N-Tau and Reelin joint detection product and application thereof

Abstract

The invention discloses an N-Tau and Reelin joint detection product and application thereof, and belongs to the technical field of medicines. The product contains magnetic bead particles coupled with N-Tau and Reelin capture antibodies and N-Tau and Reelin detection antibodies labeled by acridinium ester or alkaline phosphatase, the two magnetic bead particles are respectively preserved in a magnetic bead preserving fluid, and the magnetic bead preserving fluid comprises 50mM Tris with the pH value of 7.4, 0.9% of NaCl, 2% of BSA, 2% of sucrose, 0.05% of Tween-20 and 0.1% of Proclin300. According to the invention, early detection can be carried out on AD, and the diagnosis efficiency is very high.

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 application thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive dysfunction. Traditional AD diagnostic methods mainly rely on imaging examinations, neuropsychological assessments, and cerebrospinal fluid testing, which may not be able to make early diagnoses or are highly invasive. Early diagnosis of AD helps early intervention and delay disease progression, and biochemical changes occur 20 years before clinical symptoms appear, so the detection of blood biomarkers is an important tool for early diagnosis of AD.

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

[0004] Reelin protein is an extracellular matrix serine tyrosine protease composed of 3461 amino acids. Reelin protein mainly consists of three parts: the N-terminus is mainly composed of the F-Spondin sequence and a typical signal peptide; the middle is a repeating sequence of 8 Reelin domains; and the last is a small, highly conserved C-terminal sequence. The classic signaling pathways of Reelin are the Reelin-cofolin pathway and the Reelin-Tau pathway, which regulate the cessation of brain neuron migration and the migration of cortical neurons and the formation of laminar structures, respectively. Abnormal expression of Reelin protein can lead to abnormal phosphorylation of Tau protein, which is closely related to the occurrence of AD.

[0005] N-Tau and Reelin proteins are related to 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. At present, the biomarkers for hematological Alzheimer's disease detection mainly 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 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 State Food and Drug Administration has not yet obtained relevant product registration information for chemiluminescence testing of N-Tau and Reelin proteins, so it is of great significance to develop a chemiluminescence detection method for N-Tau and Reelin. Summary of the invention

[0007] One of the purposes of the present invention is to provide a N-Tau and Reelin combined 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 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] The second object of the present invention is to provide a N-Tau and Reelin combined detection product, which contains magnetic beads coupled to N-Tau and Reelin capture antibodies and N-Tau and Reelin detection antibodies labeled with alkaline phosphatase.

[0012] The 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 preparing a diagnostic product for Alzheimer's disease.

[0013] Preferably, the reagents for detecting the concentrations of N-Tau and Reelin in plasma include magnetic beads coupled to 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 sample to be tested, 50uL of magnetic beads coupled to N-Tau or Reelin capture antibodies, and 50uL of N-Tau or Reelin detection antibodies labeled with acridinium ester or alkaline phosphatase into the reaction cup. Mix thoroughly and incubate for 30 minutes. After the reaction is completed, 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, and 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 N-Tau and Reelin combined detection product, which is suitable for 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% Proclin300.

[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] Take out the 10mM NHS-LC-biotin solution dissolved in DMF, and calculate the amount of biotin by molar ratio (labeled antibody: biotin = 1:40). Dilute the concentration of the antibody to be labeled to 2mg / mL with labeling buffer, add the calculated amount of biotin solution, mix well, react at 25℃ in the dark for 30 minutes, use desalting centrifugal column for desalting, and the purification buffer is the labeling buffer. The desalted biotin-labeled antibody is for standby use. Pipette 300nm streptavidin magnetic beads at the ratio of magnetic beads: antibody = 1mg: 40ug, wash 3 times with coating buffer, add desalted biotin-labeled antibody, rotate and mix at 25℃ for 60 minutes, wash the coated magnetic beads 3 times with magnetic bead washing solution, and store the washed magnetic beads with magnetic bead storage solution. The concentration of magnetic bead stock solution is 10mg / mL. After labeling, store at 2-8℃ for standby use.

[0035] 3. Preparation of acridinium ester labeled N-Tau and Reelin detection antibodies:

[0036] First, take out the 1 mg / mL acridinium ester solution dissolved in DMF, and then calculate the amount of acridinium ester in molar ratio (labeled antibody: acridinium ester = 1:20). Dilute the concentration of the antibody to be labeled to 1 mg / mL with labeling buffer, add the calculated amount of acridinium ester solution, mix well, and react at 25°C in the dark for 60 minutes. Finally, use a desalting centrifugal column for purification, and the purification buffer is the labeling buffer. The desalted acridinium ester labeled antibody is stored in acridinium ester storage solution and diluted to 0.1 mg / mL. After labeling, store at 2-8°C for 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 different concentrations. The kit in Example 1 was used to detect N-Tau and Reelin calibrators. Each concentration was tested twice, and the average luminescence value (RLU) was calculated. Each concentration of calibrators and reagents were placed in a fully automatic chemiluminescence immunoassay analyzer. The instrument automatically added 50uL of calibrators, 50uL of magnetic beads coupled with N-Tau or Reelin capture antibodies, and 50uL of N-Tau or Reelin detection antibodies labeled with acridinium ester to the reaction cup, mixed, incubated and washed, and then the luminescence intensity values ​​corresponding to each calibrator were read respectively. The standard curve was obtained by fitting with the 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] The high-value samples close to the upper limit of the linear range are diluted to at least 5 concentrations according to a certain ratio, among which the low-value concentration samples must be close to the lower limit of the linear range. Each concentration sample is tested 3 times, and the average value is calculated. The average value and the dilution ratio are fitted with a linear least square method, and the linear correlation coefficient (r) is calculated. The linear correlation coefficient r should be ≥0.99. The results are shown in Figure 3 and Figure 4 .

[0048] 3. Repeatability

[0049] Use 2 plasma samples with different concentration levels (high value and low value), and repeat the test 10 times for each concentration sample. Calculate the mean (M) and standard deviation (SD) of the 10 measurement results, and then calculate the coefficient of variation (CV) according to the formula CV = SD / M × 100%, and the CV should be within ≤8.0%.

[0050] Table 3

[0051]

[0052]

[0053] 4. Accuracy

[0054] Using two accuracy references at the concentration levels of N-Tau and Reelin, repeat the measurement three times and calculate the relative deviation between the average value (denoted as M) and the labeled value (denoted as T). The relative deviation B of the measured concentration is calculated according to the formula B = (MT) / T × 100%, and the relative deviation B should be within ≤15%.

[0055] Table 4

[0056]

[0057] 5. Blank limit

[0058] The luminescence value (RLU) of the zero-concentration calibrator was measured 20 times in parallel, and its mean value (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] According to the clinical diagnosis results, 50 plasma samples from patients with Alzheimer's disease and 50 healthy control samples were collected. The concentrations of N-Tau and Reelin in the above samples were detected using the kit in Example 1. The AUC, specificity and sensitivity of a single marker were analyzed by ROC curve.

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

[0069]

[0070] Among them, p is the probability of AD occurrence, N-Tau and Reelin are the detected concentration values, β0 is the natural constant obtained by regression, β1 and β2 are the regression coefficients of N-Tau and Reelin, and the analysis of Medcalc software showed that β0, β1, and β2 were -8.050, 0.304, and 0.028, respectively. Through ROC curve analysis, the AUC value of the model was 0.960, the specificity was 90%, and the sensitivity was 96%, which was better than the performance of single indicator detection.

[0071] Marker AUC, specificity, sensitivity:

[0072] Table 7

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

[0074] The marker AUC is Figure 5 shown.

[0075] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

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

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 to the N-Tau and Reelin capture antibodies are respectively stored in magnetic bead storage solutions, and the composition of the magnetic bead storage solution is as follows: 50mMTris pH 7.4, 0.9% NaCl, 2% BSA, 2% sucrose, 0.05% Tween-20, and 0.1% Proclin300.

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 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. Use of a reagent for detecting the concentration of N-Tau and Reelin in plasma in the preparation of Alzheimer's disease diagnostic products.

7. The use according to claim 6, characterized in that: 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.

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

9. The use according to claim 8, characterized in that: The method of using the kit is as follows: 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 sample to be tested, 50uL of magnetic beads coupled to N-Tau or Reelin capture antibodies, and 50uL of N-Tau or Reelin detection antibodies labeled with acridinium ester or alkaline phosphatase into the reaction cup. Mix thoroughly and incubate for 30 minutes. After the reaction is completed, 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, and calculate the concentration value based on the standard curve of the test kit.

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