performed, and the results are as follows: Figure 3 As shown, the AUC is 0.956.

[0074] The detection method for plasma neural-derived exosomes p-Tau217 was performed according to the method described in Example 2, including total exosome extraction, neural-derived specific exosome capture, lysis, and p-Tau217 detection. The results from 30 AD samples and 30 healthy controls are shown in Table 2. ROC curve analysis was performed, and the results are as follows: Figure 2 As shown, the AUC is 0.989.

[0075] For the detection of p-Tau217 in plasma samples, following the chemiluminescence method described in Example 2, 50 μL of plasma sample was directly used as the sample for p-Tau217 detection without any other pretreatment. Alternatively, an equal volume of 50 μL plasma sample was sent to a service company for detection using highly sensitive single-molecule immunoassay. The results of the detection in 30 AD samples and 30 healthy samples are shown in Table 2. The results showed that p-Tau217 in the plasma samples was below the detection limit and could not be detected by chemiluminescence. ROC curve analysis of the results from the single-molecule detection device yielded the following results: Figure 4 As shown, the AUC is 0.868.

[0076] The detection method for total exosomes p-Tau217 in urine was performed as follows: 20 mL of urine sample was concentrated to 2 mL of urine concentrate using a 100 kD ultrafiltration tube. Total exosome extraction was performed according to the method described in Example 2, with the only difference being that plasma was replaced with urine concentrate. After extraction, the total exosomes were concentrated to 0.25 mL using a 100 kD ultrafiltration tube, and 0.25 mL of lysis buffer was added for lysis. The lysed exosomes were directly used as samples for detection using the p-Tau217 detection method described in Example 2. The detection results from 30 AD samples and 30 healthy samples are shown in Table 2. ROC curve analysis was performed, and the results are as follows: Figure 5 As shown, the AUC is 0.944.

[0077] The detection method for p-Tau217 in urinary neurodegenerative exosomes was performed as follows: 20 mL of urine sample was concentrated to 2 mL of urine concentrate using a 100 kD ultrafiltration tube. Total exosome extraction, neurodegenerative exosome capture, lysis, and p-Tau217 detection were performed according to the method described in Example 2, with the only difference being that plasma was replaced with urine concentrate. The detection results from 30 AD samples and 30 healthy samples are detailed in Table 2. ROC curve analysis was performed, and the results are as follows: Figure 6 As shown, the AUC is 0.966.

[0078] The results of the tests on 60 samples are shown in Table 2. ROC analysis showed that the AUC of total plasma exosomes was 0.956, that of plasma neurally derived exosomes was 0.989, that of total urine exosomes was 0.944, and that of urine neurally derived exosomes was 0.966. The AUCs of all four sample types were higher than those of the undiluted plasma sample (0.868). Therefore, exosomes from different sources, whether total or neurally derived, showed superior clinical performance compared to the undiluted plasma sample.

[0079] Table 2. Detection results of p-Tau217 in samples from different sources.

[0080]

[0081]

[0082]

[0083] Example 5

[0084] A study on reference values ​​for an Alzheimer's disease detection kit: Plasma samples from 50 AD patients, plasma samples from 80 healthy individuals, and plasma samples from 20 individuals with other types of dementia (including vascular dementia, Lewy body dementia, frontotemporal dementia, and Parkinson's disease dementia) were collected from hospitals. Plasma neurodegenerative exosomes pTau217 were detected according to the method described in Example 2. Figure 7 The ROC curves plotted based on the test results are shown. The cutoff value of the kit was determined to be 51.62 pg / mL based on the Youden index of the sample test results. Under these conditions, the specificity was 100% and the sensitivity was 96.7%, as shown in Table 3. The area under the ROC curve was 0.994, indicating high diagnostic value.

[0085] Table 3 Statistical indicators of the reagent kit

[0086] Sample size Reference value Sensitivity Specificity 150 cases >51.62 pg / mL 96.7％ 100％

[0087] Example 6

[0088] Clinical performance validation of an Alzheimer's disease (AD) detection kit: 150 plasma samples from different stages of AD were collected from a hospital, including 100 samples from the dementia stage and 50 samples from the MCI stage; 200 plasma samples from healthy individuals and 50 plasma samples from other types of dementia (including vascular dementia, Lewy body dementia, frontotemporal dementia, and Parkinson's disease dementia). The detection was performed according to the method described in Example 2.

[0089] Sample concentration detection results are as follows Figure 8 As shown, the concentration of biomarkers in samples from the Alzheimer's disease (AD) stage is significantly higher than that in samples from the Mild Cognitive Impairment (MCI) stage, and the concentration in samples from the MCI stage is higher than that in healthy individuals and other types of dementia. The biomarker concentrations show an increasing trend with disease progression, suggesting that this kit has significant potential for disease monitoring.

[0090] Statistical indicators are shown in Table 4. The kit's detection rate for MCI stage samples was 88%, for dementia stage samples it was 97%, the specificity for healthy samples was 100%, and the specificity for other types of dementia samples was 100%. These statistical indicators demonstrate that the kit not only has extremely high specificity for detecting dementia in healthy individuals and other types of dementia, and high sensitivity for samples in the AD dementia stage, but also a high detection rate for early AD (MCI stage). This indicates that the kit has high value in disease diagnosis, especially in early diagnosis.

[0091] Table 4. Statistical indicators of the kit for different types of samples.

[0092]

[0093] Example 7

[0094] Validation of the consistency between Alzheimer's disease detection kit results and amyloid PET imaging results: Plasma samples with amyloid PET imaging results at different stages were collected, including 60 plasma samples from AD patients (20 in the MCI stage and 40 in the dementia stage), 50 plasma samples from healthy individuals, and 10 plasma samples from patients with other types of dementia (including vascular dementia, Lewy body dementia, frontotemporal dementia, and Parkinson's disease dementia). The detection was performed according to the method described in Example 2, using 51.62 pg / mL as the cutoff value. The consistency between the kit results and the amyloid PET imaging results was analyzed. Statistical results are shown in Table 5: the positive concordance rate was 98.4%, the negative concordance rate was 100%, and the overall concordance rate was 99.2%. The results indicate that the Alzheimer's disease detection kit results have a high consistency with the amyloid PET imaging results, and the kit's detection results may be able to characterize brain amyloid deposition.

[0095] Table 5. Statistics of reagent kit detection results and amyloid PET imaging results

[0096]

[0097] Example 8

[0098] Comparison of exosomes from different neural origins: Plasma samples from 30 AD patients and plasma samples from 30 healthy individuals were collected. Total exosomes were extracted, neural-derived specific exosomes were captured and lysed, and p-Tau217 was detected according to the method described in Example 2. Subsequently, three other types of neurally derived exosomes were captured and their biomarkers were detected as described in Example 2, with the difference being that the biotinylated CD171 was replaced with biotinylated GALST antibody (GLAST antibody (Thermo Scientific, MA5-38203) and biotin (Thermo Scientific, 21343) reacted at a 1:20 molar ratio at 25°C for 1 h, followed by dialysis to remove free biotin), biotinylated PLP11 antibody (PLP1 antibody (Thermo Scientific, MA5-37605) and biotin (Thermo Scientific, 21343) reacted at a 1:20 molar ratio at 25°C for 1 h, followed by dialysis to remove free biotin), and biotinylated Iba1 antibody (Iba1 antibody (Thermo Scientific, MA5-43700) and biotin (Thermo Scientific, 21343) reacted at a 1:20 molar ratio at 25°C for 1 h, followed by dialysis to remove free biotin). The results of ROC curve analysis of exosomes from four different neural cell sources are as follows: Figure 9-12 As shown, the AUC of CD171-positive neural-derived exosomes was 0.991 ( Figure 9 The AUC of GALST-positive neural-derived exosomes was 0.963. Figure 10 The AUC of PLP1-positive neurogenic exosomes was 0.973. Figure 11 The AUC of Iba1-positive neurogenic exosomes was 0.967. Figure 12 All four different types of neurogenic exosomes showed good differentiation between AD group samples and healthy human samples.

[0099] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. Use of an Alzheimer's disease detection marker in the manufacture of an Alzheimer's disease detection reagent, characterized in that, The marker is p-Tau217 protein obtained after cleavage treatment of exosomes, the exosomes are total exosomes or nerve-derived specific exosomes, the exosomes are plasma exosomes or urine exosomes.

2. Use according to claim 1, characterized in that, The nerve-derived specific exosomes are exosomes in which any one of membrane surface CD171, GLAST, PLP1, Ibal is expressed.

3. Use according to any one of claims 1-2, characterized in that, The reagent comprises a p-Tau217 detection reagent, an exosome enrichment reagent and an exosome cleavage reagent.

4. Use according to claim 3, characterized in that, The p-Tau217 detection reagent comprises a detection antibody, which is a Tau antibody.

5. Use according to claim 4, characterized in that, The detection antibody further comprises a p-Tau217 antibody.

6. Use according to claim 4 or 5, characterized in that, The detection antibody is labeled with acridinone.

7. Use according to claim 3, characterized in that, The exosome enrichment reagent comprises a total exosome extraction reagent and / or a nerve-derived specific exosome capture reagent.

8. Use according to claim 7, characterized in that, The nerve-derived specific exosome capture reagent comprises a solid phase carrier labeled with one or more of CD171 antibody, GLAST antibody, PLP1 antibody, Ibal antibody.

9. Use according to claim 3, characterized in that, The reagent further comprises a pre-priming solution and a priming solution and a washing solution.

10. An Alzheimer's disease detection kit, characterized by, The kit is composed of the reagents contained in the application of any one of claims 3-9. The kit is composed of the reagents contained in the application of any one of claims 3-9.

Images