Magnetic particle chemiluminescence detection kit for detecting human non-phosphorylated Tau217 protein and preparation method thereof

Abstract

The invention discloses a magnetic particle chemiluminiscence detection kit for human non-phosphorylated Tau217 protein and a preparation method of the magnetic particle chemiluminiscence detection kit. The kit comprises a micro magnetic particle coupled with a human non-phosphorylated Tau217 protein capture antibody, an acridinium ester labeled human non-phosphorylated Tau217 protein detection antibody, a human non-phosphorylated Tau217 protein calibration product and a quality control product. The direct chemiluminescence method established by the invention has the advantages of high sensitivity, strong specificity, accuracy, rapidness and short detection time, the detection result has higher accuracy and repeatability, and the kit can be used for various luminescence detection instruments.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay analysis, and specifically relates to a magnetic particle chemiluminescence detection kit for human non-phosphorylated Tau217 protein and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a common progressive neurodegenerative disease in the elderly, with memory impairment, cognitive decline and abnormal behavior as the main clinical features, accounting for 60%-80% of all cognitive impairments in the elderly. AD is a continuous disease spectrum, including preclinical AD, AD-induced mild cognitive impairment (MCI), and AD-induced dementia. Clinical diagnosis is complex, and commonly used detection methods include molecular positron emission tomography (PET) and cerebrospinal fluid biomarker detection. Molecular PET detection is expensive, and only a few hospitals have the equipment and can carry out it, with poor clinical accessibility; cerebrospinal fluid biomarker detection requires lumbar puncture sampling, patient compliance is cross-cutting, and it may cause infection. The above two methods are neither suitable for early screening nor for continuous detection of the disease and concomitant diagnosis of medication. Therefore, it is still necessary to seek an objective, reliable, simple, cheap, easy to follow-up and continuous observation method as a supplementary means. At the same time, abnormal Tau protein phosphorylation levels, such as increased levels of Tau protein phosphorylated at amino acid position 181 (p-Tau181) and Tau protein phosphorylated at amino acid position 217 (p-Tau217), can effectively predict the incidence of AD and provide a basis for diagnosis, and are a hot topic in current clinical research and diagnostic biomarker research.

[0003] The main pathological characteristics of AD are Aβ deposition to form neuritic plaques and neurofibrillary tangles caused by abnormal phosphorylation of Tau protein. Tau protein is the most abundant microtubule-associated protein in neurons. The cellular function of Tau in normal neurons is to bind to tubulin to promote the formation of microtubules and maintain their stability. p-Tau217 is a specific tau protein phosphorylation marker, which refers to the form of phosphorylation at amino acid position 217 of tau protein. Studies have shown that p-Tau217 is significantly increased in the brain, cerebrospinal fluid and plasma of patients with Alzheimer's disease. It can not only be used for early detection of the disease, but also to assess the degree of cognitive decline. np-Tau217 refers to the state in which tau protein is not phosphorylated at amino acid position 217. Unlike p-Tau217, np-Tau217 has relatively good function and stability in the nervous system and can effectively bind to microtubules, thereby supporting the structure and function of nerve cells. The level of np-Tau217 can understand the health status of normal neurons. By comparing p-Tau217 and np-Tau217, the health status of neurons, disease progression and efficacy of medication can be more comprehensively evaluated clinically.

[0004] So far, the methods used to detect human non-phosphorylated Tau217 protein (np-pTau217) are mainly mass spectrometry. However, the detection methods for related Alzheimer's disease biomarkers include: single molecule array technology (SimoA), direct chemiluminescence, enzymatic chemiluminescence, and electrochemiluminescence. WO2019199871A1 disclosed a detection kit for detecting human neurofilament light chain protein in 2019, specifically a single molecule immunoarray detection technology for detecting NfL, a marker related to Alzheimer's disease and other progressive neurodegenerative diseases. The main advantage of this kit is ultra-high sensitivity, but the main disadvantages are cumbersome operation, lengthy process, more prone to large CV, low throughput, and high price. It can neither meet the needs of rapid detection in large hospitals nor the needs of patients with controllable detection costs. There are currently no commercial methods and diagnostic reagents for np-pTau217. Therefore, it is of great significance to establish an effective, rapid, simple, highly sensitive and highly specific method for detecting human non-phosphorylated Tau217 protein (np-Tau217). Summary of the invention

[0005] The object of the present invention is to provide a magnetic particle chemiluminescence detection kit for human non-phosphorylated Tau217 protein (np-Tau217) with high sensitivity, short reaction time, simple operation and high specificity and a preparation method thereof.

[0006] To achieve the above object, the present invention adopts the following technical solution: A capture antibody for human non-phosphorylated Tau217 protein, wherein the heavy chain amino acid sequence of the capture antibody is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2; the capture antibody is a monoclonal antibody.

[0007] A detection antibody for human non-phosphorylated Tau217 protein, wherein the heavy chain amino acid sequence of the detection antibody is shown in SEQ ID NO: 3, the light chain amino acid sequence is shown in SEQ ID NO: 4, and the detection antibody is a monoclonal antibody.

[0008] A magnetic particle chemiluminescence detection kit for human non-phosphorylated Tau217 protein, the kit comprising a magnetic particle suspension of human non-phosphorylated Tau217 protein capture antibody, an acridinium ester-labeled human non-phosphorylated Tau217 protein detection antibody, a quality control product and a standard product; the magnetic particle suspension of human non-phosphorylated Tau217 protein capture antibody comprises a capture antibody and magnetic particles, and the capture antibody can bind to the 217 amino acid site of human non-phosphorylated Tau protein.

[0009] The amino acid sequence of the heavy chain of the capture antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 2; the capture antibody is a monoclonal antibody.

[0010] The amino acid sequence of the heavy chain of the above detection antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain is shown in SEQ ID NO: 4. The detection antibody is a monoclonal antibody.

[0011] The concentration of the capture antibody is 0.1-2.0 mg / mL; the concentration of the detection antibody is 0.1-0.6 mg / L.

[0012] The inner core of the magnetic particle is ferroferric oxide or ferrous oxide, and the surface modification groups of the magnetic particle are one or more of carboxyl, amino, p-toluenesulfonyl or synavidin.

[0013] The micromagnetic particles are directly coupled to the capture antibody, or the magnetic particles are coupled to streptavidin, and the capture antibody is labeled with biotin.

[0014] The molar ratio of the acridinium ester to the human non-phosphorylated Tau217 protein detection antibody is 100:1-10:1; the acridinium ester is NSP-DMAE-NHS or NSP-SA-NHS.

[0015] The calibrator and quality control product are prepared by adding a pure human non-phosphorylated Tau217 protein to a buffer containing 0.1-10% BSA, and the buffer is one of PBS, Tris-NaCl, MSE, and HEPES.

[0016] The concentrations of the calibration solutions are 0, 100, 500, 1000, and 2000 pg / mL, respectively.

[0017] The concentrations of the quality control solutions are 100 and 500 pg / mL, respectively.

[0018] The preparation method of the magnetic particle chemiluminescence detection kit for detecting non-phosphorylated Tau217 protein comprises the following steps: (1) Prepare a suspension of magnetic microparticles coupled with capture antibodies: evenly mix the magnetic microparticles and capture antibodies and incubate at room temperature for 30 min; then add the coupling reagent EDC and incubate at room temperature for 2 h, and finally add the blocking solution and incubate at room temperature for 1 h. After the incubation, discard the supernatant; (2) Preparation of acridinium ester-labeled human non-phosphorylated Tau217 protein detection antibody reagent: Place the human non-phosphorylated Tau217 protein detection antibody in a dialysis bag for dialyzing. After the dialysis, react with acridinium ester at room temperature for 1 hour. After adding BSA or glycine to terminate the reaction, separate the labeled substance and free acridinium ester by chromatography column.

[0019] A method for detecting human non-phosphorylated Tau217 protein for non-diagnostic purposes, using the kit according to claim 1 to detect human non-phosphorylated Tau217 protein in a sample.

[0020] The principle of the present invention is to combine the high specificity of antibody-antigen reaction with the high sensitivity of acridinium ester luminescence, and use acridinium ester to capture the photons generated by the reaction to detect the concentration of the product.

[0021] The beneficial effects of the present invention are: (1) The present invention uses a double antibody sandwich method combined with magnetic particle chemiluminescence technology to determine the content of human non-phosphorylated Tau217 protein (np-Tau217).

[0022] (2) Acridinium esters have obvious advantages as markers in direct chemiluminescence. The reaction does not require a catalyst and can proceed in an alkaline environment. The reaction is rapid and can completely capture the photons generated by the reaction. The background luminescence is low, the signal-to-noise ratio is high, there are few interference factors, the reagent stability is good, the system is simple, and the cost of the excitation solution is low. Acridinium esters are easy to bind to proteins, and the photon yield does not decrease after binding.

[0023] The magnetic microparticle chemiluminescence established by the present invention has high sensitivity, strong specificity, accuracy and rapidity, short detection time, and the detection results have higher accuracy and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the linear range of the magnetic microparticle chemiluminescent detection kit for human non-phosphorylated Tau217 protein. DETAILED DESCRIPTION

[0025] In order to make the technical solutions and advantages of the present invention more clear, the invention contents of the present invention are described in detail through the following examples, but they should not be regarded as limiting the scope of the present invention.

[0026] The methods used in the implementation of the following cases are conventional methods unless otherwise specified, and the materials used are commercially available unless otherwise specified.

[0027] The present invention provides a magnetic particle chemiluminescence detection kit for human non-phosphorylated Tau217 protein (np-Tau217) and a preparation method thereof. The kit for detecting human non-phosphorylated Tau217 protein (np-Tau217) by a micromagnetic particle chemiluminescence method provided by the present invention adopts micromagnetic particles coupled with soluble human non-phosphorylated Tau217 protein (np-Tau217) specific site recognition capture antibodies, and acridinium ester-labeled soluble human non-phosphorylated Tau217 protein (np-Tau217) detection antibody liquid phase reagent, and the kit also includes soluble human non-phosphorylated Tau217 protein (np-Tau217) calibrators and quality control products.

[0028] Specifically, the magnetic particle coupling suspension is composed of magnetic particles of soluble human non-phosphorylated Tau217 protein (np-Tau217) specific site recognition capture antibody and coupled antibody buffer, wherein the concentration of soluble human non-phosphorylated Tau217 protein (np-Tau217) capture antibody is 0.1-2.0 mg / mL.

[0029] Specifically, the core of the magnetic microparticles is ferroferric oxide, and the magnetic microparticles can be directly coupled with human non-phosphorylated Tau217 protein (np-Tau217) capture antibodies, or the magnetic microparticles can be coupled with streptavidin, and biotin is used to label soluble human non-phosphorylated Tau217 protein (np-Tau217) capture antibodies. Before the use of magnetic microparticles, incomplete sedimentation of the solid phase will affect the accuracy, so magnetic beads with good dispersibility and slow sedimentation speed should be selected.

[0030] Specifically, when preparing the magnetic particle suspension, the coupled antibody buffer is 20-200 mM PBS buffer (pH 6.0-8.0).

[0031] Specifically, when preparing the magnetic particle suspension, the blocking buffer used is a buffer containing 0.2-5% BSA.

[0032] Specifically, when preparing acridinium ester-labeled soluble human non-phosphorylated Tau217 protein (np-Tau217) detection antibody liquid phase reagent, the labeling buffer used is Tris-NaCl or PBS buffer containing BSA, casein or glycine, pH 6.0-8.0, and a concentration of 20-200mM.

[0033] Specifically, the calibrator used is prepared by adding pure human non-phosphorylated Tau217 protein (np-Tau217) to PBS, Tris-NaCl, MSE, or HEPES buffer containing 0.1-10% protective protein (one or more of BSA, casein, and calf serum) as the matrix liquid, and the calibrator is in the form of liquid or solid.

[0034] Example 1: Construction and preparation method of magnetic microparticle chemiluminescence kit for human non-phosphorylated Tau217 protein (np-Tau217) 1. Kit Components A magnetic particle chemiluminescence kit for detecting human non-phosphorylated Tau217 protein (np-Tau217) comprises the following components: magnetic particle-coupled human non-phosphorylated Tau217 protein (np-Tau217) capture antibody; acridinium ester-labeled human non-phosphorylated Tau217 protein (np-Tau217) detection antibody; human non-phosphorylated Tau217 protein (np-Tau217) series standards and quality control products; This embodiment provides a human non-phosphorylated Tau217 protein (np-Tau217) specific site recognition capture antibody, whose heavy chain amino acid sequence is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2; This embodiment also provides a human non-phosphorylated Tau217 protein (np-Tau217) detection antibody, the amino acid sequence of its heavy chain is shown in SEQ ID NO:3, and the amino acid sequence of its light chain is shown in SEQ ID NO:4.

[0035] 2. Source of reagents Table 1 3. Preparation of magnetic microparticle suspension of human non-phosphorylated Tau217 protein (np-Tau217) capture antibody (1) Take 1 mg of carboxyl magnetic particles (1.5 μm) and place it in a 0.5 mL centrifuge tube. Add 200 μL of 0.1 mol / L MES buffer (pH 6.0). Vortex to mix. Place it on a magnetic rack and let it stand for 5 min to separate the magnetic particles from the liquid. Discard the supernatant. Wash three times. Add 200 μL of MES buffer (pH 6.0) and vortex.

[0036] (2) Add 15 μg of human non-phosphorylated Tau217 protein (np-Tau217) capture antibody to the carboxyl magnetic particles obtained in step (1) so that the molar ratio of carboxyl to antibody is 150:1. Vortex, rotate the reaction tube, and incubate at room temperature for 30 min.

[0037] (3) Add 10 μL of 10 mg / mL coupling reagent EDC to step (2), vortex on a rotary reactor, and incubate at room temperature for 2 h.

[0038] (4) Add 200 μL of 1% BSA solution for blocking for 1 h.

[0039] (5) Remove the supernatant, add 200 μL of blocking buffer (PBS solution containing 2% BSA), and wash three times.

[0040] (6) Place the prepared magnetic particle suspension in 500 μL of storage solution (PBS solution containing 2% BSA) and store at 2-8°C.

[0041] 3. Preparation of acridinium ester-labeled human non-phosphorylated Tau217 protein (np-Tau217) detection antibody liquid phase reagent (1) Purification of human non-phosphorylated Tau217 protein (np-Tau217) detection antibody: Place 100 μg of human non-phosphorylated Tau217 protein (np-Tau217) detection antibody in a dialysis bag, and place the dialysis bag (molecular weight cutoff 10kD) in no less than 1 L of labeling buffer for dialysis. During the dialysis, the labeling buffer was changed 5 times, with the last dialysis being overnight. The labeling buffer was 100 mM PBS buffer (pH 7.4).

[0042] (2) Weigh 1.7 mg of acridinium ester NSP-DMAE-NHS and dissolve it in 447 μL of anhydrous dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) to prepare a 6.5 mmol / L NSP-DMAE-NHS solution in DMF or DMSO.

[0043] (3) Place the dialyzed human non-phosphorylated Tau217 protein (np-Tau217) detection antibody solution in a 500 μL centrifuge tube, add 200 μL labeling buffer, and then add 759 μL of 6.5 mmol / L NSP-DMAE-NHS DMF (reaction in dark) to make the molar ratio of acridinium ester to human non-phosphorylated Tau217 protein (np-Tau217) detection antibody 100:1 (can be selected according to the system signal value requirements), shake to mix, react at room temperature for 1 h, add 100 μL of 1% BSA, let stand for 15 min, terminate the labeling reaction and obtain the labeled substance NSP-DMAE-NHS-Ab.

[0044] (4) The labeled NSP-DMAE-NHS-Ab and free NSP-DMAE-NHS were separated by a Sephadex G-50 column (1×25 cm), and the column was equilibrated and eluted with PBS buffer at a pH of 7.4 and a concentration of 0.1 mol / L.

[0045] (5) During the separation process, the protein peak was detected by a chromatograph, and the chemiluminescence intensity and 280 nm absorbance of the effluent were measured respectively.

[0046] (6) Collect the eluate with high photometric value and high absorbance, add 1% BSA, and then divide into aliquots and freeze for storage.

[0047] 4. Preparation of human non-phosphorylated Tau217 protein (np-Tau217) calibrators and quality controls The pure human non-phosphorylated Tau217 protein (np-Tau217) was prepared with Tris-HCl buffer (pH 8.0) containing 2% BSA to prepare a gradient of calibrators with a total of 5 concentrations of 0 / 100 / 500 / 1000 / 2000 pg / ml and a quality control with a total of 2 concentrations of 100 / 500 pg / ml.

[0048] Example 2 Detection of human non-phosphorylated Tau217 protein (np-Tau217) using a magnetic microparticle chemiluminescence detection kit The following steps are involved: (1) Add 50 μL of the sample (EDTA-plasma) to the reaction cup, then add 40 μL of the magnetic particle coupling suspension, shake to mix, and incubate at 37°C for 10 min.

[0049] (2) Separate and wash three times, and shake the reaction container thoroughly after washing to disperse the magnetic particles.

[0050] (3) Add 100 μL of acridinium ester marker (NSP-DMAE-NHS-Ab) to the reaction cup, shake to mix, and incubate at 37°C for 5-10 min.

[0051] (4) Separate and wash three times, and shake the reaction container thoroughly after washing to disperse the magnetic particles.

[0052] (5) Add 100 μL of chemiluminescent pre-excitation solution A, and then add 100 μL of chemiluminescent excitation solution B after 1 second. Measure the relative luminescence intensity. The content of human non-phosphorylated Tau217 protein (np-Tau217) in the sample is proportional to its relative luminescence intensity.

[0053] Example 3 Performance indicators of the kit (1) Analytical sensitivity Using 0 pg / mL calibrator as the sample to be tested, repeat the measurement 20 times, obtain the RLU value (relative luminescence value) of the 20 measurement results, calculate its mean (M) and standard deviation (SD), and obtain the RLU value corresponding to M+2SD. Perform two-point regression fitting based on the concentration-RLU value results between the zero concentration calibrator and the adjacent calibrator to obtain a linear equation, substitute the RLU value of M+2SD into the above equation, and calculate the corresponding concentration value. According to the minimum detection limit detection method in the detection scheme, repeat the experiment 3 times, and finally calculate that the analytical sensitivity of this kit for soluble human non-phosphorylated Tau217 protein (np-Tau217) is 10pg / mL.

[0054] (2) Precision The human non-phosphorylated Tau217 protein (np-Tau217) kit was used to test samples with concentrations of (100.0±10.0) ng / mL and (500.0±50.0) ng / mL. Each concentration was tested 10 times and the precision of the kit was calculated. The results are shown in Table 2, indicating that the CV of the kit was less than 5%.

[0055] Table 2 Precision results of the kit (3) Stability The human non-phosphorylated Tau217 protein (np-Tau217) detection kit was subjected to routine storage stability test, and the test was performed at 2-8°C for 1, 3, 5, 7, 9, 11, 12, 13, 14, and 15 months; the stability test after opening the lid was performed at 2-8°C for 0 days, 7 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, and 32 days. The results showed that the soluble human non-phosphorylated Tau217 protein (np-Tau217) detection kit was stored at 2-8°C in a light-proof environment with a shelf life of 12 months. The shelf life is 30 days when the lid is opened and stored at 2-8°C in a light-proof environment.

[0056] (4) Specificity The phosphorylated Tau441 of the interfering substance of different concentrations was added to the high and low value samples, and the cross-reaction rate was calculated. The results are shown in Table 3. When 1000 pg / ml of phosphorylated tau441 was added, the cross-reaction rate was <0.5%, and there was basically no cross-reaction.

[0057] Table 3 Kit specificity results (5) Linear range The high and low value samples were prepared into linear samples in different proportions, and the correlation coefficient between the theoretical concentration and the measured concentration was calculated. The conclusion was: the correlation coefficient r>0.99, and the linearity was good in the range of 0.5-690pg / mL.

[0058] Table 4 Linearity results of the kit It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0059] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A capture antibody for human non-phosphorylated Tau217 protein, characterized in that: The amino acid sequence of the heavy chain of the capture antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 2; the capture antibody is a monoclonal antibody.

2. A detection antibody for human non-phosphorylated Tau217 protein, characterized in that: The amino acid sequence of the heavy chain of the detection antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain is shown in SEQ ID NO:

4. The detection antibody is a monoclonal antibody.

3. A magnetic microparticle chemiluminescent detection kit for human non-phosphorylated Tau217 protein, characterized in that: The kit comprises a magnetic microparticle suspension of a human non-phosphorylated Tau217 protein capture antibody, an acridinium ester-labeled human non-phosphorylated Tau217 protein detection antibody, a quality control product and a standard product; the magnetic microparticle suspension of the human non-phosphorylated Tau217 protein capture antibody comprises a capture antibody and magnetic microparticles, and the capture antibody binds to the 217 amino acid site of the human non-phosphorylated Tau protein.

4. The magnetic microparticle chemiluminescence detection kit for human non-phosphorylated Tau217 protein according to claim 3, characterized in that: The concentration of the capture antibody is 0.1-2.0 mg / mL; the concentration of the detection antibody is 0.1-0.6 mg / L.

5. The magnetic microparticle chemiluminescence detection kit for human non-phosphorylated Tau217 protein according to claim 1, characterized in that: The inner core of the magnetic particle is ferroferric oxide or ferrous oxide, and the surface modification groups of the magnetic particle are one or more of carboxyl, amino, p-toluenesulfonyl or synavidin.

6. The magnetic microparticle chemiluminescence detection kit for human non-phosphorylated Tau217 protein according to claim 3, characterized in that: The micromagnetic particles are directly coupled with the capture antibody; or the magnetic particles are coupled with streptavidin and the capture antibody is labeled with biotin.

7. The magnetic microparticle chemiluminescence detection kit for human non-phosphorylated Tau217 protein according to claim 3, characterized in that: The molar ratio of the acridinium ester to the human non-phosphorylated Tau217 protein detection antibody is 100:1-10:1; the acridinium ester is NSP-DMAE-NHS or NSP-SA-NHS.

8. The magnetic microparticle chemiluminescence detection kit for human non-phosphorylated Tau217 protein according to claim 3, characterized in that: The calibrator and quality control product are prepared by adding a pure human non-phosphorylated Tau217 protein to a buffer containing 0.1-10% BSA, and the buffer is one of PBS, Tris-NaCl, MSE, and HEPES.

9. The method for preparing the magnetic microparticle chemiluminescence detection kit for detecting non-phosphorylated Tau217 protein according to any one of claims 3 to 8, characterized in that: The following steps are involved: (1) Prepare a suspension of magnetic microparticles coupled with capture antibodies: Evenly mix the magnetic microparticles and capture antibodies and incubate at room temperature for 30 min; then add the coupling reagent EDC and incubate at room temperature for 2 h; finally, add the blocking solution and incubate at room temperature for 1 h. After the incubation, discard the supernatant; (2) Preparation of acridinium ester-labeled human non-phosphorylated Tau217 protein detection antibody reagent: Place the human non-phosphorylated Tau217 protein detection antibody in a dialysis bag for dialyzing. After the dialysis, react with acridinium ester at room temperature for 1 hour. After adding BSA or glycine to terminate the reaction, separate the labeled substance and free acridinium ester by chromatography column.

10. A method for detecting human non-phosphorylated Tau217 protein for non-diagnostic purposes, characterized in that: The kit according to claim 1 is used to detect human non-phosphorylated Tau217 protein in a sample.

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