A combined diagnostic kit for Alzheimer's disease and its application

Abstract

This invention discloses a combined diagnostic kit for Alzheimer's disease and its application, belonging to the field of medical diagnostic technology. The kit includes a first reagent for detecting neurogranule protein (NRGN), a second reagent for detecting β-amyloid 42 (Aβ42), and a third reagent for detecting phosphorylated tau protein 217 (p-tau217). By jointly detecting the levels of these three biomarkers—NRGN, Aβ42, and p-tau217—and comprehensively evaluating them according to the criteria of NRGN < 100 pg / mL, Aβ42 < 800 pg / mL, and p-tau217 > 0.5 pg / mL, this invention significantly improves the sensitivity (95.8%) and specificity (92.2%) of Alzheimer's disease diagnosis, with an area under the ROC curve (AUC) of 0.956, superior to any single biomarker. The kit uses peripheral blood, urine, and cerebrospinal fluid as test samples, is non-invasive, convenient, and cost-effective, and is suitable for large-scale screening and clinical auxiliary diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of medical diagnostic technology, and in particular relates to a combined detection kit for Alzheimer's disease and its application. Background Technology

[0002] The core pathology of neurodegenerative diseases (such as Alzheimer's disease AD) is not only Aβ plaques and tau tangles, but also synaptic damage and loss, which are key early events and directly associated with cognitive decline. Currently used clinical biomarkers (Aβ42, tau, p-tau217) mostly reflect mid-to-late stage pathology, lack synaptic damage specificity, have insufficient early sensitivity, and are invasive due to the use of cerebrospinal fluid (CSF).

[0003] Neurogranin (NRGN, also known as RC3) is a calmodulin-binding protein specific to the postsynaptic compactum. It is highly expressed in the brain, primarily located in dendritic spines, and is released into the CSF and peripheral blood upon synaptic injury. Recent studies have shown that CSF-NRGN levels are significantly elevated in AD patients, preceding tau / Aβ abnormalities, and are positively correlated with cognitive decline, making it a highly specific biomarker for synaptic dysfunction. However, no patents disclose the systematic application, combined detection protocols, or matching kits of NRGN as a biomarker for broad-spectrum neurodegenerative diseases, and its diagnostic value in frontotemporal dementia, Parkinson's disease dementia, and ALS remains unclear. Summary of the Invention

[0004] To address the above technical problems, this invention provides a combined detection kit for Alzheimer's disease, the kit comprising: The first test reagent is used to detect the level of NRGN; The second test reagent is used to detect the level of Aβ42; The third test reagent is used to detect the level of p-tau217.

[0005] Preferably, the first detection reagent, the second detection reagent, and the third detection reagent are each independently selected from one of the following: ELISA detection reagent, chemiluminescent immunoassay reagent, flow cytometry detection reagent, fluorescent immunoassay reagent, colloidal gold immunochromatographic detection reagent, and latex immunochromatographic detection reagent.

[0006] More preferably, the determination rule for the reagent kit is: A positive result is defined as NRGN concentration <100 pg / mL, Aβ42 concentration <800 pg / mL, or p-tau217 concentration >0.5 pg / mL.

[0007] More preferably, the test sample is peripheral blood plasma.

[0008] More preferably, the test sample is cerebrospinal fluid or urine.

[0009] The present invention also provides the application of the above-mentioned first detection reagent, second detection reagent and third detection reagent in the preparation of Alzheimer's disease detection products.

[0010] Preferably, the product is a reagent kit.

[0011] More preferably, the first detection reagent, the second detection reagent, and the third detection reagent are each independently selected from one of the following: ELISA detection reagent, chemiluminescent immunoassay reagent, flow cytometry detection reagent, fluorescent immunoassay reagent, colloidal gold immunochromatographic detection reagent, and latex immunochromatographic detection reagent.

[0012] More preferably, the test sample is peripheral blood plasma.

[0013] More preferably, the test sample is cerebrospinal fluid or urine.

[0014] The detection of NRGN, Aβ42, and p-tau217 in the sample can be performed using any of the following methods: (1) Enzyme-linked immunosorbent assay (ELISA) A double-antibody sandwich ELISA was used: NRGN, Aβ42, and p-tau217 specific antibodies were used as coating antibodies and enzyme-labeled detection antibodies, and standard wells, sample wells, and quality control wells were set up. Incubation, washing, and color development were completed according to the kit procedure. The OD value was measured at 450 nm using an ELISA reader. A standard curve was plotted with the standard concentration as the x-axis and the OD value as the y-axis, and the concentrations of each marker in urine, plasma, and cerebrospinal fluid were calculated.

[0015] (2) Chemiluminescent immunoassay (CLIA) The double-antibody sandwich chemiluminescence method was used: the antibody was captured and coated with magnetic microparticles, and the antibody-labeled acridinium ester, luminol or horseradish peroxidase was detected; the sample was incubated with the magnetic microparticles and labeled antibody to form an immune complex; a luminescence initiation reagent was added, and the relative luminescence units (RLU) were detected by a chemiluminescence analyzer; the concentrations of each marker in urine, plasma and cerebrospinal fluid were quantitatively calculated according to the standard curve.

[0016] (3) Conventional fluorescence immunoassay The double-antibody sandwich fluorescence immunoassay was used: the capture antibody was coated on a solid-phase carrier, and the antibody-labeled FITC and Cy3 were detected; after the sample and antibody were fully bound, the fluorescence intensity was detected by a fluorescence immunoassay analyzer; the levels of NRGN, Aβ42, and p-tau217 in urine, plasma, and cerebrospinal fluid were quantified according to the standard curve.

[0017] (4) Time-resolved fluorescence immunoassay (TRFIA) A double-antibody sandwich time-resolved fluorescence assay was used: with europium (Eu) 3+ ), Terbium (Tb 3+ Lanthanide elements such as lanthanides are used as fluorescent markers to label detection antibodies; the captured antibodies are coated with a solid-phase carrier; after the immune reaction is completed, the fluorescence signal is detected by delay using a time-resolved fluorescence analyzer to eliminate background fluorescence interference; the concentration of each marker in urine, plasma and cerebrospinal fluid is quantified according to the standard curve.

[0018] (5) Immunoassay of fluorescent microspheres The fluorescent microsphere labeling immunoassay method is used: specific antibodies against NRGN, Aβ42, and p-tau217 are conjugated to the surface of fluorescently encoded microspheres or fluorescein-stained microspheres; after the sample binds to the fluorescent microsphere antibodies, the fluorescence signal of the microspheres is read by flow cytometry or fluorescence immunoassay analyzer; this method enables simultaneous quantitative detection of multiple indicators and is suitable for urine, plasma, and cerebrospinal fluid samples.

[0019] (6) Colloidal gold / latex immunochromatography The double-antibody sandwich immunochromatography method is adopted: the detection line is coated with specific capture antibodies against NRGN, Aβ42, and p-tau217, and the conjugate pad is sprayed with colloidal gold or latex-labeled detection antibodies; urine, plasma, and cerebrospinal fluid samples are added and subjected to capillary chromatography, and qualitative or semi-quantitative determination is made by the color intensity of the detection line.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The first public disclosure of the systematic application of NRGN as a marker for a broad spectrum of neurodegenerative diseases, covering AD, frontotemporal dementia, Parkinson's disease dementia, and ALS; (2) Elevated NRGN occurs earlier than Aβ / tau abnormalities and can be used for screening in the preclinical / mild cognitive impairment (MCI) stage; (3) NRGN specifically reflects synaptic damage and is directly related to cognitive decline, with a specificity ≥85%; (4) It can detect peripheral blood and urine, and the compliance rate is much higher than that of CSF; (5) The combined detection sensitivity of NRGN+Aβ42+p-tau217 is ≥92%, which is significantly better than that of a single marker; (6) The kit can be developed quickly, the cost is controllable, and it is suitable for large-scale screening. Attached Figure Description

[0021] Figure 1 This is the ROC curve from Example 1. Detailed Implementation

[0022] Example 1 I. Screening criteria for AD patients and control samples 1. Inclusion criteria for AD patients (1) Age 65-85, right-handed, primary school education or above; (2) Meets the 2011 National Institute on Aging and Alzheimer's Disease Association (NIA-AA) diagnostic criteria for AD, with a clinical stage of moderate AD; (3) MMSE score of 10-20, MoCA score <18, indicating clear memory and executive function impairment; (4) Head MRI / CT showed medial temporal lobe atrophy, without infarction, hemorrhage, tumor or other organic brain lesions; (5) No serious liver or kidney dysfunction, malignant tumors, autoimmune diseases, severe infections, or other diseases that may interfere with the detection; (6) No medications that affect central neurotransmitters or synaptic function were taken.

[0023] 2. Inclusion criteria for healthy controls (1) Age, gender, and education level were strictly matched with the AD group; (2) No chief complaint of cognitive decline, MMSE score ≥28, MoCA score ≥26; (3) No history of neurodegenerative diseases, mental illnesses, or cerebrovascular diseases; (4) No abnormalities were found in physical examination, laboratory tests, or cranial imaging.

[0024] II. Complete Experimental Process 1. Sample Collection and Processing (1) Collect peripheral venous blood from subjects on an empty stomach, let stand at room temperature for 30 min, centrifuge at 3000 r / min for 10 min, separate the plasma and store at -80℃ for later use; (2) Strict aseptic technique is used to collect cerebrospinal fluid (CSF) to avoid hemolysis. Centrifuge at 1500 r / min for 10 min, and store the supernatant at -80℃. Avoid repeated freeze-thaw cycles.

[0025] 2. Detection Method (1) Using the specific antibodies of NRGN, Aβ42, and p-tau217 of this invention, a double-antibody sandwich ELISA method was established for detection. The kit instructions were strictly followed, and standard wells, sample wells, and quality control wells were set up. The absorbance (OD value) was measured at a wavelength of 450 nm using an ELISA reader. A standard curve was plotted with the standard concentration as the x-axis and the OD value as the y-axis, and the concentration of each marker in the sample was calculated.

[0026] (2) Testing instruments and quality control Instrument: Fully automated enzyme-linked immunosorbent assay (ELISA) analyzer; Quality control: The CV value of the internal quality control products is <10%, and the intra-batch and inter-batch differences meet the clinical immunoassay testing standards.

[0027] III. Joint Testing and Judgment Criteria 1. Critical values ​​for individual biomarkers (determined based on ROC curves) The test samples were peripheral venous plasma. Based on the test data of the AD group (80 cases) and the healthy control group (70 cases), ROC curves of NRGN, Aβ42, and p-tau217 were plotted with the concentration of each biomarker as the test variable and the clinical diagnosis result as the state variable. The concentration value corresponding to the maximum Youden index (sensitivity + specificity - 1) was used as the optimal diagnostic cutoff value for each biomarker. The results are as follows: (1) NRGN < 100 pg / mL is positive (suggesting synaptic damage); (2) Aβ42 < 800 pg / mL is positive (suggesting Aβ plaque deposition); (3) p-tau217 > 0.5 pg / mL is positive (indicating tau protein hyperphosphorylation).

[0028] 2. Joint Judgment Rules If any one of the three tests is positive (NRGN < 100 pg / mL, Aβ42 < 800 pg / mL, or p-tau217 > 0.5 pg / mL), it strongly suggests AD; if all three tests are negative, AD is ruled out.

[0029] IV. Independent Queue Validation Results 1. Verification queue AD validation group: 60 cases (meeting the above NIA-AA criteria); Health verification group: 50 cases (age and gender matched).

[0030] 2. Verify the test results The combined detection showed a sensitivity of 95.8% and a specificity of 92.2%. ROC curve analysis showed an AUC of 0.956 and a 95% CI of 0.912–0.981, which was significantly better than that of single biomarkers (NRGN: AUC = 0.92; Aβ42: AUC = 0.81; p-tau217: AUC = 0.91).

[0031] 3. Verification Conclusion Independent cohort validation showed that the combined diagnostic criteria of peripheral venous blood plasma NRGN+Aβ42+p-tau217 are stable and accurate, and can be used for precise clinical detection of AD.

[0032] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A diagnostic kit for Alzheimer's disease, characterized in that, The kit includes: The first test reagent is used to detect the level of NRGN; The second test reagent is used to detect the level of Aβ42; The third test reagent is used to detect the level of p-tau217.

2. The combined detection kit according to claim 1, characterized in that, The first, second, and third detection reagents are each independently selected from one of the following: ELISA detection reagents, chemiluminescent immunoassay reagents, flow cytometry detection reagents, fluorescent immunoassay reagents, colloidal gold immunochromatographic detection reagents, and latex immunochromatographic detection reagents.

3. The combined detection kit according to claim 1 or 2, characterized in that, The determination rule for the reagent kit is as follows: A positive result is defined as NRGN concentration < 100 pg / mL, Aβ42 concentration < 800 pg / mL, or p-tau217 concentration > 0.5 pg / mL.

4. The combined detection kit according to claim 1 or 2, characterized in that, The test sample is peripheral blood plasma or urine.

5. The combined detection kit according to claim 1 or 2, characterized in that, The sample tested was cerebrospinal fluid.

6. The use of the first, second, and third detection reagents as described in claim 1 in the preparation of Alzheimer's disease detection products.

7. The application according to claim 6, characterized in that, The product in question is a reagent kit.

8. The application according to claim 6 or 7, characterized in that, The first, second, and third detection reagents are each independently selected from one of the following: ELISA detection reagents, chemiluminescent immunoassay reagents, flow cytometry detection reagents, fluorescent immunoassay reagents, colloidal gold immunochromatographic detection reagents, and latex immunochromatographic detection reagents.

9. The application according to claim 8, characterized in that, The test sample is peripheral blood plasma or urine.

10. The application according to claim 8, characterized in that, The sample tested was cerebrospinal fluid.

Images