A N-Tau and Reelin combined detection product and its application

Abstract

The present invention discloses a combined N-Tau and Reelin detection product and its application, belonging to the field of medical technology. The product contains magnetic beads coupled with N-Tau and Reelin capture antibodies and N-Tau and Reelin detection antibodies labeled with acridinium ester or alkaline phosphatase. The two magnetic beads are separately stored in a magnetic bead storage solution composed of 50mM Tris pH 7.4, 0.9% NaCl, 2% BSA, 2% sucrose, 0.05% Tween-20, and 0.1% Proclin 300. The present invention can detect AD early with high diagnostic efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an N-Tau and Reelin combined detection product and its application. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and cognitive impairment. Traditional AD diagnostic methods rely primarily on imaging, neuropsychological assessments, and cerebrospinal fluid testing, which can be inadequate for early diagnosis or can be highly invasive. Early diagnosis of AD facilitates early intervention and slows disease progression. Biochemical changes can occur up to 20 years before clinical symptoms appear, making blood biomarker testing a crucial tool for early AD diagnosis.

[0003] Tau protein is a highly soluble, naturally disordered protein belonging to the microtubule-associated protein family. It consists of four structural domains: an N-terminal region, a proline-rich region, a microtubule-binding region, and a C-terminal region. Neurofibrillary tangles formed by tau protein are one of the two major pathological hallmarks of AD, and the N-terminal region is closely associated with the formation of pathological tau aggregates. Studies have shown that plasma N-terminal tau (N-Tau) can predict tau aggregation, as well as neurodegeneration and cognitive impairment, and is also associated with abnormal β-amyloid protein. Therefore, plasma N-Tau can be used as a biomarker for the early diagnosis and monitoring of AD.

[0004] Reelin is an extracellular matrix serine tyrosine protease composed of 3461 amino acids. Reelin consists of three main components: the N-terminus is primarily composed of the F-spondin sequence and a typical signal peptide; the middle portion is a repeating sequence of eight Reelin domains; and the final portion is a small, highly conserved C-terminal sequence. The classic Reelin signaling pathways are the Reelin-cofolin pathway and the Reelin-Tau pathway, which regulate the cessation of neuronal migration in the brain and the migration and laminar formation of cortical neurons, respectively. Abnormal expression of Reelin leads to abnormal phosphorylation of Tau, which is closely associated with the development of Alzheimer's disease.

[0005] N-Tau and Reelin proteins are associated with the pathological changes in Alzheimer's patients and are significantly elevated in the blood. Combined measurement of N-Tau and Reelin can aid in the early diagnosis of Alzheimer's disease. Regular monitoring of these biomarkers can also track the progression of the disease and the effectiveness of therapeutic interventions.

[0006] Chemiluminescent immunoassay (CLIA) is a method of bioanalysis using chemiluminescence technology. It has the advantages of high sensitivity, high specificity and high throughput. It is widely used in clinical diagnosis and can be used to detect markers of various diseases. Currently, the main biomarkers for Alzheimer's disease detection based on hematology include Aβ40, Aβ42, T-tau, p-Tau181, p-Tau217, NFL, etc., and usually a single indicator is used, which has a certain probability of missed detection and false positive. N-Tau and Reelin are the first Alzheimer's disease biomarkers created in China, and they use a joint detection method. They have high sensitivity and specificity in distinguishing Alzheimer's patients from healthy people. At present, the National Medical Products Administration has not yet obtained relevant product registration information for chemiluminescence testing of N-Tau and Reelin proteins. Therefore, it is of great significance to develop a chemiluminescence detection method for N-Tau and Reelin. Summary of the Invention

[0007] One of the objectives of the present invention is to provide a combined N-Tau and Reelin detection product, which contains magnetic beads coupled to N-Tau and Reelin capture antibodies and acridinium ester-labeled N-Tau and Reelin detection antibodies.

[0008] Preferably, the product is a kit.

[0009] More preferably, the magnetic beads coupled to the N-Tau and Reelin capture antibodies are respectively stored in a magnetic bead storage solution, and the composition of the magnetic bead storage solution is as follows: 50mM Tris pH 7.4, 0.9% NaCl, 2% BSA, 2% sucrose, 0.05% Tween-20, 0.1% Proclin300.

[0010] More preferably, the N-Tau and Reelin detection antibodies are respectively stored in an acridinium ester storage solution, and the composition of the acridinium ester storage solution is as follows: 50 mM citric acid, 0.9% NaCl, 1% BSA, 0.1% Proclin 300, 0.05% Tween-20, pH 6.0.

[0011] A second object of the present invention is to provide a combined detection product for N-Tau and Reelin, which contains magnetic beads coupled with N-Tau and Reelin capture antibodies and alkaline phosphatase-labeled N-Tau and Reelin detection antibodies.

[0012] A third object of the present invention is to provide a reagent for detecting the concentration of N-Tau and Reelin in plasma and its use in the preparation of Alzheimer's disease diagnostic products.

[0013] Preferably, the reagents for detecting the concentrations of N-Tau and Reelin in plasma include magnetic beads coupled with N-Tau and Reelin capture antibodies and N-Tau and Reelin detection antibodies labeled with acridinium ester or alkaline phosphatase.

[0014] Preferably, the product is a kit.

[0015] More preferably, the method for using the kit is:

[0016] Place the test kit and the sample to be tested into the fully automatic chemiluminescence immunoassay analyzer. Use the instrument's sample needle to sequentially add 50uL of the test sample, 50uL of magnetic beads coupled to N-Tau or Reelin capture antibody, and 50uL of N-Tau or Reelin detection antibody labeled with acridinium ester or alkaline phosphatase into the reaction cup. Mix thoroughly and incubate for 30 minutes. After the reaction is complete, perform magnetic separation and cleaning, add 100uL of substrate, and then read the luminescence intensity value corresponding to each sample to be tested by the instrument's optical system. Calculate the concentration value based on the standard curve of the test kit.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a combined detection product of N-Tau and Reelin, which is suitable for the early detection of AD and has high diagnostic efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the N-Tau standard curve in Example 2.

[0020] Figure 2 This is the Reelin standard curve in Example 2.

[0021] Figure 3 This is the linear curve of N-Tau in Example 2.

[0022] Figure 4 This is the linear curve of Reelin in Example 2.

[0023] Figure 5 This is the AUC graph of the markers in Example 3. DETAILED DESCRIPTION

[0024] The N-Tau capture antibody used in the following examples was purchased from Xiamen Kangji Biotechnology with the catalog number KA0081; the Reelin capture antibody was purchased from biorbyt with the catalog number orb113311; the N-Tau detection antibody was purchased from Xiamen Kangji Biotechnology with the catalog number KA0082; the Reelin detection antibody was purchased from biorbyt with the catalog number orb238737; the N-Tau antigen was purchased from biorbyt with the catalog number orb392121; and the Reelin antigen was purchased from biorbyt with the catalog number orb754941.

[0025] Example 1 Preparation of the kit

[0026] 1. Solution preparation:

[0027] (1) Labeling buffer: PBS, pH 7.4.

[0028] (2) Coating buffer: 10 mM Tris, 1 M NaCl, 0.05% Tween-20, pH 7.4.

[0029] (3) Magnetic bead washing solution: PBS, 0.05% Tween-20, pH 7.4.

[0030] (4) Magnetic bead storage solution: 50 mM Tris pH 7.4, 0.9% NaCl, 2% BSA, 2% sucrose, 0.05% Tween-20, 0.1% Proclin 300.

[0031] (5) Labeling buffer: PBS, pH 7.4.

[0032] (6) Acridinium ester storage solution: 50 mM citric acid, 0.9% NaCl, 1% BSA, 0.1% Proclin 300, 0.05% Tween-20, pH 6.0.

[0033] 2. Preparation of magnetic beads coupled with N-Tau and Reelin capture antibodies:

[0034] Dissolve the 10mM NHS-LC-biotin solution in DMF and calculate the amount of biotin to be used (labeled antibody:biotin = 1:40) by molar ratio. Dilute the labeled antibody to a concentration of 2mg / mL with labeling buffer, add the calculated amount of biotin solution, mix thoroughly, and incubate at 25°C in the dark for 30 minutes with shaking. Desalt the antibody using a desalting spin column, using the labeling buffer as the purification buffer. The desalted biotin-labeled antibody is ready for use. Pipette 300nm streptavidin magnetic beads at a ratio of 1mg:40ug. Wash three times with coating buffer, add the desalted biotin-labeled antibody, rotate and coat at 25°C for 60 minutes. Wash the coated beads three times with magnetic bead washing buffer, store the washed beads in magnetic bead storage buffer, and store the stock solution at a concentration of 10mg / mL. Label the beads and store at 2-8°C until ready for use.

[0035] 3. Preparation of Acridinium Ester-labeled N-Tau and Reelin Detection Antibodies:

[0036] First, prepare a 1 mg / mL acridinium ester solution dissolved in DMF and calculate the amount of acridinium ester to be used based on a molar ratio (labeled antibody: acridinium ester = 1:20). Dilute the labeled antibody to a concentration of 1 mg / mL with labeling buffer, add the calculated amount of acridinium ester solution, mix thoroughly, and incubate at 25°C in the dark for 60 minutes with shaking. Finally, purify using a desalting spin column, using the labeling buffer as the purification buffer. Store the desalted acridinium ester-labeled antibody in acridinium ester storage buffer and dilute to 0.1 mg / mL. After labeling, store at 2-8°C until ready to use.

[0037] Example 2 Performance Evaluation

[0038] 1. Standard curve

[0039] N-Tau and Reelin antigens were diluted with standard diluent and prepared into calibrators of varying concentrations. The N-Tau and Reelin calibrators were tested using the kit described in Example 1, with each concentration tested twice and the average luminescence unit (RLU) calculated. Each calibrator and reagent were placed in a fully automated chemiluminescence immunoassay. The instrument automatically added 50 μL of calibrator, 50 μL of magnetic beads conjugated to an N-Tau or Reelin capture antibody, and 50 μL of acridinium ester-labeled N-Tau or Reelin detection antibody to the reaction cup. Mixing, incubation, and washing were performed. The luminescence intensity corresponding to each calibrator was then read. A standard curve was generated using concentration as the horizontal axis and the average luminescence intensity as the vertical axis.

[0040] N-Tau standard curve ( Figure 1 ):

[0041] Table 1

[0042] Concentration (pg / mL) RLU 0 145 5 1890 20 6366 100 28366 500 142978 2000 625232

[0043] Reelin standard curve ( Figure 2 ):

[0044] Table 2

[0045] Concentration (ng / mL) RLU 0 233 2 992 10 5041 50 26303 200 108510 1000 482873

[0046] 2. Linear

[0047] Dilute the high-value sample close to the upper limit of the linear range into at least 5 different concentrations according to a certain ratio, among which the low-value concentration sample must be close to the lower limit of the linear range. Repeat the test for each concentration sample 3 times, calculate the average value, and use the least squares method to perform a linear fit between the average value and the dilution ratio, and calculate the linear correlation coefficient (r). The linear correlation coefficient r should be ≥0.99. The results are shown in Figure 3 and Figure 4 .

[0048] 3. Repeatability

[0049] Use two plasma samples at different concentration levels (high and low), and test each concentration sample 10 times. Calculate the mean (M) and standard deviation (SD) of the 10 measurements. Then, calculate the coefficient of variation (CV) using the formula CV = SD / M × 100%. The CV should be within 8.0%.

[0050] Table 3

[0051]

[0052]

[0053] 4. Accuracy

[0054] Using two accuracy reference standards (N-Tau and Reelin) at different concentrations, perform three replicate measurements and calculate the relative deviation between the mean value (denoted as M) and the labeled value (denoted as T). Calculate the relative deviation (B) of the measured concentration using the formula B = (MT) / T x 100%. The relative deviation (B) should be ≤ 15%.

[0055] Table 4

[0056]

[0057] 5. Blank limit

[0058] The luminescence value (RLU) of the zero-concentration calibrator was measured in parallel 20 times, and the mean (M) and standard deviation (SD) were calculated to obtain the RLU value corresponding to M+2SD. A linear equation was obtained by performing a two-point regression fit based on the concentration-RLU between the zero-concentration calibrator and the adjacent calibrator. The RLU value of M+2SD was substituted into the above equation to obtain the corresponding concentration value, which is the blank limit.

[0059] N-Tau blank limit:

[0060] Table 5

[0061]

[0062]

[0063] Reelin blank limit:

[0064] Table 6

[0065]

[0066] Example 3 Early Diagnosis of Alzheimer's Disease

[0067] Based on clinical diagnosis, plasma samples were collected from 50 Alzheimer's patients and 50 healthy controls. N-Tau and Reelin concentrations in these samples were measured using the kit described in Example 1. Receiver-operating characteristic (ROC) curves were used to analyze the AUC, specificity, and sensitivity of the individual markers.

[0068] A logistic regression model was used to predict the occurrence of AD. The model formula is as follows:

[0069]

[0070] Where p is the probability of AD, N-Tau and Reelin are the detected concentrations, β0 is the natural constant obtained from regression, β1 and β2 are the regression coefficients for N-Tau and Reelin, respectively. Analysis with Medcalc software revealed β0, β1, and β2 to be -8.050, 0.304, and 0.028, respectively. Receiver-operating characteristic (ROC) curve analysis showed an AUC of 0.960, a specificity of 90%, and a sensitivity of 96%, surpassing the performance of single-marker tests.

[0071] Marker AUC, specificity, sensitivity:

[0072] Table 7

[0073] Landmark name AUC Specificity (%) Sensitivity (%) N-Tau 0.881 86 80 Reelin 0.879 78 88 N-Tau+Reelin 0.960 92 96

[0074] Marker AUC Figure 5 shown.

[0075] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A combined N-Tau and Reelin detection product, characterized in that: The product contains magnetic beads coupled with N-Tau and Reelin capture antibodies and acridinium ester-labeled N-Tau and Reelin detection antibodies.

2. The N-Tau and Reelin combined detection product according to claim 1, characterized in that: The product is a test kit.

3. The N-Tau and Reelin combined detection product according to claim 2, characterized in that: The magnetic beads coupled with the N-Tau and Reelin capture antibodies were respectively stored in a magnetic bead storage solution, the composition of which was as follows: 50 mM Tris pH 7.4, 0.9% NaCl, 2% BSA, 2% sucrose, 0.05% Tween-20, and 0.1% Proclin 300.

4. The N-Tau and Reelin combined detection product according to claim 3, characterized in that: The N-Tau and Reelin detection antibodies were respectively stored in an acridinium ester storage solution, and the composition of the acridinium ester storage solution was as follows: 50 mM citric acid, 0.9% NaCl, 1% BSA, 0.1% Proclin 300, 0.05% Tween-20, pH 6.

0.

5. The N-Tau and Reelin combined detection product according to claim 1, characterized in that: Alkaline phosphatase-labeled N-Tau and Reelin detection antibodies were used instead of acridinium ester-labeled N-Tau and Reelin detection antibodies.

6. Application of a reagent for detecting N-Tau and Reelin concentrations in plasma in the preparation of Alzheimer's disease diagnostic products The reagents for detecting the concentrations of N-Tau and Reelin in plasma include magnetic beads coupled with N-Tau and Reelin capture antibodies and N-Tau and Reelin detection antibodies labeled with acridinium ester or alkaline phosphatase.

7. The use according to claim 6, characterized in that The product is a test kit.

8. The use according to claim 7, characterized in that The method of using the kit is as follows: Place the test kit and the sample to be tested into a fully automatic chemiluminescence immunoassay analyzer. Use the instrument's sample needle to sequentially add 50µL of the test sample, 50µL of magnetic beads coupled to N-Tau or Reelin capture antibody, and 50µL of acridinium ester- or alkaline phosphatase-labeled N-Tau or Reelin detection antibody into the reaction cup. Mix thoroughly and incubate for 30 minutes. After the reaction is complete, perform magnetic separation and cleaning, add 100µL of substrate, and then read the luminescence intensity value corresponding to each sample to be tested using the instrument's optical system. Calculate the concentration value based on the standard curve of the kit.

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