Detection methods, kits, and applications for 15N-p-Tau protein, p-Tau protein, or fragments thereof.

Abstract

A method for detecting N-p-Tau 217 protein, p-Tau protein or fragments thereof, a kit and application 15 The application relates to the technical field of protein spectrum detection, and relates to a method for detecting N-p-Tau 217 protein, p-Tau protein or fragments thereof, a kit and application. 15 The N-p-Tau-217 protein can be used for accurate quantification of Tau-217 protein or fragments thereof, the detection method steps are simple, the detection time is short, the sensitivity is high, the precision is good, the detection result is high in reliability, the mass spectrum detection pretreatment can be completed only by 5 steps, the required sample amount is reduced, and the application value and significance are important.

Description

Technical Field

[0001] This invention relates to the field of protein spectrum detection technology, specifically to a... 15 Methods, kits, and applications for detecting Np-Tau 217 protein, p-Tau protein, or fragments thereof. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with insidious onset and complex pathology. Clinical features include irreversible brain damage, cognitive impairment, and a decline in the ability to perform daily activities, ultimately leading to dementia and death. With the increasing aging of the global population, the prevalence and socioeconomic burden of AD continue to rise, becoming one of the major challenges facing global public health. It has become the most common form of dementia and a leading cause of increasing disability among the elderly worldwide. Currently, clinical diagnosis of AD mainly relies on imaging techniques combined with cognitive tests, and it is usually diagnosed at an advanced stage. Although there is currently no effective treatment for AD, early diagnosis and intervention can still help reduce neurological damage, slow disease progression, and thus improve patients' quality of life.

[0003] Currently, the main diagnostic methods for Alzheimer's disease (AD) include neuropsychological scales, imaging examinations, and biomarker testing. Neuropsychological scale assessments often result in highly variable results due to differences in participants' education levels and individual comprehension abilities. Neuroimaging examinations, such as magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT), are also strong evidence for AD diagnosis. CT can directly show brain atrophy in AD patients, but its value is limited due to poor soft tissue resolution. MRI and PET are expensive and not suitable for population-based screening.

[0004] The main pathological features of Alzheimer's disease (AD) patients are the extracellular deposition of β-amyloid (Aβ) in the brain, forming senile plaques, and the hyperphosphorylation of Tau protein causing neurofibrillary tangles within brain nerve cells. In the very early stages of AD, the levels of certain protein markers in cerebrospinal fluid (CSF), such as Aβ40, Aβ42, total Tau protein (T-Tau), and hyperphosphorylated Tau protein (p-Tau), change. However, clinically, detecting these markers in CSF often requires lumbar puncture. This method is invasive, causes significant harm to patients, and is expensive, limiting patient acceptance and hindering early AD diagnosis. Studies have found that damage to the blood-brain barrier in AD patients allows certain protein markers in CSF to cross the blood-brain barrier and enter the peripheral blood circulation in the early stages of the disease, resulting in significant changes in blood protein marker levels even in the very early stages of the disease. Therefore, non-invasive detection techniques for AD protein markers in peripheral blood, due to their convenient sample collection and minimal trauma, have become the most promising early screening and follow-up methods, bringing new hope for early AD diagnosis.

[0005] In the early stages of Alzheimer's disease (AD), only a small number of protein biomarkers could cross the blood-brain barrier from the cerebral cortex (CSF) into the peripheral circulation, and their abundance in the blood was extremely low (fM level). Extracting the target protein from the blood matrix involved a series of procedures, inevitably leading to losses and resulting in poor reproducibility both in vitro and in vitro, as well as significant deviations from the actual concentration. Therefore, there was an urgent need to develop a high-precision and accurate absolute quantification method to ensure that the measured value was as consistent as possible with the actual concentration of the target protein. Isotope-labeled proteins share the same amino acid sequence, immunoenrichment effect, partition behavior, liquid chromatography retention time, mass spectrometry ionization efficiency, and secondary fragmentation ions as the target protein. Due to their different mass numbers, the ratio of their peak intensities can be obtained on mass spectrometry. The amount of the corresponding hydrolyzed peptide in the sample can then be calculated from the amount of isotope-labeled peptide, and finally, the amount of the corresponding protein can be obtained based on the stoichiometry of the peptide. However, methods that can simulate the binding, extraction, denaturation, and detection processes of endogenous proteins have not been widely used for absolute protein quantification due to their difficulty in obtaining the necessary data. Furthermore, there are no reports on the preparation and application of p-Tau217 isotope-labeled proteins. Summary of the Invention

[0006] In view of this, the main objective of the present invention is to provide a 15 The invention aims to provide detection methods, kits, and applications for Np-Tau 217 protein, p-Tau protein, or fragments thereof, with the intention of at least partially resolving the aforementioned technical problems.

[0007] To achieve the above objectives, as a first aspect of the present invention, a method is provided. 15 Np-Tau protein, the 15Np-Tau protein was prepared by the following method:

[0008] 1) Add GSK3β diluted with kinase dilution buffer I to a pre-chilled centrifuge tube, then add GSK3β diluted with kinase detection buffer. 15 N-Tau;

[0009] 2) Add ATP diluted with kinase detection buffer, mix thoroughly, and incubate.

[0010] 3) Add GSK3β inhibitor to terminate the reaction, purify, and obtain the desired product. 15 Np-Tau protein.

[0011] Preferably, the GSK3β diluted with kinase dilution buffer I in step 1) is specifically:

[0012] Dilute GSK3β to 0.1 μg / μl with kinase dilution buffer I, adding 5 μL-50 μL.

[0013] Preferably, the kinase detection buffer diluted in step 1) 15 N-Tau specifically refers to:

[0014] Use kinase detection buffer to... 15 N-Tau is diluted to 1 mg / mL and added in volumes of 5 μL to 50 μL.

[0015] Preferably, the ATP diluted with kinase detection buffer in step 2) is specifically:

[0016] Dilute the 0.1M ATP stock solution to 250μM with kinase detection buffer, and add 2.5μL-12.5μL.

[0017] Preferably, step 3) involves adding a GSK3β inhibitor aqueous solution with a concentration of 100 ug / mL, in an amount of 0.75 μL to 7.5 μL.

[0018] Secondly, the present invention provides a method for detecting Tau protein or a fragment thereof, the method comprising adding, as described in the first aspect, during sample pretreatment. 15 Steps for producing Np-Tau protein.

[0019] Preferably, the method includes at least the following steps:

[0020] 1) Sample pretreatment: Add sample diluent to the sample, then add the above-mentioned... 15 Np-Tau protein was used to obtain solution A;

[0021] 2) Immunoenrichment: The nanomagnetic microsphere-p-Tau 217 antibody complex was added to solution A and incubated. After passing through a magnetic rack, the purified immunoenriched complex (i.e., the target protein-Tau 217 antibody-nanomagnetic microsphere complex) was obtained.

[0022] 3) Washing: The immunoenriched complex was washed with a nonionic surfactant (such as NP40) to obtain the washed complex.

[0023] 4) The cleaned complex was reconstituted with NH4HCO3, and then digested and terminated.

[0024] 5) Desalination, vacuum concentration, and mass spectrometry injection;

[0025] 6) Perform mass spectrometry detection and output the results.

[0026] Preferably, the sample is a CSF, blood, or plasma sample;

[0027] Preferably, the sample diluent in step 1) includes: Tris-HCl, NaCl, EDTA (ethylenediaminetetraoxic acid), protease inhibitor, and phosphatase inhibitor; and / or

[0028] The sample diluent in step 1) also includes NP40 and / or Tween 20 (a nonionic surfactant made from polyether sulfate).

[0029] Preferably, the sample diluent in step 1) includes: 20-80 mM Tris-HCl, 30-80 mM NaCl, 1-10 mM EDTA (ethylenediaminetetraoxic acid), and 0.02%-0.2% protease inhibitor and 0.02%-0.2% phosphatase inhibitor by mass fraction; wherein, the concentration of Tris-HCl can be 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, or all values ​​within the range, because Due to space limitations, these will not be listed individually; the concentration of NaCl can be 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, or any value within the range mentioned above, and will not be listed individually due to space limitations; the concentration of EDTA can be 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, or any value within the range mentioned above, and will not be listed individually due to space limitations. The functions of the sample diluent are: to reduce the viscosity of the plasma, allowing the antibody microspheres to be evenly distributed in the plasma, increasing the probability of target protein capture; to create a buffer system and the presence of a non-ionic surfactant, which helps in the binding of antigen and antibody and reduces the non-specific adsorption of microspheres to non-target proteins; and to inhibit the enzymatic hydrolysis of target proteins by bioactive enzymes, as blood contains a series of biologically active enzymes that can enzymatically hydrolyze proteins in vitro, by adding protease inhibitors and phosphatase inhibitors.

[0030] And / or the sample diluent in step 1) further includes 0.1%-1% by mass of NP40 and / or Tween 20, wherein the mass fraction of NP40 and / or Tween 20 can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or all values ​​within the range described above; due to space limitations, they will not be listed individually. And / or

[0031] Step 1) p-Tau- 15 The N-peptide fragment is 0.5-5 pg / mL. 15 Np-Tau217 aqueous solution, p-Tau- 15 The concentration of the N-peptide can be 0.5 pg / mL, 1 pg / mL, 1.5 pg / mL, 2 pg / mL, 2.5 pg / mL, 3 pg / mL, 3.5 pg / mL, 4 pg / mL, 4.5 pg / mL, 5 pg / mL, or any value within the range described above. Due to space limitations, they will not be listed one by one.

[0032] Preferably, step 2) includes: adding the magnetic nanosphere-p-Tau 217 antibody complex to solution A, incubating at room temperature by rotation for at least 2 hours, passing through a magnetic rack to obtain the purified immuno-enriched complex (i.e., the target protein-p-Tau 217 antibody-magnetic nanosphere complex);

[0033] The nanomagnetic microsphere-p-Tau 217 antibody complex contains a monoclonal antibody that recognizes the p-Tau 217 site. Its purpose is to capture the target protein (p-Tau 217) in plasma, yielding an immuno-enriched complex (i.e., the target protein-p-Tau 217 antibody-nanomagnetic microsphere complex). The superparamagnetic nanospheres are easily demagnetized, facilitating subsequent separation and efficient washing. Even after multiple magnetizations, the superparamagnetic nanospheres maintain their properties and exhibit good reproducibility. The built-in magnets in the magnetic rack allow the magnetic nanosphere-p-Tau 217 antibody complex to be fixed to the container wall, achieving solid-liquid separation and facilitating automated conversion. Utilizing its binding to specific antibodies, separation of the solution and the nanomagnetic microsphere-p-Tau 217 antibody complex can be achieved without centrifugation, simplifying the extraction process.

[0034] Preferably, step 3) includes: washing the immunoenriched complex at least three times with PBS solution containing 0.1%-0.5% NP40 by mass.

[0035] The nonionic surfactant (NP40 in PBS) serves to remove nonspecific adsorption on the surface of the magnetic nanosphere-antibody complex. The mass fraction of NP40 in the PBS solution can be 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or any value within the range described above; due to space limitations, these will not be listed individually.

[0036] Preferably, step 4) includes: reconstitute the washed complex with NH4HCO3 at a concentration of 20-200 mM, then add trypsin for digestion for at least 8 hours, and finally terminate the digestion with a 10% (v / v) formic acid aqueous solution. The main function of ammonium bicarbonate (NH4HCO3) is to provide an alkaline hydrolysis environment to ensure the highest activity of trypsin (optimal pH 8-9), decomposing the p-Tau217 protein into specific peptides that can be detected by mass spectrometry. The concentration of NH4HCO3 can be 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or any value within the range mentioned above. Due to space limitations, these values ​​will not be listed individually.

[0037] Preferably, the desalination in step 5) includes:

[0038] ① Add 200 μL of methanol to the C18 centrifuge column and centrifuge at 2500 rpm for 5 min, then discard the filtrate;

[0039] ② Add 200 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0040] ③ Add 200 μL of 1% formic acid aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0041] ④ Add the acidified sample to a C18 centrifuge column and centrifuge at 1800 rpm for 10 min, then discard the filtrate;

[0042] ⑤ Add 80 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and collect the filtrate;

[0043] In the aforementioned steps, residual salt ions may remain, which can interfere with mass spectrometry analysis and potentially damage the chromatographic column. Since the stationary phase on a C18 column is composed of silica gel particles modified with octyl (C18) silane compounds with a carbon chain length of 18 atoms, its strong hydrophobicity can effectively retain peptides and remove salt ions during the desalting step.

[0044] Preferably, the vacuum concentration and mass spectrometry injection in step 5) includes: passing the desalted sample through a vacuum concentration centrifuge and drying it; the mass spectrometry is an orbital ion trap mass spectrometer;

[0045] Preferably, the orbital ion trap mass spectrometry injection in step 5) includes: reconstituted the dried sample with an aqueous solution of 0.1% formic acid by volume, followed by centrifugation and injection.

[0046] Preferably, the following is added to the reconstituted sample before mass spectrometry injection: 15 Np-Tau 217 stock solution, shake to mix for 1 min, briefly centrifuge for 30 s, and place in mass spectrometer.

[0047] Preferably, the method includes at least the following steps:

[0048] 1) Sample pretreatment: Add sample diluent to the sample to obtain solution A; the sample diluent includes: 0.1%-1% NP40, 0.1%-1% Tween 20, 20-80mM Tris-HCl, 30-80mM NaCl, 0.02%-0.2% protease inhibitor, and 0.02%-0.2% phosphatase inhibitor; the sample is CSF, blood, or plasma; wherein, p-Tau- 15The N-peptide segment is 0.5-5 pg / mL. 15 Np-Tau217 aqueous solution;

[0049] 2) Immunoenrichment: The nanomagnetic microsphere-antibody complex was added to solution A and incubated at room temperature by rotation for at least 2 hours. The mixture was then passed through a magnetic rack to obtain the purified immunoenriched complex.

[0050] 3) Washing: The immunoenriched complex was washed at least 3 times with 0.1%-0.5% NP40 PBS solution to obtain the washed complex.

[0051] 4) Digestion: The washed complex was reconstituted in 20-200mM NH4HCO3, then digested with trypsin for at least 8 hours, and the digestion was terminated by adding 10% formic acid aqueous solution.

[0052] 5) Desalination:

[0053] ① Add 200 μL of methanol to the C18 centrifuge column and centrifuge at 2500 rpm for 5 min, then discard the filtrate;

[0054] ② Add 200 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0055] ③ Add 200 μL of 1% formic acid aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0056] ④ Add the acidified sample to a C18 centrifuge column and centrifuge at 1800 rpm for 10 min, then discard the filtrate;

[0057] ⑤ Add 80 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and collect the filtrate;

[0058] 6) Dry the desalted sample using a vacuum concentrator, then reconstitute the dried sample with 0.1% formic acid solution, centrifuge with shaking, and inject the sample.

[0059] 7) Use mass spectrometry for detection and output the results.

[0060] The principle of this invention is as follows: This invention uses immunoenrichment to specifically adsorb the target protein (p-Tau217) in the sample onto immunomicrospheres (nanomagnetic p-Tau 217 antibody). The nanomagnetic microspheres are superparamagnetic, so when the immunoenriched complex is placed on a magnetic rack with a magnetic field, the immunomicrosphere complex with the adsorbed target protein can be attracted, while various impurities that cannot be attracted by the immunomicrospheres cannot remain in the magnetic field and can therefore be washed away. After thorough washing, the adsorbed antigen-antibody complex is digested with trypsin to separate it. The antibody binds to the nanomagnetic microspheres, and the target protein is released. Subsequently, after steps such as termination, desalting, drying, and centrifugation, the target protein is completely separated. The isotopically labeled protein and the target protein have the same amino acid sequence, immunoenrichment effect, partition behavior, liquid chromatography retention time, mass spectrometry ionization efficiency, and secondary fragmentation ions. Due to their different mass numbers, the ratio of their peak intensities can be obtained on mass spectrometry. The amount of the corresponding hydrolyzed peptide in the sample can be calculated by the amount of isotopically labeled peptide. Finally, the amount of the corresponding protein can be obtained based on the stoichiometry of the peptide. This method can be used to diagnose and / or treat subjects with or at risk of developing neurological or neurodegenerative diseases.

[0061] It should be noted that the raw data output from the orbital ion trap mass spectrometer needs to be imported into the Skyline software for secondary processing in order to convert the mass spectrometry signal data into content results.

[0062] Thirdly, the present invention provides a detection kit for p-Tau 217 protein or a fragment thereof, the kit comprising:

[0063] 1) Preprocessing module: including at least the sample diluent and the solution described in the first aspect. 15 Np-Tau217 protein, wherein the sample dilution includes: 0.1%-1% by mass of NP40 and / or Tween 20, 20-80 mM of Tris-HCl, 30-80 mM of NaCl, 0.02%-0.2% by mass of protease inhibitor, and 0.02%-0.2% by mass of phosphatase inhibitor.

[0064] 15 Np-Tau protein is 0.5-5 pg / mL 15 Np-Tau 217 aqueous solution.

[0065] 2) Immunoenrichment module: including at least the nanomagnetic microsphere-p-Tau 217 antibody complex;

[0066] 3) Cleaning the digestion module: This includes at least 0.1%-0.5% NP40 PBS solution and 20-200mM NH4HCO3 solution;

[0067] 4) Desalination module: includes at least methanol, 80% acetonitrile aqueous solution, and 1% formic acid aqueous solution;

[0068] 5) Drying and injection module: includes at least 1% formic acid aqueous solution by volume.

[0069] Fourthly, the present invention provides a kit for the use of p-Tau 217 protein or fragments thereof as described in the third aspect in the early diagnosis of Alzheimer's disease.

[0070] Based on the above technical solution, it can be seen that the p-Tau 217- of the present invention... 15 The detection methods, kits, and applications for N-peptides, p-Tau proteins, or fragments thereof, compared to existing technologies, possess at least one of the following advantages:

[0071] This invention incorporates stable isotope labeling during pretreatment. 15 Np-Tau217 protein can be used for the accurate quantification of p-Tau 217 protein or its fragments. Because the isotopically labeled protein and the target protein share the same amino acid sequence, immunoenrichment effect, partition behavior, liquid chromatography retention time, mass spectrometry ionization efficiency, and secondary fragmentation ions, and because they differ in mass number, the ratio of their peak intensities can be obtained on mass spectrometry. The amount of the corresponding hydrolyzed peptide in the sample can then be calculated from the amount of the isotopically labeled peptide, and finally, the amount of the corresponding protein can be obtained based on the stoichiometry of the peptide. Especially when added during pretreatment, the entire process (including sample dilution, immunoenrichment, washing, digestion, desalting, etc.) can be monitored, effectively overcoming the losses caused by the extraction of p-Tau 217 from blood matrix, as well as problems such as poor reproducibility in and out of the laboratory and large deviations from the true content.

[0072] Furthermore, the detection method for phosphorylated Tau-217 protein or its fragments of the present invention is simple in procedure, short in detection time, highly sensitive, precise, and reliable in results. Only five steps are required for mass spectrometry pretreatment, reducing the required sample volume by approximately 75%. The washing step is simplified, omitting acid precipitation, acid washing, and solid-phase extraction steps found in existing technologies, and significantly shortening the digestion and incubation time. This invention solves the problems of existing Alzheimer's disease detection kits, such as complex operation, long processing time, low sensitivity, poor precision, and lack of accurate predictive ability for Alzheimer's disease, and has significant application value and significance. Attached Figure Description

[0073] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0074] Figure 1 This is the p-Tau 217IP-MS flowchart;

[0075] Figure 2 This is a graph showing the concentration difference of p-Tau 217 between the AD patient group and the healthy population (control group) in Example 4;

[0076] Figure 3 This is the calibration curve of p-Tau 217 protein in Comparative Example 3. Detailed Implementation

[0077] Existing technologies for biomarker detection of Alzheimer's disease involve cumbersome pretreatment steps, complex procedures, long processing times, and low sensitivity. The inventors of this invention, through dedicated research, have simplified and optimized these steps, achieving higher detection accuracy and detection limits through simpler operation. Specifically, as a first aspect of this invention, a method is provided... 15 Np-Tau protein, the 15 Np-Tau protein was prepared by the following method:

[0078] 1) Add GSK3β diluted with kinase dilution buffer I to a pre-chilled centrifuge tube, then add GSK3β diluted with kinase detection buffer. 15 N-Tau;

[0079] 2) Add ATP diluted with kinase detection buffer, mix thoroughly, and incubate.

[0080] 3) Add GSK3β inhibitor to terminate the reaction, purify, and obtain the desired product. 15 Np-Tau protein.

[0081] As a preferred embodiment, the GSK3β diluted with kinase dilution buffer I in step 1) is specifically as follows:

[0082] Dilute GSK3β to 0.1 μg / μl with Kinase Dilution Buffer I (Kinase Dilution Buffer I), adding 5-50 μL.

[0083] In a preferred embodiment, step 1) involves diluting the sample with the kinase detection buffer. 15 N-Tau specifically refers to:

[0084] Use kinase detection buffer to... 15 N-Tau is diluted to 1 mg / mL and added in volumes of 5 μL to 50 μL.

[0085] In a preferred embodiment, the ATP diluted with the kinase detection buffer in step 2) specifically refers to:

[0086] Dilute the 0.1M ATP stock solution to 250μM with kinase detection buffer, and add 2.5μL-12.5μL.

[0087] In a preferred embodiment, step 3) of adding the GSK3β inhibitor specifically involves adding an aqueous solution of the GSK3β inhibitor at a concentration of 100 ug / mL, in an amount of 0.75 μL to 7.5 μL.

[0088] Secondly, the present invention provides a method for detecting Tau protein or fragments thereof, the method comprising adding, as described in the first aspect, during sample pretreatment. 15 The steps for producing Np-Tau protein.

[0089] In a preferred embodiment, the method includes at least the following steps:

[0090] 1) Sample pretreatment: Add sample diluent to the sample, then add the above-mentioned... 15 Np-Tau protein was used to obtain solution A;

[0091] 2) Immunoenrichment: The nanomagnetic microsphere-p-Tau 217 antibody complex was added to solution A and incubated. After passing through a magnetic rack, the purified immunoenriched complex (i.e., the target protein-Tau 217 antibody-nanomagnetic microsphere complex) was obtained.

[0092] 3) Washing: The immunoenriched complex was washed with a nonionic surfactant (such as NP40) to obtain the washed complex.

[0093] 4) The cleaned complex was reconstituted with NH4HCO3, and then digested and terminated.

[0094] 5) Desalination, vacuum concentration, and mass spectrometry injection;

[0095] 6) Perform mass spectrometry detection and output the results.

[0096] In a preferred embodiment, the sample is a CSF, blood, or plasma sample;

[0097] In a preferred embodiment, the sample diluent in step 1) includes: Tris-HCl, NaCl, EDTA (ethylenediaminetetraoxic acid), protease inhibitor, and phosphatase inhibitor; and / or

[0098] The sample diluent in step 1) also includes NP40 and / or Tween 20 (a nonionic surfactant made from polyether sulfate).

[0099] In a preferred embodiment, the sample dilution in step 1) comprises: 20-80 mM Tris-HCl, 30-80 mM NaCl, 1-10 mM EDTA (ethylenediaminetetraoxic acid), and 0.02%-0.2% by mass of protease inhibitor and 0.02%-0.2% by mass of phosphatase inhibitor; and / or

[0100] The sample diluent in step 1) also includes 0.1%-1% by mass of NP40 and / or Tween 20; and / or

[0101] Preferably, in step 1) 15 Np-Tau protein concentration: 0.5-5 pg / mL 15 Np-Tau217 aqueous solution.

[0102] In a preferred embodiment, step 2) includes: adding the nanomagnetic-p-Tau 217 antibody microsphere complex to solution A, incubating at room temperature by rotation for at least 2 hours, passing through a magnetic rack to obtain the purified immuno-enriched complex (i.e., the target protein-Tau 217 antibody-nanomagnetic microsphere complex).

[0103] The nanomagnetic microsphere-p-Tau 217 antibody complex contains a monoclonal antibody that recognizes the p-Tau 217 site. Its purpose is to capture the target protein (p-Tau 217) in plasma, yielding an immuno-enriched complex (i.e., the target protein-p-Tau 217 antibody-nanomagnetic microsphere complex). The superparamagnetic nanospheres are easily demagnetized, facilitating subsequent separation and efficient washing. Even after multiple magnetizations, the superparamagnetic nanospheres maintain their properties and exhibit good reproducibility. The built-in magnets in the magnetic rack allow the nanomagnetic microsphere-p-Tau 217 antibody complex to be fixed to the container wall, achieving solid-liquid separation and facilitating automated conversion. Utilizing its binding to specific antibodies, separation of the solution and the nanomagnetic microsphere-p-Tau 217 antibody complex can be achieved without centrifugation, simplifying the extraction process.

[0104] In a preferred embodiment, step 3) includes washing the immunoenriched complex at least three times with 0.1%-0.5% NP40 PBS solution.

[0105] In a preferred embodiment, step 4) includes: reconstitute the washed complex with NH4HCO3 at a concentration of 20-200 mM, then add trypsin for digestion for at least 8 hours, and terminate the digestion by adding a 10% (v / v) formic acid aqueous solution; the main function of ammonium bicarbonate (NH4HCO3) is to provide an alkaline hydrolysis environment to ensure the highest activity of trypsin (optimal pH 8-9), and to decompose the p-Tau217 protein into specific peptides that can be detected by mass spectrometry.

[0106] In a preferred embodiment, the desalination in step 5) includes:

[0107] ① Add 200 μL of methanol to the C18 centrifuge column and centrifuge at 2500 rpm for 5 min, then discard the filtrate;

[0108] ② Add 200 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0109] ③ Add 200 μL of 1% formic acid aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0110] ④ Add the acidified sample to a C18 centrifuge column and centrifuge at 1800 rpm for 10 min, then discard the filtrate;

[0111] ⑤ Add 80 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and collect the filtrate;

[0112] In the aforementioned steps, residual salt ions may remain, which can interfere with mass spectrometry analysis and potentially damage the chromatographic column. Since the stationary phase on a C18 column is composed of silica particles modified with octyl (C18) silane compounds with a carbon chain length of 18 atoms, its strong hydrophobicity can effectively retain peptides and remove salt ions during the desalting step.

[0113] In a preferred embodiment, step 5) of vacuum concentration and mass spectrometry injection includes: passing the desalted sample through a vacuum concentration centrifuge and drying it; the mass spectrometry is an orbital ion trap mass spectrometer.

[0114] In a preferred embodiment, the orbital ion trap mass spectrometry injection in step 5) includes: reconstituted the dried sample with an aqueous solution of 0.1% formic acid by volume, followed by centrifugation and injection.

[0115] In a preferred embodiment, the following is added to the reconstituted sample before mass spectrometry injection: 15 Np-Tau 217 stock solution, shake to mix for 1 min, briefly centrifuge for 30 s, and place in mass spectrometer.

[0116] In a preferred embodiment, the method includes at least the following steps:

[0117] 1) Sample pretreatment: Add sample diluent to the sample to obtain solution A; the sample diluent includes: 0.1%-1% NP40, 0.1%-1% Tween 20, 20-80mM Tris-HCl, 30-80mM NaCl, 0.02%-0.2% protease inhibitor, and 0.02%-0.2% phosphatase inhibitor; the sample is CSF, blood, or plasma; wherein, 15 Np-Tau protein is 0.5-5 pg / mL 15 Np-Tau217 aqueous solution;

[0118] 2) Immunoenrichment: The nanomagnetic microsphere-antibody complex was added to solution A and incubated at room temperature by rotation for at least 2 hours. The mixture was then passed through a magnetic rack to obtain the purified immunoenriched complex.

[0119] 3) Washing: Wash the immunoenriched complex at least 3 times with 0.1-0.5% NP40 PBS solution to obtain the washed complex;

[0120] 4) Digestion: The washed complex was reconstituted in 20-200mM NH4HCO3, then digested with trypsin for at least 8 hours, and the digestion was terminated by adding 10% formic acid aqueous solution.

[0121] 5) Desalination:

[0122] ① Add 200 μL of methanol to the C18 centrifuge column and centrifuge at 2500 rpm for 5 min, then discard the filtrate;

[0123] ② Add 200 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0124] ③ Add 200 μL of 1% formic acid aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0125] ④ Add the acidified sample to a C18 centrifuge column and centrifuge at 1800 rpm for 10 min, then discard the filtrate;

[0126] ⑤ Add 80 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and collect the filtrate;

[0127] 6) Dry the desalted sample using a vacuum concentrator, then reconstitute the dried sample with 0.1% formic acid solution, centrifuge with shaking, and inject the sample.

[0128] 7) Use mass spectrometry for detection and output the results.

[0129] The principle of this invention is as follows: This invention uses immunoenrichment to specifically adsorb the target protein (p-Tau217) in the sample onto immunomicrospheres (magnetic nanospheres-Tau 217 antibody). The magnetic nanospheres are superparamagnetic, so when the immunoenriched complex is placed on a magnetic rack with a magnetic field, the immunomicrosphere complex with the adsorbed target protein can be attracted, while various impurities that cannot be attracted by the immunomicrospheres cannot remain in the magnetic field and can therefore be washed away. After thorough washing, the adsorbed antigen-antibody complex is digested with trypsin to separate it. The antibody binds to the magnetic nanospheres, and the target protein is released. Subsequently, after steps such as termination, desalting, drying, and centrifugation, the target protein is completely separated. The isotopically labeled protein and the target protein have the same amino acid sequence, immunoenrichment effect, partition behavior, liquid chromatography retention time, mass spectrometry ionization efficiency, and secondary fragmentation ions. Due to their different mass numbers, the ratio of their peak intensities can be obtained on mass spectrometry. The amount of the corresponding hydrolyzed peptide in the sample can be calculated by the amount of isotopically labeled peptide. Finally, the amount of the corresponding protein can be obtained based on the stoichiometry of the peptide. This method can be used to diagnose and / or treat subjects with or at risk of developing neurological or neurodegenerative diseases.

[0130] Thirdly, the present invention provides a detection kit for p-Tau 217 protein or a fragment thereof, the kit comprising:

[0131] 2) Preprocessing module: including at least the sample diluent and the solution described in the first aspect. 15 Np-Tau217 protein, wherein the sample dilution includes: 0.1%-1% by mass of NP40 and / or Tween 20, 20-80 mM of Tris-HCl, 30-80 mM of NaCl, 0.02%-0.2% by mass of protease inhibitor, and 0.02%-0.2% by mass of phosphatase inhibitor.

[0132] p-Tau- 15 The N-peptide segment is 0.5-5 pg / mL. 15 Np-Tau 217 aqueous solution.

[0133] 2) Immunoenrichment module: including at least the nanomagnetic microsphere-p-Tau 217 antibody complex;

[0134] 3) Cleaning the digestion module: This includes at least 0.1%-0.5% NP40 PBS solution and 20-200mM NH4HCO3 solution;

[0135] 4) Desalination module: includes at least methanol, 80% acetonitrile aqueous solution, and 1% formic acid aqueous solution;

[0136] 5) Drying and injection module: includes at least 1% formic acid aqueous solution by volume.

[0137] Fourthly, the present invention provides a kit for the use of p-Tau 217 protein or fragments thereof as described in the third aspect in the early diagnosis of Alzheimer's disease.

[0138] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0139] Unless otherwise specified, all conventional reagents and equipment used in this invention are commercially available.

[0140] Plasma sample collection: Peripheral blood from Alzheimer's patients and healthy individuals was collected from the blood bank of a municipal hospital in Anhui Province. After extracting plasma from each peripheral blood sample, it was stored at -80℃ for later use.

[0141] Example 1: Preparation of Immunospheres (Magnetic Nanospheres - Tau 217 Antibody)

[0142] Two 10 μL aliquots of the magnetic nanosphere suspension (10 mg / mL 0.1% BSA + 0.02% NaN3) were placed in 1.5 mL EP tubes and washed three times with 2 mM EDTA in PBS solution, 3 min / time, 1 mL / time. After removing the supernatant, 4 μL of 100 μg / mL p-Tau 217 antibody was added to the experimental group, and no p-Tau 217 antibody was added to the blank background group. 500 μL of PBS solution was then added, and the mixture was incubated at 4 °C for 8 h to obtain immunospheres (magnetic nanosphere-p-Tau 217 antibody complex). The BCA method was used, with the blank background group as the zero point for subtraction. Protein levels in the solutions before and after conjugation were tested to evaluate antibody conjugation efficiency. The experiment was repeated three times. The evaluation results for the experimental group are shown in Table 1. The blank group had no results and was automatically subtracted as background. As can be seen from the data in Table 1, the coupling efficiency between p-Tau 217 antibody and magnetic nanospheres is above 75%, indicating a good coupling effect.

[0143] Table 1. Evaluation of antibody conjugation efficiency

[0144] 1 0.112 0.025 77.68％ 2 0.111 0.027 75.68％ 3 0.107 0.025 76.63％

[0145] Example 2 15 Preparation of Np-Tau 217 protein

[0146] 1) Reagent preparation

[0147] Reagent 1: Dilute GSK3β to 0.1 μg / μl (10 μg + 100 μL) with Kinase Dilution Buffer I;

[0148] Reagent 2: Use kinase detection buffer to... 15 N-Tau diluted to 1 mg / mL (50 μg) 15 N-Tau + 50 μL kinase detection buffer);

[0149] Reagent 3: Dilute 0.1M ATP stock solution to 250μM with kinase detection buffer (1mL water + 2.5μL 0.1M ATP);

[0150] Reagent 4: 100 μg / mL SB415286 aqueous solution.

[0151] 2) Phosphorylation reaction: Add 10 μL of reagent 1 to a pre-cooled microcentrifuge tube, then add 10 μL of reagent 2 to make the initial reaction volume reach 20 μL. Vortex mix for 1 min, centrifuge briefly for 30 s, add 5 μL of reagent 3, mix and incubate at 30℃ for 30 min.

[0152] 3) Reaction termination: Add 1.5 μL of 100 ug / mL SB415286 to terminate the reaction, vortex mix for 2 min, and then briefly detach for 30 s.

[0153] 4) Concentration determination: Take 1 μL of the reaction solution and dilute it with 379 μL of 100 mM NH4HCO3. Vortex mix for 30 s to prepare a 1 μg / mL solution. Take another 100 μL of the solution and place it in a low-adsorption EP tube. Add 1 μL of 20 μg / mL trypsin and shake for 30 s. Sonicate for 30 s and place on a constant temperature shaker for 8 h at 37℃ and 1000 rpm. Then add 10% formic acid solution and vortex for 5 min to terminate digestion. Desalt the solution and perform mass spectrometry detection using DDA (data-dependent acquisition) mode to obtain the mass spectral signal intensity of each phosphorylated protein. The results show that... 15 The phosphorylation efficiency of N-Tau was 72.73%, with threonine (T) phosphorylation efficiency at 54.55%. After quantification, 15 The concentration of Np-Tau 217 was 20 μg / mL.

[0154] Example 3: p-Tau 217 mass spectrometry detection

[0155] (1) Reagent preparation:

[0156] 1) Sample dilution preparation: Mix 0.2 mL NP40, 2 mL 1 mM Tris-HCl, 0.14 g NaCl, 58.5 mg EDTA, 40 μL protease inhibitor, 40 μL phosphatase inhibitor and 37.8 mL water to prepare the sample dilution.

[0157] 2) 15 Preparation of Np-Tau217 working solution: Dilute with purified water 15 Np-Tau 217 stock solution to 1 μg / mL;

[0158] 3) Preparation of cleaning solution: Mix 0.05 mL of NP40 with 100 mL of PBS to obtain a PBS solution containing 0.5% NP40;

[0159] 4) Preparation of digestion solution: 197.64 mg of NH4CHO3 was mixed with 50 mL of purified water to obtain NH4CHO3 with a concentration of 50 mM;

[0160] 5) Preparation of trypsin solution: Mix 20 μg of trypsin with 1 mL of digestion solution;

[0161] 6) Preparation of reconstituted solution: Mix 10 μL of formic acid with 1 mL of purified water to prepare a 0.1% (v / v) formic acid aqueous reconstituted solution.

[0162] (2) Basic processing steps:

[0163] 1) Sample pretreatment: Add sample diluent to 0.25-1 mL of sample, then add 10 μL of sample diluent. 15 Np-Tau 217 was mixed by rotation to obtain solution A.

[0164] 2) Immunoenrichment: The nanomagnetic microsphere-antibody complex was added to solution A and incubated at room temperature for 2 hours to obtain the immunoenriched complex, namely the protein-antibody-microsphere complex.

[0165] 3) Washing: The protein-antibody-microsphere complex from 2) was placed on a magnetic rack for 1 min to allow solid-liquid separation. After solid-liquid separation, the supernatant was removed, and 0.5% NP40 in PBS was added. The complex was then washed by rotation at a rate of 1 ml / time and 5 min / time. This process was repeated 3 times to obtain the washed complex.

[0166] 4) Digestion: Add 1 mL of 50 mM NH4HCO3 solution to the complex washed in step 3), and perform a vortex wash at a rate of 5 min / time. Repeat the operation 3 times, replace the solvent, add 1 μL of 20 μg / ml trypsin, shake for 30 s, sonicate for 30 s, place on a constant temperature shaker, and digest at 37℃ and 1000 rpm for 8 h. Then add 10% formic acid solution and vortex for 5 min to terminate the digestion.

[0167] 5) Desalination:

[0168] ① Add 200 μL of methanol to the C18 centrifuge column and centrifuge at 2500 rpm for 5 min, then discard the filtrate;

[0169] ② Add 200 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0170] ③ Add 200 μL of 1% formic acid aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0171] ④ Add the acidified sample to a C18 centrifuge column and centrifuge at 1800 rpm for 10 min, then discard the filtrate;

[0172] ⑤ Add 80 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and collect the filtrate;

[0173] 6) Place in a vacuum freeze dryer and centrifuge for 30 minutes to remove the solvent.

[0174] 7) Add 10 μL of reconstitution solution before mass spectrometry injection, shake to mix for 5 min, and briefly incubate for 30 s to prepare for mass spectrometry detection; then perform mass spectrometry detection. The mass spectrometry parameters are set as follows: Isolation window: 0.7; HCD: 30%; Orbitrap Resolution: 45000; Scan range: 120-1500; RF lens: 50%; AGC (Automatic Gain Control): 100%; Maximum injection time: 110. Chromatographic analysis time: 30 min. Injection volume: 5 μL; chromatographic parameters are shown in Table 2.

[0175] Table 2 Chromatographic parameters

[0176] 0 0.5 4.0 5 1.8 0.5 4.5 5 2.0 0.3 5.0 5 11 0.3 20.0 5 27 0.3 35.0 5 27.5 0.5 55.0 5 28 0.5 99.0 5 30 0.5 99.0 5

[0177] 8) Result calculation: The peak area of ​​the target peptide is compared with the peak area of ​​the isotope peptide, and then multiplied by the concentration of the isotope peptide to obtain the concentration of the target peptide. The concentration of the target peptide is then converted into the concentration of the target protein.

[0178] (3) Sample dilution ratio screening: Plasma samples were mixed with sample dilutions at ratios of 1:1, 1:2, 1:3, and 1:4, with other steps consistent with step (2). The peak area ratio of the target protein to the isotope-labeled protein and the peak height of the target protein to the baseline ratio (S / N) 1 min after the peak were read using an orbital ion trap mass spectrometer to determine the optimal dilution ratio. The results are shown in Table 3. The data in the table show that the sample-to-dilution ratio is p-Tau 217 / 15 NP-Tau217 has the highest value and a high signal-to-noise ratio, therefore the sample-to-dilution ratio was determined to be 1:2.

[0179] Table 3 Results of the determination of the ratio of different samples to diluent

[0180]

[0181] (4) Sample dilution ratio: The sample dilution formulations were designed as follows: 1) 0.5% NP40 + 60mM NaCl + 5mM EDTA + 0.1% protease inhibitor + 0.1% phosphatase inhibitor, with Tris-HCl concentrations of 20mM, 40mM, 60mM, and 80mM; 2) 0.5% NP40 + 50mM Tris-HCl + 5mM EDTA + 0.1% protease inhibitor + 0.1% phosphatase inhibitor, with NaCl concentrations of 30mM, 50mM, 70mM, and 80mM; 3) 0.5% NP40 + 50mM Tris-HCl + 60mM NaCl + 0.1% protease inhibitor + 0.1% phosphatase inhibitor, with EDTA concentrations of 2mM, 4mM, 6mM, and 8mM, and other parameters consistent with step 2), were used. The peak area ratio of the target protein to the isotopically labeled protein, and the peak height of the target protein to the baseline ratio (S / N) 1 min after the peak were read using an orbital ion trap mass spectrometer to determine the optimal dilution ratio. The results are shown in Table 4. The data in the table show that when the Tris-HCl concentration in the sample dilution is within the range of 20mM-80mM, the NaCl concentration is within the range of 30mM-80mM, and the EDTA concentration is within the range of 2mM-8mM, the detection concentration is basically stable, and the signal-to-noise ratio is greater than 10, meeting the quantitative requirements.

[0182] Table 4 Comparison of different concentrations of Tris-HCl, NaCl, and EDTA

[0183]

[0184] (5) Cleaning solution formulation: The cleaning solution was formulated into four different formulations: 0.5% NP40PBS, 0.1% NP40PBS, PBS, and 0.1N NaCl. Other parameters were the same as in step 2). The peak area ratio of the target protein to the isotope-labeled protein, and the peak height of the target protein to the baseline ratio (S / N) 1 min after the peak were read using an orbital ion trap mass spectrometer to determine the optimal dilution ratio. The results are shown in Table 5. The data in the table show that the cleaning solution formulations with 0.5% NP40PBS and 0.1% NP40PBS were basically consistent with the theoretical concentration. However, the PBS and 0.1N NaCl solutions deviated from the theoretical concentration by more than 15%. Regarding the signal-to-noise ratio, the 0.5% NP40PBS formulation had the highest signal-to-noise ratio. Since the cleaning solution formulation was set to 0.5% NP40PBS, 0.1% NP40PBS could be used as an alternative solution.

[0185] Table 5

[0186]

[0187] Example 4: Application of the p-Tau217 mass spectrometry detection kit

[0188] Case samples were selected from patients treated in Chongqing, including 20 cases of Alzheimer's disease and 20 healthy individuals. The kit of this invention was used for testing, and the results are shown in Table 6. Figure 2 As shown; from Figure 2 It can be seen that the p-Tau 217 content differs between the AD patient group (AD1-20) and the healthy population (control group, control1-20).

[0189] Table 6. Actual Sample Detection Results

[0190]

[0191]

[0192] Comparative Example 1

[0193] The difference from Example 3 is that no additives are used during sample pretreatment. 15 Np-Tau 217 protein. This comparative example evaluates the repeatability, spike recovery, and clinical performance of the analytical method for reagent sample detection.

[0194] (1) Linearity Validation: Recombinant phosphorylated Tau protein at final concentrations of 0.1 pg / mL, 1 pg / mL, 5 pg / mL, and 30 pg / mL was added to 1 mL of blank plasma to prepare calibration curves. The sample processing and detection were performed according to the procedure in Example 4. Linear fitting was performed using the ratio of the signal intensity of the target protein's corresponding peptide to the signal intensity of the isotopic peptide (peak area ratio) as the ordinate and the target protein concentration as the abscissa. The results were obtained... Figure 3 The calibration curve shown in the figure indicates that the linear correlation coefficient R0 2 =0.9674, indicating that without isotopic peptide signal correction, p-Tau 217 exhibits poor linearity in the concentration range of 0.1-30 pg / ml.

[0195] (2) Repeatability test: Actual samples of different age groups (with known differences in target proteins) were tested separately. The samples were processed in parallel three times, following the processing procedure of Comparative Example 1. The peak area ratio was substituted into the calibration curve equation to calculate the concentration of the actual sample. The results are shown in Table 7. It can be seen that the precision CV of the low concentration sample in Comparative Example 1 is 19.14%, which is greater than 15%.

[0196] Table 7 shows the precision test results of Comparative Example 1.

[0197] 1 0.97 0.11 2 1.13 0.089 3 0.86 0.086 mean 0.99 0.10 CV, % 13.76 19.14

[0198] (3) Accuracy determination: Recombinant phosphorylated Tau protein was added to plasma at final concentrations of 0.15 pg / mL and 0.8 pg / mL, respectively. The plasma was processed and detected according to the operating steps of the kit of this invention. The peak area ratio was substituted into the calibration curve equation to calculate the concentration of the actual sample. The accuracy was calculated as: Accuracy = (Spiked detection result - Background concentration) / Theoretical concentration * 100%. The results are shown in Table 8. It can be seen that the accuracy of Comparative Example 1 is <80%.

[0199] Table 8. Accuracy test results of Example 1

[0200] Test results after spiking 0.23 0.744 Background concentration 0.12 0.12 Accuracy, % 73.33 78.00

[0201] In summary, this invention incorporates stable isotope-labeled p-Tau- during pretreatment. 15The N-peptide can be used for the accurate quantification of Tau-217 protein or its fragments. Because the isotopically labeled protein and the target protein share the same amino acid sequence, immunoenrichment effect, partition behavior, liquid chromatography retention time, mass spectrometry ionization efficiency, and secondary fragmentation ions, and due to their different mass numbers, the ratio of their peak intensities can be obtained on mass spectrometry. Furthermore, the amount of the corresponding hydrolyzed peptide in the sample can be calculated from the amount of the isotopically labeled peptide, and finally, the amount of the corresponding protein can be obtained based on the stoichiometry of the peptide. Especially when added during pretreatment, the entire process (including sample dilution, immunoenrichment, washing, digestion, desalting, etc.) can be monitored, effectively overcoming the losses caused by the extraction of p-Tau217 from the blood matrix, as well as problems such as poor reproducibility in and out of the laboratory and large deviations from the true content.

[0202] Furthermore, the detection method for p-Tau-217 protein or its fragments provided by this invention is simple in procedure, short in detection time, highly sensitive, precise, and reliable in results. Only five steps are required for mass spectrometry pretreatment, reducing the required sample volume by approximately 75%. The washing step is simplified, omitting acid precipitation, acid washing, and solid-phase extraction steps found in existing technologies, and significantly shortening the digestion and incubation time. This invention solves the problems of existing Alzheimer's disease detection kits, such as complex operation, long processing time, low sensitivity, poor precision, and lack of accurate predictive ability for Alzheimer's disease, and has significant application value and significance.

[0203] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting p-Tau 217 protein or a fragment thereof, characterized in that, Includes the following steps: 1) Sample pretreatment: Add sample diluent to the sample at a volume ratio of 1:2, then add... 15 Np-Tau 217 protein was used to obtain solution A; 2) Immunoenrichment: The nanomagnetic microsphere-p-Tau 217 antibody complex was added to solution A and incubated, then passed through a magnetic rack to obtain the purified immunoenriched complex. 3) Washing: The immunoenriched complex was washed with a nonionic surfactant to obtain the washed complex. 4) The cleaned complex was reconstituted with NH4HCO3, and then digested and terminated. 5) Desalination, vacuum concentration, and mass spectrometry injection; 6) Perform mass spectrometry detection and output the results; The above 15 The preparation method of Np-Tau 217 protein includes the following steps: a) Add GSK3β diluted with kinase dilution buffer I to a pre-chilled centrifuge tube, then add GSK3β diluted with kinase detection buffer. 15 N-Tau protein; b) Add ATP diluted with kinase detection buffer, mix thoroughly, and incubate; c) Add GSK3β inhibitor to terminate the reaction, purify, and obtain the desired product. 15 Np-Tau 217 protein; In step a) of the preparation method, the kinase dilution buffer I is used to dilute GSK3β to 0.1 μg / μL, and the added amount is 5 μL-50 μL. The kinase detection buffer is used to dilute... 15 N-Tau concentration is 1 mg / mL, and the added volume is 5 μL-50 μL. In step b) of the preparation method, the ATP diluted with kinase detection buffer is 250 μM, and the added amount is 2.5 μL-12.5 μL. After thorough mixing, it is incubated at 30°C for 30 min. The specific steps of preparation method c) involve adding GSK3β inhibitor as follows: adding an aqueous solution of GSK3β inhibitor SB415286 with a concentration of 100 μg / mL, in an amount of 0.75 μL to 7.5 μL.

2. The detection method according to claim 1, characterized in that, The sample dilution in step 1) includes: 20-80 mM Tris-HCl, 30-80 mM NaCl, 1-10 mM EDTA, and 0.02%-0.2% (w / w) protease inhibitor and 0.02%-0.2% (w / w) phosphatase inhibitor; and / or The sample diluent in step 1) also includes 0.1%-1% by mass of NP40 and / or Tween 20; and / or The steps described in step 1) 15 Np-Tau 217 protein at a concentration of 0.5-5 pg / mL 15 Np-Tau 217 aqueous solution.

3. The detection method according to claim 1, characterized in that, Step 2) includes: adding the nanomagnetic microsphere-p-Tau217 antibody complex to solution A, incubating at room temperature by rotation for at least 2 hours, passing through a magnetic rack to obtain the purified immuno-enriched complex.

4. The detection method according to claim 1, characterized in that, Step 3) includes washing the immunoenriched complex at least three times with 0.1%-0.5% NP40 PBS solution.

5. The detection method according to claim 1, characterized in that, Step 4) includes: adding the washed complex to NH4HCO3 at a concentration of 20-200 mM for reconstitution, then adding trypsin for digestion for at least 8 hours, and finally adding formic acid aqueous solution to terminate the digestion.

6. A detection kit for p-Tau 217 protein or a fragment thereof, characterized in that, include: 1) Pretreatment module: including at least sample diluent and as described in claim 1 15 Np-Tau 217 protein, wherein the sample dilution solution includes: 0.1%-1% NP40, 0.1%-1% Tween 20, 20-80 mM Tris-HCl, 30-80 mM NaCl, 0.02%-0.2% protease inhibitor, and 0.02%-0.2% phosphatase inhibitor; wherein, 15 Np-Tau 217 protein at a concentration of 0.5-5 pg / mL 15 Np-Tau 217 aqueous solution; 2) Immunoenrichment module: including the nanomagnetic microsphere-p-Tau 217 antibody complex; 3) Cleaning and digestion module: including 0.1%-0.5% NP40 PBS solution and 20-200 mM NH4HCO3 solution; 4) Desalination module: includes methanol, 80% acetonitrile aqueous solution, and 1% formic acid aqueous solution; 5) Drying and injection module: includes a 1% (v / v) formic acid aqueous solution.

7. The application of the detection kit for p-Tau 217 protein or fragment thereof according to claim 6 in the preparation of early diagnostic products for Alzheimer's disease.

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