Tau protein antibody-cnbr complex, preparation method, detection method, kit and application thereof

Abstract

The application relates to a Tau protein antibody-CNBr complex and a preparation method, a detection method, a kit and an application thereof, and relates to the field of protein spectrum detection. The detection method of the phosphorylated Tau protein or the fragment thereof comprises the following steps: sample pretreatment, immunological enrichment, cleaning, NH4HCO3 resuspension, desalination, vacuum concentration, mass spectrum sampling and obtaining a detection result. Compared with the prior art, the method has the advantages of simple steps, high sensitivity, good precision, high reliability of a detection result, and the like. The mass spectrum detection pretreatment can be completed only by five steps, the required sample amount is reduced by about 75%, and the problems of the existing Alzheimer's disease detection kit, such as complex operation, high cost, low sensitivity, poor precision, lack of accurate prediction ability for Alzheimer's disease and the like are solved, and the application has important application value and significance.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry detection technology, specifically to a Tau protein antibody-CNBr complex and its preparation method, detection method, kit, and application. Background Technology

[0002] Alzheimer's disease (AD), also known as primary dementia, is a fatal neurodegenerative disease and the most common type of dementia, accounting for approximately 50%-70% of all dementia cases. Its main symptoms include memory loss, cognitive decline, behavioral impairment, and reduced ability to perform daily activities. It can also lead to various complications such as depression and anxiety, posing a significant threat to health. Like other central nervous system (CNS) degenerative diseases, AD is characterized by a disruption in the production, accumulation, and clearance of proteins. In AD, the metabolic disorder of the protein amyloid-β (Aβ) manifests as the massive accumulation of this protein in the brain of patients as amyloid plaques. Additionally, the protein tau accumulates in the brain as tau tangles. AD leads to memory loss, cognitive decline, and ultimately loss of autonomy, resulting in death. This disease imposes a heavy personal and economic burden on patients, families, and society. Due to the severity and increasing prevalence of this disease in the population, there is an urgent need to develop better diagnostic and treatment methods.

[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] Cerebrospinal fluid (CSF) biomarkers are currently recognized as a source of Alzheimer's disease (AD) biomarkers. CSF examination can be used to rule out rare, reversible cognitive impairment diseases and can also help in the active molecular diagnosis of AD. However, CSF isolation and invasive procedures limit the use of these biomarkers in the early diagnosis of AD. Currently, research on blood biomarkers involved in the pathogenesis of AD involves multiple biomarkers, including Aβ, Tau protein, and p-Tau protein; these biomarkers can also be measured more quickly and with less plasma, and can provide important evidence for the early diagnosis of Alzheimer's disease.

[0005] However, existing Alzheimer's disease testing kits often suffer from the following problems: complex operating procedures, high cost, long testing time, low sensitivity, poor precision, narrow linear range, inability to automate and batch testing, lack of specificity detection, and insufficient ability to accurately predict Alzheimer's disease. For example, an article published by Laia Montoliu-Gaya et al. in *Nature Aging* volume 3, 661–669 (2023) discloses a mass spectrometry detection method for p-Tau 217 protein, which includes ten steps such as washing, acid precipitation, acid washing, solid-phase extraction, and digestion incubation. Among them, the washing step alone includes three steps, the digestion incubation time is as long as 18 hours, and the minimum required plasma volume is 1000 μL. It can be seen that the above method is very cumbersome, time-consuming, and has limited sensitivity, making it completely unsuitable for clinical diagnosis and having very limited clinical application significance.

[0006] Antibody-microbead complexes, as the core component of immunoenrichment, largely determine the effectiveness of the immunoenrichment process. Currently, commercially available microbeads mainly include agarose gel microspheres and magnetic microspheres. Although their automation in conversion is limited, agarose beads, due to their larger diameter and sponge-like structure, have a larger surface area and greater binding capacity than magnetic beads. Without antibody saturation, agarose beads can effectively bind a very large amount of target protein. Moreover, when the target protein content is low, such as in early Alzheimer's disease (AD), where only a small number of protein markers can cross the blood-brain barrier from CSF into the peripheral circulation, and their abundance in the blood is extremely low (fM level), requiring increased sample volume (>2ml) to improve sensitivity, agarose gel microspheres offer a significant advantage in ensuring protein enrichment due to the limited capacity of magnetic separation equipment. Furthermore, agarose gel microspheres are more cost-effective, making them indispensable in immunoenrichment. However, due to their large specific surface area and porous structure, the portions of agarose beads not covered by antibodies can freely bind to any adherable substance. This increased non-specific binding leads to an increase in background signal, at which point the "high loading advantage" becomes a "high loading disadvantage." Therefore, reducing non-specific adsorption and ensuring stable application in the detection of phosphorylated Tau protein during immunoenrichment is a pressing issue that needs to be addressed. Summary of the Invention

[0007] In view of this, the main objective of the present invention is to provide a Tau protein antibody-CNBr agarose gel complex and its preparation method, a detection method, kit and application for phosphorylated Tau protein or its fragments, in order to at least partially solve the above-mentioned technical problems.

[0008] To achieve the above objectives, as a first aspect of the present invention, a method for preparing a Tau protein antibody-CNBr agarose gel complex (CNBr-antibody complex) is provided, comprising the following steps:

[0009] 1) Microsphere soaking: Freeze-dried microspheres are mixed with strong acid and soaked to obtain soaked microspheres;

[0010] 2) Washing 1: Wash the soaked microspheres with purified water;

[0011] 3) Washing 2: Wash the soaked microspheres with coupling buffer;

[0012] 4) Coupling: The washed and foamed microspheres and phosphorylated Tau protein antibody were mixed, and coupling buffer and NP40 were added to couple them to obtain phosphorylated Tau protein antibody-CNBr agarose gel complex.

[0013] 5) Blocking 1: Glycine solution was mixed with phosphorylated Tau protein antibody-CNBr agarose gel complex and incubated to occupy the CNBr active site of unbound antibody;

[0014] 6) Blocking 2: Add the BSA to the phosphorylated Tau protein antibody-CNBr agarose gel complex that has undergone the blocking 1 treatment step and mix and incubate to increase non-specific adsorption and occupy well sites, thereby obtaining the Tau protein antibody-CNBr agarose gel complex.

[0015] Preferably, the strong acid in step 1) is any one or a combination of at least two of hydrochloric acid, sulfuric acid, or nitric acid; and / or

[0016] In step 1), the freeze-dried microspheres are mixed with a strong acid and then expanded by rotation at room temperature to obtain expanded microspheres; and / or

[0017] Wash at least 3 times in step 2); and / or

[0018] Wash at least 3 times in step 3); and / or

[0019] Step 3) The coupling buffer has a pH of 8.0-9.0, and the formulation of the coupling buffer includes 0.5-1M NaCl and 0.1-0.5M NaHCO3; and / or

[0020] The phosphorylated Tau protein antibody mentioned in step 4) is a p-Tau 217 protein antibody and / or a p-Tau181 protein antibody; and / or

[0021] Step 4) of the coupling process specifically involves: mixing the foamed microspheres after two washes with the phosphorylated Tau protein antibody, adding coupling buffer and NP 40, and rotating at room temperature for coupling; then centrifuging, removing the supernatant, to obtain the phosphorylated Tau protein antibody-CNBr agarose gel complex; and / or

[0022] Step 5) of the blocking process specifically involves: mixing glycine solution with the phosphorylated Tau protein antibody-CNBr agarose gel complex, and incubating overnight at 2-8°C by rotation to occupy the unbound antibody-active CNBr sites; and / or

[0023] Step 6) Blocking 2 process is as follows: Add BSA PBS solution to the phosphorylated Tau protein antibody-CNBr agarose gel complex that has been blocked 1, and incubate at room temperature by rotation for at least 4 hours to increase non-specific adsorption and occupy well sites.

[0024] Preferably, the method further includes 7) storage: the blocked phosphorylated Tau protein antibody-CNBr agarose gel complex is washed at least 3 times with PBS, then added to the preservation solution and stored at a temperature of 2-8°C. The pH of the preservation solution in step 7) is 7.0-7.5, and the formulation of the preservation solution includes: 0.05% (v / v) ProClin 300 (a preservative produced by Merck), 0.05% (v / v) bovine serum albumin, and 50% (w / w) glycine.

[0025] This invention addresses the problem that the detection sensitivity of phosphorylated Tau protein cannot be improved by increasing the amount of sample due to the low content of phosphorylated Tau protein in blood and the limitations of magnetic microspheres in terms of magnetic separation equipment and cost. It utilizes a CNBr-activated agarose gel antibody complex suitable for large-sample protein detection for immunoenrichment. However, existing CNBr-activated agarose gel antibody complexes exhibit significant non-specific adsorption during target protein capture, meaning a large amount of non-phosphorylated Tau protein is also adsorbed, resulting in poor target protein enrichment. Based on the covalent interaction and spatial structure of CNBr and amino groups on CNBr-activated agarose gel, and by incubating under different conditions according to the antibody-amino acid-BSA sequence, a novel method for preparing the CNBr-activated agarose gel antibody complex is creatively proposed. This method effectively reduces the non-specific adsorption of the CNBr-activated agarose gel antibody complex during the immunoenrichment process of the sample, thereby improving the enrichment effect of phosphorylated Tau protein.

[0026] In a second aspect, the present invention provides a phosphorylated Tau protein antibody-CNBr agarose gel complex prepared by the preparation method described in the first aspect.

[0027] Thirdly, the present invention provides a detection kit for phosphorylated Tau protein or fragments thereof, the kit comprising the Tau protein antibody-CNBr agarose gel complex described in the first aspect.

[0028] Preferably, the kit comprises:

[0029] 1) Pretreatment module: including at least a sample diluent, the sample diluent having the following formulation: 20-80 mM Tris-HCl (trimethylolaminomethane hydrochloride, trimethylamine hydrochloride), 30-80 mM NaCl, and 1-10 mM EDTA (ethylenediaminetetraoxic acid), and 0.02-0.2% by mass of a protease inhibitor, 0.02-0.2% by mass of a phosphatase inhibitor, 0.1%-1% by mass of NP-40 (4-nonylphenyl-polyethylene glycol) and / or Tween 20;

[0030] 2) Immunoenrichment module: including at least a phosphorylated Tau protein antibody-CNBr agarose gel complex;

[0031] 3) Cleaning module: includes at least 10-50mM Tris-HCl, 100-300mM NaCl and 0.5-2mM EDTA;

[0032] 4) Digestion module: includes at least 25-200 mM NH4HCO3, trypsin, and a 5-20% formic acid aqueous solution;

[0033] 5) Desalination module: includes at least methanol, acetonitrile aqueous solution with a volume fraction of 50-80%, and formic acid aqueous solution with a volume fraction of 0.5-1%;

[0034] 6) Drying and injection module: including at least 0.05-0.1% formic acid water by volume, and isotopic peptides as shown in Seq No.1 and / or isotopic peptides as shown in Seq No.2.

[0035] Fourthly, the present invention provides a method for detecting phosphorylated Tau (p-Tau) protein or fragments thereof, comprising the following steps:

[0036] 1) Sample pretreatment: Add sample diluent to the sample to obtain solution A;

[0037] 2) Immunoenrichment: The Tau protein antibody-CNBr agarose gel complex described in the second aspect was added to solution A and incubated by rotation at room temperature to obtain the immunoenriched complex.

[0038] 3) Washing: The immunoenriched complex was washed with Tris-HCl, NaCl and EDTA respectively, and then washed with NH4HCO3 to obtain the washed complex.

[0039] 4) The cleaned complex was reconstituted with NH4HCO3, and then digested and terminated.

[0040] 5) Desalting, vacuum concentration, and mass spectrometry injection;

[0041] 6) Use mass spectrometry for detection and output the results.

[0042] Preferably, the phosphorylated Tau protein may be p-Tau 217 and / or p-Tau 181; and / or

[0043] The sample dilution 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, 0.1%-1% NP-40 and / or Tween 20; and / or

[0044] The purpose of the sample diluent is to reduce the viscosity of the plasma, allowing antibody microspheres to be evenly distributed in the plasma and increasing the capture probability of the target protein; to create a buffer system and the presence of nonionic surfactants, which helps 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 adding protease inhibitors and phosphatase inhibitors, since blood contains a series of biologically active enzymes that can enzymatically hydrolyze proteins in vitro.

[0045] The sample is a CSF, blood, or plasma sample; and / or

[0046] Step 2) includes: adding 10-40 μL of CNBr-activated agarose gel antibody complex (CNBr-antibody) to solution A and incubating by rotation at room temperature for at least 2 hours; and / or

[0047] The CNBr-antibody microsphere complex contains a monoclonal antibody that specifically recognizes phosphorylated Tau protein sites. Its purpose is to capture target proteins in plasma, yielding an immuno-enriched complex (i.e., the target protein-antibody-CNBr microsphere complex). The CNBr microspheres have a large diameter and a sponge-like structure, allowing for efficient capture of the target protein without antibody saturation. Separation of the solution and the target protein-antibody-CNBr microsphere complex is achieved by centrifugation.

[0048] The washing solution in step 3) includes: 10-50 mM Tris-HCl, 100-300 mM NaCl, and 0.5-2 mM EDTA. The immunoenriched complex is washed at least three times with this washing solution. The purpose of washing is to maintain ideal protein interactions, remove non-specific adsorption on the surface of the CNBr-microsphere complex, and / or

[0049] Step 4) includes at least the following: washing the washed complex at least twice with 25-200 mM NH4HCO3, replacing the solvent; reconstituted with 25-200 mM NH4HCO3, then adding trypsin and shaking to digest, and finally terminating the digestion with formic acid solution; the main function of the digestion solution is to provide an alkaline hydrolysis environment to ensure the highest activity of trypsin (optimal pH 8-9), breaking down phosphorylated Tau protein into specific peptides that can be detected by mass spectrometry; and / or

[0050] Step 5) includes desalination, which includes:

[0051] ① Add 200 μL of methanol to the C18 centrifuge column and centrifuge at 2500 rpm for 5 min, then discard the filtrate;

[0052] ② Add 200 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0053] ③ Add 200 μL of 1% formic acid aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0054] ④ Add the acidified sample to a C18 centrifuge column and centrifuge at 1800 rpm for 10 min, then discard the filtrate;

[0055] ⑤ Add 80 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and collect the filtrate; and / or

[0056] Step 5) involves vacuum concentration and mass spectrometry injection, which includes drying the desalted sample using a vacuum concentration centrifuge; and / or

[0057] Step 5) of the mass spectrometry injection includes: reconstituted the dried sample with 0.1% formic acid solution, centrifuged by shaking, and then injected; and / or

[0058] Step 6) Add the isotopic peptide fragment as shown in Seq No. 1 during mass spectrometry injection: TPSLP T PPT R (Among them, amino acid T at position 6 (see bold and underlined) is phosphorylated, and all C atoms of amino acid R at position 10 (see bold and underlined) are isotopically labeled.) 13C-labeled, all N atoms of amino acid R at position 10 (see bold and underlined) are isotopically... 15 N-labeled) and / or isotopic peptides as shown in Seq No. 2: TPPAPKTPPSSGEPPK (where all C atoms of amino acid K at position 6 are isotopically labeled) 13 C-labeled, all N atoms of amino acid K at position 6 are isotopically... 15 N-labeled, amino acid T at position 7 is phosphorylated, and all C atoms of amino acid K at position 16 are isotopically labeled. 13 C-labeled, all N atoms of amino acid K at position 16 are isotopically... 15 (N-marker). and / or

[0059] The mass spectrometry used in this invention is orbital ion trap mass spectrometry.

[0060] The principle of this invention is as follows: This invention utilizes immunoenrichment to specifically adsorb the target protein in a sample onto immunobeads (CNBr-antibody). Cyanogen bromide (CNBr) reacts with the hydroxyl groups on agarose (Sepharose) to form activated cyanate ester groups, which bind to the specific antibody. Furthermore, the agarose beads have a large diameter, providing a larger surface area and binding capacity, enabling efficient antibody capture. These immunobeads are then placed in a biological matrix for immunoprecipitation. Due to their sponge-like structure, they can still capture a large amount of target protein even when the antibody is unsaturated. A washing solution with a certain ionic strength and surfactant removes some non-specific adsorbed impurities, while the target protein bound to the antibody and covalently bound is retained. Then, trypsin digests the antigen-antibody complex cross-linked in the agarose, separating them. The antibody binds to CNBr, and the target protein is released. Subsequently, after digestion, desalting, and reconstitution steps, the target protein is completely separated and converted into the target peptide. The target peptide (a specific peptide of the target protein) and the stable isotope-labeled peptide have the same physical or chemical properties. Except for slight differences in mass, they fragment in the same way during mass spectrometry, thus allowing for effective monitoring and correction of signal fluctuations caused by matrix influences during mass spectrometry detection. Furthermore, since the mass spectrometry signal is proportional to the analyte concentration, the concentration of the target peptide in the sample can be calculated from the signal of a known concentration of the stable isotope-labeled peptide in the mass spectrometer, and further, the concentration of the target protein in the sample can be calculated. Therefore, using the technique provided herein, endogenous proteins and peptide fragments can be quantified and can be used for the diagnosis and / or treatment of subjects with or at risk of developing neurological or neurodegenerative diseases.

[0061] Fifthly, the application of a phosphorylated Tau protein antibody-CNBr agarose gel complex as described in the second aspect in the early diagnosis of Alzheimer's disease.

[0062] In a sixth aspect, the present invention provides a detection kit for phosphorylated Tau protein or fragments thereof as described in the third aspect for the early diagnosis of Alzheimer's disease.

[0063] Based on the above technical solutions, it can be seen that the detection method, kit, and application of phosphorylated Tau protein of the present invention have at least one of the following beneficial effects compared with the prior art:

[0064] The detection method for phosphorylated Tau protein or its fragments provided by this invention is simple, highly sensitive, precise, and provides highly reliable results. It requires only five steps 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. Furthermore, the digestion and incubation time is significantly shortened. This invention can be used for the analysis and detection of early diagnostic biomarkers for Alzheimer's disease, effectively improving the capture efficiency of phosphorylated Tau protein and enhancing the specificity and sensitivity of early Alzheimer's disease diagnosis and screening. This invention solves the problems of existing Alzheimer's disease detection kits, such as complex operation, high cost, low sensitivity, poor precision, and lack of accurate predictive ability for Alzheimer's disease, and has significant application value and significance. Attached Figure Description

[0065] Figure 1A , 1B These are the calibration curves for p-Tau181 and p-Tau217 in Example 1, respectively.

[0066] Figure 2A , 2B The calibration curves for p-Tau181 and p-Tau217 in Comparative Example 1 are shown respectively.

[0067] Figure 3A , 3B The calibration curves are for p-Tau181 and p-Tau217 in Comparative Example 2, respectively. Detailed Implementation

[0068] 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 operations. Specifically, as a first aspect of this invention, a method for preparing a Tau protein antibody-CNBr agarose gel complex (CNBr-antibody complex) is provided, comprising the following steps:

[0069] 1) Microsphere soaking: The freeze-dried microspheres are soaked in hydrochloric acid to obtain soaked microspheres;

[0070] 2) Washing 1: Wash the soaked microspheres with purified water;

[0071] 3) Washing 2: Wash the soaked microspheres with coupling buffer;

[0072] 4) Coupling: The washed and foamed microspheres are mixed with phosphorylated Tau protein antibody and coupled with coupling buffer and NP40 to obtain phosphorylated Tau protein antibody-CNBr agarose gel complex.

[0073] 5) Blocking 1: Glycine solution was mixed with phosphorylated Tau protein antibody-CNBr agarose gel complex and incubated to occupy the CNBr active site of unbound antibody;

[0074] 6) Blocking 2: Add BSAPBS solution to the phosphorylated Tau protein antibody-CNBr agarose gel complex that has undergone Blocking 1 and incubate to increase non-specific adsorption and occupy well sites, thereby obtaining the Tau protein antibody-CNBr agarose gel complex.

[0075] In a preferred embodiment, the strong acid in step 1) is any one or a combination of at least two of hydrochloric acid, sulfuric acid, or nitric acid; and / or

[0076] In step 1), the freeze-dried microspheres are mixed with a strong acid and then expanded by rotation at room temperature to obtain expanded microspheres; and / or

[0077] Wash at least 3 times in step 2); and / or

[0078] Wash at least 3 times in step 3); and / or

[0079] Step 3) The coupling buffer has a pH of 8.0-9.0, and the formulation of the coupling buffer includes 0.5M NaCl and 0.1M NaHCO3; and / or

[0080] The phosphorylated Tau protein antibody is p-Tau 217 polyclonal sheep anti-rabbit protein antibody and / or p-Tau 181 polyclonal sheep anti-rabbit protein antibody; and / or

[0081] Step 4) of the coupling process specifically involves: mixing the foamed microspheres after two washes with the phosphorylated Tau protein antibody, adding coupling buffer and NP 40, and rotating at room temperature for coupling; then centrifuging, removing the supernatant, to obtain the phosphorylated Tau protein antibody-CNBr agarose gel complex; and / or

[0082] Step 5) of the blocking process specifically involves: mixing glycine solution with the phosphorylated Tau protein antibody-CNBr agarose gel complex, and incubating overnight at 2-8°C by rotation to occupy the unbound antibody-active CNBr sites; and / or

[0083] Step 6) of the blocking 2 treatment specifically involves adding BSA PBS solution to the phosphorylated Tau protein antibody-CNBr agarose gel complex that has undergone blocking 1, and incubating at room temperature by rotation for at least 4 hours to increase non-specific adsorption and occupancy of well sites; and / or

[0084] Step 7) The pH of the preservation solution is 7.0-7.5, and the formulation of the preservation solution includes: 0.05% ProClin 300 by volume, 0.05% bovine serum albumin by volume, and 50% glycine by mass.

[0085] In a second aspect, the present invention provides a phosphorylated Tau protein antibody-CNBr agarose gel complex prepared by the preparation method described in the first aspect.

[0086] Thirdly, the present invention provides a phosphorylated Tau protein detection kit, the kit comprising at least the Tau protein antibody-CNBr agarose gel complex described in the first aspect.

[0087] In a preferred embodiment, the kit includes at least:

[0088] 1) Pretreatment module: including at least a sample diluent, the sample diluent having the following formulation: 20-80 mM Tirs-HCl, 30-80 mM NaCl, and 1-10 mM EDTA (ethylenediaminetetraoxic acid), and 0.02-0.2% by mass of protease inhibitor and phosphatase inhibitor, 0.1%-1% by mass of NP-40 (4-nonylphenyl-polyethylene glycol) and / or Tween 20;

[0089] 2) Immunoenrichment module: including at least a phosphorylated Tau protein antibody-CNBr agarose gel complex;

[0090] 3) Cleaning module: includes at least 10-50mM Tris-HCl, 100-300mM NaCl and 0.5-2mM EDTA;

[0091] 4) Digestion module: includes at least 25-200 mM NH4HCO3, trypsin, and a 5-20% formic acid aqueous solution;

[0092] 5) Desalination module: includes at least methanol, acetonitrile aqueous solution with a volume fraction of 50-80%, and formic acid aqueous solution with a volume fraction of 0.5-1%;

[0093] 6) Drying and injection module: including at least 0.05-0.1% formic acid water by volume, and isotopic peptides as shown in Seq No.1 and / or isotopic peptides as shown in Seq No.2.

[0094] Fourthly, the present invention provides a method for detecting phosphorylated Tau (p-Tau) protein or fragments thereof, comprising at least the following steps:

[0095] 1) Sample pretreatment: Add sample diluent to the sample to obtain solution A;

[0096] 2) Immunoenrichment: The Tau protein antibody-CNBr agarose gel complex described in the second aspect was added to solution A and incubated by rotation at room temperature to obtain the immunoenriched complex.

[0097] 3) Washing: The immunoenriched complex was washed with Tris-HCl, NaCl and EDTA respectively, and then washed with NH4HCO3 to obtain the washed complex.

[0098] 4) The cleaned complex was reconstituted with NH4HCO3, and then digested and terminated.

[0099] 5) Desalting, vacuum concentration, and mass spectrometry injection;

[0100] 6) Use mass spectrometry for detection and output the results.

[0101] In a preferred embodiment, the phosphorylated Tau protein may be p-Tau 217 and / or p-Tau181; and / or

[0102] The sample dilution in step 1) includes: 20-80 mM Tirs-HCl, 30-80 mM NaCl, 1-10 mM EDTA (ethylenediaminetetraoxic acid), 0.02%-0.2% protease inhibitor, 0.02%-0.2% phosphatase inhibitor, and 0.1%-1% NP-40 and / or Tween 20; and / or

[0103] The sample is a CSF, blood, or plasma sample; and / or

[0104] Step 2) includes: adding 10-40 μL of CNBr-activated agarose gel antibody complex (CNBr-antibody) to solution A and incubating by rotation at room temperature for at least 2 hours; and / or

[0105] Step 3) involves washing with a solution comprising 10-50 mM Tris-HCl, 100-300 mM NaCl, and 0.5-2 mM EDTA. The immunoenriched complex is washed at least three times with this solution. The purpose of washing is to maintain ideal protein interactions, remove non-specific adsorption from the surface of the CNBr-microsphere complex, and / or

[0106] Step 4) includes: washing the washed complex at least twice with 25-200 mM NH4HCO3, replacing the solvent; reconstituted with 25-200 mM NH4HCO3, then adding trypsin and shaking to digest, and finally terminating the digestion with formic acid solution; the main function of the digestion solution is to provide an alkaline hydrolysis environment to ensure the highest activity of trypsin (optimal pH 8-9), breaking down phosphorylated Tau protein into specific peptides that can be detected by mass spectrometry; and / or

[0107] Step 5) includes desalination, which includes:

[0108] ① Add 200 μL of methanol to the C18 centrifuge column and centrifuge at 2500 rpm for 5 min, then discard the filtrate;

[0109] ② Add 200 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0110] ③ Add 200 μL of 1% formic acid aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0111] ④ Add the acidified sample to a C18 centrifuge column and centrifuge at 1800 rpm for 10 min, then discard the filtrate;

[0112] ⑤ Add 80 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and collect the filtrate; and / or

[0113] Step 5) involves vacuum concentration and mass spectrometry injection, which includes drying the desalted sample using a vacuum concentration centrifuge; and / or

[0114] Step 5) of the mass spectrometry injection includes: reconstituted the dried sample with 0.1% formic acid solution, centrifuged by shaking, and then injected; and / or

[0115] Step 6) Add the isotopic peptides shown in Seq No. 1 and Seq No. 2 during mass spectrometry injection. And / or

[0116] The mass spectrometry used in this invention is orbital ion trap mass spectrometry.

[0117] Fifthly, the application of a phosphorylated Tau protein antibody-CNBr agarose gel complex as described in the second aspect in the early diagnosis of Alzheimer's disease.

[0118] In a sixth aspect, the present invention provides a detection kit for phosphorylated Tau protein or fragments thereof as described in the third aspect for the early diagnosis of Alzheimer's disease.

[0119] 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.

[0120] 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 plasma was extracted from each peripheral blood sample, it was stored at -80℃ for later use.

[0121] The important reagents used in the specific embodiments of this invention are shown in Table 1. Mass spectrometry was performed using an Exploris 480 (Thermofisher Scientific) orbital ion trap mass spectrometer with the following instrument parameters: Isolation window: 0.7; HCD: 30%; Orbitrap Resolution: 45000; Scan range: 120-1500; RF lens: 50%; AGC: 100%; Maximum injection time: 110; Chromatographic analysis time: 30 min; Injection volume: 0.5 μL.

[0122] Table 1. Reagents used in specific embodiments of the present invention.

[0123]

[0124]

[0125] Example 1

[0126] This study used plasma samples from healthy individuals as the research subject and employed CNBr-activated agarose gel antibody complex to perform immunoenrichment detection of phosphorylated Tau protein in the samples.

[0127] (1) Phosphorylated Tau protein antibody-CNBr agarose gel complex and its preparation

[0128] 1) Microsphere soaking: Weigh 0.01-0.1g of dry microspheres into a 15mL Corning centrifuge tube, then add 5-15mL of 3mM hydrochloric acid (pH=2.5) and soak at room temperature for 60min by rotation.

[0129] 2) Washing 1: Wash 3 times with 5-15mL purified water each time (rotate and wash for 3min, centrifuge at 3000rpm for 3min);

[0130] 3) Wash 2: 5 mL coupling buffer (0.5 M NaCl, 0.1 M NaHCO3, pH 8.3) 3 times, 5-15 mL each time (rotate and wash for 3 min, centrifuge at 3000 rpm for 3 min);

[0131] 4) Coupling: Take 5-50 μL of phosphorylated Tau antibody and 10-100 μL of microspheres and place them in a 1.5 mL EP tube. Then add 250 μL of coupling buffer and 2 μL of NP40 and rotate to couple at room temperature (at least 2 h). After coupling, centrifuge at 2500 rpm for 5 min at 28 °C, remove the supernatant, and obtain the microbead antibody complex.

[0132] 5) Blocking 1: Add 0.5-5 mL of 0.5 M glycine solution to the microbead antibody complex and incubate overnight (>12 h) at 2-8 °C by rotation to occupy the CNBr active site of the unbound antibody;

[0133] 6) Blocking 2: Add 0.5-5 mL of 0.1% BSA in PBS solution to the microbead antibody complex after blocking 1, and incubate at room temperature for 4 h by rotation to increase non-specific adsorption and occupancy of pore sites;

[0134] 7) Storage: After washing the blocked microbead antibody complex three times with 1 ml PBS, add it to the preservation solution. The preservation solution formula is: 0.05% (v / v) ProClin 300 + 0.05% (v / v) bovine serum albumin (BSA) + 50% (w / v) glycine (Gly) pH=7.3. Store at 2-8 degrees Celsius for 2 weeks.

[0135] (2) Detection method for phosphorylated Tau (p-Tau) protein or its fragments

[0136] 1) Sample pretreatment: Add sample diluent to 4 mL of sample at a 1:1 ratio to obtain solution A; the diluent includes: 0.5% NP40 + 50 mM Tris-HCl + 60 mM NaCl + 0.1% protease inhibitor + 0.1% phosphatase inhibitor mixed solution;

[0137] 2) Immunoenrichment: Add 20 μL of immunobeads (CNBr-antibody) to solution A and incubate at room temperature by rotation for at least 2 h to obtain the immunoenriched protein-antibody-microsphere complex;

[0138] 3) Washing: The immunoenriched complex was washed three times with 1 mL of 25 mM Tris-HCl + 150 mM NaCl + 1 mM EDTA to obtain the washed complex.

[0139] 4) Wash the washed complex twice with 1 mL of 50 mM NH4HCO3, replacing the solvent. Redissolve the washed complex in 100 μL of 50 mM NH4HCO3, then add 1 μL of trypsin and digest at 37℃ and 1000 rpm for 8 h with shaking. Finally, add 10 μL of 10% formic acid aqueous solution to stop the digestion.

[0140] 5) Desalination, including:

[0141] ① Add 200 μL of methanol to the C18 centrifuge column and centrifuge at 2500 rpm for 5 min, then discard the filtrate;

[0142] ② Add 200 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0143] ③ Add 200 μL of 1% formic acid aqueous solution, centrifuge at 2500 rpm for 5 min, and discard the filtrate;

[0144] ④ Add the acidified sample to a C18 centrifuge column and centrifuge at 1800 rpm for 10 min, then discard the filtrate;

[0145] ⑤ Add 80 μL of 80% acetonitrile aqueous solution, centrifuge at 2500 rpm for 5 min, and collect the filtrate;

[0146] 6) Vacuum concentration and mass spectrometry injection, including: drying the desalted sample using a vacuum concentration centrifuge;

[0147] 7) Mass spectrometry injection, including: reconstitute the dried sample with 10 μL of 0.1% formic acid water containing isotopic peptides as shown in Seq No.1 and isotopic peptides as shown in Seq No.2, vortex mix for 3 min, incubate for 10 s and then inject the sample.

[0148] In this embodiment, the performance of the above-mentioned CNBr agarose gel-phosphorylated Tau protein antibody complex in the detection of phosphorylated Tau in plasma was investigated. The investigated items included linearity, repeatability, and accuracy. The results are as follows:

[0149] 1) Linearity Validation: Recombinant phosphorylated Tau 181 protein at final concentrations of 1 pg / mL, 5 pg / mL, 10 pg / mL, and 20 pg / mL, and recombinant phosphorylated Tau 217 protein at concentrations of 0.15 pg / mL, 1 pg / mL, 2 pg / mL, and 10 pg / mL, were added to 1 mL of blank matrix to prepare calibration curves. The sample processing and detection were performed according to the procedure in Example 1. The ratio of the signal intensity of the target protein's corresponding peptide to the signal intensity of the isotopic peptide (peak area ratio) was used as the ordinate, and the concentration of the target protein was used as the abscissa for linear fitting. The calibration curves shown in Figure 1 were obtained. As can be seen from the figure, the linear correlation coefficient Rp-Tau 181 is [missing value].2 =0.9977, indicating that p-Tau 181 has good linearity in the concentration range of 1-20 pg / ml; the linear correlation coefficient R of p-Tau 217 is 0.9977. 2 =0.9986, indicating that p-Tau 217 exhibits good linearity in the concentration range of 0.15-10 pg / ml.

[0150] 2) Repeatability verification: p-Tau 217 at final concentrations of 0.25 pg / mL and 1 pg / mL, and p-Tau 181 recombinant phosphorylated Tau protein at final concentrations of 4 pg / mL and 16 pg / mL were added to plasma, respectively. The treatment was carried out according to the procedure in Example 1, and 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 2. It can be seen that the precision CV of the determination results in Example 1 is <15%.

[0151] Table 2 Repeatability verification data from Example 1

[0152]

[0153] 3) Accuracy Verification: Recombinant phosphorylated Tau protein at final concentrations of p-Tau 217 (0.25 pg / mL and 1 pg / mL) and p-Tau 181 (4 pg / mL and 16 pg / mL) was added to plasma. The plasma background and spiked plasma were processed according to the procedure in Example 1. The peak area ratio was substituted into the calibration curve equation to calculate the actual sample concentration. The accuracy was calculated as: Accuracy = (Spiked Detection Result - Background Concentration) / Theoretical Concentration * 100%. The results are shown in Table 3. It can be seen that the accuracy of Example 1 is >80%.

[0154] Table 3. Accuracy verification data for Example 1

[0155]

[0156] Example 2

[0157] The difference from Example 1 lies in the concentration of the CNBr-antibody complex during preparation, and the volume of 0.5M glycine in the blocking solution (0.5, 2.5, and 5 mL). In this example, the performance of the above-mentioned CNBr agarose gel-phosphorylated Tau protein antibody complex in the detection of phosphorylated Tau in plasma was investigated. The investigated parameters were linearity, repeatability, accuracy, and signal-to-noise ratio (S / N) near the limit of quantitation. The results are shown in Table 4: the linear correlation coefficient R between the two phosphorylated Tau proteins... 2The CV of p-Tau271 was <15%, and the CV of p-Tau181 was <8.49%, with both having an accuracy of >79%. The signal-to-noise ratio (S / N) of samples near the limit of quantitation was >15, indicating that the volume of glycine in the blocking solution of this method can meet the detection requirements in the range of 0.5-2 mL.

[0158] Table 4 Statistical table of performance index verification results in Example 2

[0159]

[0160]

[0161] Example 3

[0162] The difference from Example 1 lies in the concentration of NaCl in the washing solution, which is 100mM, 200mM, and 300mM, respectively. In this example, the performance of the above-mentioned CNBr agarose gel-phosphorylated Tau protein antibody complex in the detection of phosphorylated Tau in plasma was investigated. The investigated parameters were linearity, repeatability, accuracy, and signal-to-noise ratio (S / N) near the limit of quantitation. The results are shown in Table 5: the linear correlation coefficient R between the two phosphorylated Tau proteins... 2 The CV of p-Tau271 was >0.992, and the CV of p-Tau181 was <10%, with both having an accuracy >75%. The signal-to-noise ratio (S / N) of samples near the limit of quantitation was >20, indicating that the concentration of NaCl in the cleaning solution of this method can meet the detection requirements in the range of 100-300 mM.

[0163] Table 5 Statistical table of performance index verification results in Example 3

[0164]

[0165] Example 4

[0166] The difference from Example 1 lies in the concentration of EDTA in the washing solution, which is 0.5 mM and 2 mM, respectively. In this example, the performance of the above-mentioned CNBr agarose gel-phosphorylated Tau protein antibody complex in the detection of phosphorylated Tau in plasma was investigated, including linearity, repeatability, accuracy, and signal-to-noise ratio (S / N) near the limit of quantitation. The results are shown in Table 6: the linear correlation coefficient R between the two phosphorylated Tau proteins... 2 The CV of p-Tau271 was >0.990, % <9%, and the CV of p-Tau181 was <12%, with both having an accuracy >79%. The signal-to-noise ratio (S / N) of samples near the limit of quantitation was >10, indicating that the concentration of EDTA in the cleaning solution of this method can meet the detection requirements in the range of 0.5-2 mM.

[0167] Table 6 Statistical table of performance index verification results of Example 4

[0168]

[0169] Example 5

[0170] Example 5 differs from Example 1 in the concentration of Tris-HCl in the washing solution, which are 10, 25, and 40 mM, respectively. In this example, the performance of the above-mentioned CNBr agarose gel-phosphorylated Tau protein antibody complex in detecting phosphorylated Tau in plasma was investigated, including linearity, repeatability, accuracy, and signal-to-noise ratio (S / N) near the limit of quantitation. The results are shown in Table 7: the linear correlation coefficient R between the two phosphorylated Tau proteins... 2 The CV of p-Tau271 was >0.991, and the CV of p-Tau181 was <9%, with both having an accuracy >80%. The signal-to-noise ratio (S / N) of samples near the limit of quantitation was >14, indicating that the concentration of Tris-HCl in the cleaning solution of this method can meet the detection requirements in the range of 10-40 mM.

[0171] Table 7 Statistical table of performance index verification results in Example 5

[0172]

[0173] Comparative Example 1

[0174] The difference from Example 1 is that no secondary blocking is performed. In this comparative example, the performance of the above-mentioned CNBr agarose gel-phosphorylated Tau protein antibody complex in the detection of phosphorylated Tau in plasma was investigated. The investigated items were linearity, repeatability, accuracy, and signal-to-noise ratio (S / N) of samples near the limit of quantitation. The results are as follows:

[0175] (1) Linearity verification and sensitivity test: Recombinant phosphorylated Tau181 protein at final concentrations of 1 pg / mL, 5 pg / mL, 10 pg / mL, and 20 pg / mL, and recombinant phosphorylated Tau217 protein at concentrations of 0.15 pg / mL, 1 pg / mL, 2 pg / mL, and 10 pg / mL were added to 1 mL of blank matrix to prepare calibration curves. The sample processing and detection were performed according to the sample processing procedure in Example 1. The ratio of the signal intensity of the target protein's corresponding peptide to the signal intensity of the isotopic peptide (peak area ratio) was used as the ordinate, and the concentration of the target protein was used as the abscissa for linear fitting. The calibration curves shown in Figure 2 were obtained. As can be seen from the figure, the linear correlation coefficient R of p-Tau181 is... 2 =0.9958, indicating that p-Tau181 has good linearity in the concentration range of 1-20 pg / ml; the linear correlation coefficient R of p-Tau217 is 0.9958.2 =0.9781, indicating that p-Tau217 exhibits poor linearity in the concentration range of 0.15–10 pg / mL. Furthermore, the chromatograms of p-Tau181 and p-Tau217 in samples near the limit of quantitation in Comparative Example 1 show that p-Tau217 has a low signal-to-noise ratio at 0.15 pg / mL, failing to meet the requirements for accurate quantification.

[0176] (2) Repeatability verification: Recombinant phosphorylated Tau protein with final concentrations of p-Tau 217: 0.25 pg / mL and 1 pg / mL, and p-Tau 181: 4 pg / mL and 16 pg / mL were added to plasma, respectively. The plasma was processed according to the procedure in Example 1, and 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 8. It can be seen that the CV of low concentration p-Tau217 in Comparative Example 1 was 15.35%, and the CV of low concentration p-Tau181 was 13.71%. Without secondary blocking, the active site and surface pore size were not fully occupied. During immunoprecipitation, the target protein and impurity proteins competed with each other and the competition was random, resulting in poor repeatability between samples.

[0177] Table 8. Repeatability results data for Comparative Example 1

[0178]

[0179] (3) Accuracy verification: Recombinant phosphorylated Tau protein with final concentrations of p-Tau 217 (0.25 pg / mL and 1 pg / mL) and p-Tau 181 (4 pg / mL and 16 pg / mL) was added to plasma. The plasma background and spiked plasma were processed according to the procedure in Example 1, and the peak area ratio was substituted into the calibration curve equation to calculate the concentration of the actual sample. The accuracy was calculated as (spiked detection result - background concentration) / theoretical concentration * 100%. The results are shown in Table 9. It can be seen that the accuracy of p-Tau217 in Comparative Example 1 is only 68.8%, which is much lower than that in Example 1. Without secondary blocking, the active site and surface pore size are not fully occupied. During immunoprecipitation, the target protein and impurity proteins compete with each other randomly, resulting in a decrease in the target protein capture efficiency.

[0180] Table 9 shows the accuracy verification results for Comparative Example 1.

[0181]

[0182] Comparative Example 2

[0183] The difference from Example 1 is that the washing solution was formulated as a 1% (v / v) NP40 PBS solution. In this comparative example, the performance of the above-mentioned CNBr agarose gel-phosphorylated Tau protein antibody complex in the detection of phosphorylated Tau in plasma was investigated. The investigated parameters were linearity, repeatability, accuracy, and signal-to-noise ratio (S / N) near the limit of quantitation. The results are as follows:

[0184] (1) Linearity Validation: Recombinant phosphorylated Tau181 protein at final concentrations of 1 pg / mL, 5 pg / mL, 10 pg / mL, and 20 pg / mL, and recombinant phosphorylated Tau217 protein at concentrations of 0.15 pg / mL, 1 pg / mL, 2 pg / mL, and 10 pg / mL, respectively, were added to 1 mL of blank matrix to prepare calibration curves. The samples were processed and detected according to the sample processing procedure in Example 1. The ratio of the signal intensity of the target protein's corresponding peptide to the signal intensity of the isotopic peptide (peak area ratio) was used as the ordinate, and the concentration of the target protein was used as the abscissa for linear fitting. The calibration curves shown in Figure 3 are as follows: The linear correlation coefficient R² for p-Tau181 is 0.9909, indicating good linearity within the concentration range of 1–20 pg / mL; the linear correlation coefficient R² for p-Tau217 is 0.9906, indicating good linearity within the concentration range of 0.15–10 pg / mL. Furthermore, the chromatograms of p-Tau181 and p-Tau217 in samples near the quantitation limit in Comparative Example 2 show that the signal-to-noise ratio of p-Tau217 at 0.15 pg / mL is approximately 10, which basically meets the quantification requirements.

[0185] (2) Repeatability Validation: Recombinant phosphorylated Tau protein at final concentrations of p-Tau 217 (0.25 pg / mL and 1 pg / mL) and p-Tau 181 (4 pg / mL and 16 pg / mL) was added to plasma, and the plasma was processed according to the procedure in 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 10. It can be seen that the precision CV of the p-Tau 217 determination results in Comparative Example 2 were 19.42% and 20.40%, respectively, with CV% > 15%. Compared with the above examples, the repeatability of p-Tau 217 decreased when the washing solution formulation was changed.

[0186] Table 10. Repeatability results data for Comparative Example 2

[0187]

[0188] (3) Accuracy verification: Recombinant phosphorylated Tau protein with final concentrations of p-Tau 217 (0.25 pg / mL and 1 pg / mL) and p-Tau 181 (4 pg / mL and 16 pg / mL) was added to plasma. The plasma background and spiked plasma were processed according to the procedure in Example 1, and the peak area ratio was substituted into the calibration curve equation to calculate the concentration of the actual sample. The accuracy was calculated as (spiked detection result - background concentration) / theoretical concentration * 100%. The results are shown in Table 11. It can be seen that the accuracy of Comparative Example 2 decreased, basically to around 70%.

[0189] Table 11 Accuracy verification results for Comparative Example 2

[0190]

[0191] In summary, the detection method for phosphorylated Tau protein or its fragments provided by this invention is simple, highly sensitive, precise, and provides highly reliable results. It requires only five steps for mass spectrometry pretreatment, reducing the required sample volume by approximately 75%. The washing step is simplified by omitting acid precipitation, acid washing, and solid-phase extraction steps found in existing technologies. Furthermore, the digestion and incubation time is significantly shortened. This invention can be used for the analysis and detection of early diagnostic biomarkers for Alzheimer's disease, effectively improving the capture efficiency of phosphorylated Tau protein and enhancing the specificity and sensitivity of early Alzheimer's disease diagnosis and screening. This invention solves the problems of existing Alzheimer's disease detection kits, such as complex operation, high cost, low sensitivity, poor precision, and lack of accurate predictive ability for Alzheimer's disease, and has significant application value and significance.

[0192] 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 phosphorylated Tau protein detection kit, characterized in that, The kit includes: (1) Pretreatment module: including sample diluent, the sample diluent having the following formulation: 20-80mM Tris-HCl, 30-80mM NaCl and 1-10mM EDTA, and 0.02%-0.2% by mass of protease inhibitor, 0.02%-0.2% by mass of phosphatase inhibitor, 0.1%-1% by mass of NP40 and / or Tween 20; (2) Immunoenrichment module: including phosphorylated Tau protein antibody-CNBr agarose gel complex; (3) Cleaning module: including 10-50mM Tris-HCl, 100-300mM NaCl and 0.5-2mM EDTA; (4) Digestion module: including 25-200mM NH4HCO3, trypsin and 5-20% formic acid aqueous solution; (5) Desalination module: including methanol, acetonitrile aqueous solution with a volume fraction of 50%-80%, and formic acid aqueous solution with a volume fraction of 0.5%-1%; (6) Drying and injection module: including formic acid water with a volume fraction of 0.05%-0.1%, and isotopic peptides as shown in Seq No. 1 and / or isotopic peptides as shown in Seq No.

2. The preparation method of the phosphorylated Tau protein antibody-CNBr agarose gel complex includes the following steps: 1) Microsphere soaking: Freeze-dried microspheres are mixed with strong acid and soaked to obtain soaked microspheres; 2) Washing 1: Wash the soaked microspheres with purified water; 3) Washing 2: Wash the soaked microspheres with coupling buffer; 4) Coupling: The washed and foamed microspheres and phosphorylated Tau protein antibody were mixed, and coupling buffer and NP 40 were added to couple them to obtain phosphorylated Tau protein antibody-CNBr agarose gel complex. 5) Blocking 1: Incubate the glycine solution with the phosphorylated Tau protein antibody-CNBr agarose gel complex; 6) Blocking 2: Add PBS solution containing BSA to the phosphorylated Tau protein antibody-CNBr agarose gel complex that has undergone Blocking 1, mix and incubate to obtain the Tau protein antibody-CNBr agarose gel complex. 7) Storage: After washing, the blocked microbead antibody complex is added to the storage solution. Wherein, the strong acid mentioned in step 1) is any one or a combination of at least two of hydrochloric acid, sulfuric acid, or nitric acid; and / or In step 1), the freeze-dried microspheres are mixed with a strong acid and then expanded by rotation at room temperature to obtain expanded microspheres; and / or In step 2), wash with purified water at least 3 times; and / or In step 3), wash at least 3 times with coupling buffer; and / or The coupling buffer in step 3) has a pH of 8.0-9.0, and its formulation includes 0.5-1M NaCl and 0.1-0.5M NaHCO3; and / or The phosphorylated Tau protein antibody mentioned in step 4) is a p-Tau 217 protein antibody and / or a p-Tau 181 protein antibody; and / or Step 4) of the coupling process specifically involves: mixing the foamed microspheres (after two washes) with the phosphorylated Tau protein antibody, adding coupling buffer and NP 40, and rotating at room temperature for coupling; then centrifuging, removing the supernatant, to obtain the phosphorylated Tau protein antibody-CNBr agarose gel complex; and / or Step 5) of the blocking process specifically involves: mixing the glycine solution with the phosphorylated Tau protein antibody-CNBr agarose gel complex and incubating overnight at 2-8°C by rotation; and / or Step 6) Blocking 2 is specifically performed as follows: Add PBS solution containing BSA to the phosphorylated Tau protein antibody-CNBr agarose gel complex that has undergone Blocking 1, and incubate at room temperature by rotation for at least 4 hours. The storage process in step 7) is as follows: after washing the blocked microbead antibody complex three times with 1 ml of PBS, it is added to the preservation solution. The pH of the preservation solution is 7.0-7.

5. The preservation solution is formulated with: 0.05% ProClin 300 (volume fraction), 0.05% bovine serum albumin (volume fraction), and 50% glycine (mass fraction).

2. A method for detecting phosphorylated Tau protein or fragments thereof, characterized in that, Includes the following steps: 1) Sample pretreatment: Add sample diluent to the sample to obtain solution A; 2) Immunoenrichment: The Tau protein antibody-CNBr agarose gel complex as described in claim 1 is added to solution A and incubated by rotation at room temperature to obtain the immunoenriched complex. 3) Washing: The immunoenriched complex was washed with Tris-HCl, NaCl and EDTA respectively, and then washed with NH4HCO3 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) Use mass spectrometry for detection and output the results.

3. The detection method according to claim 2, characterized in that, The phosphorylated Tau is p-Tau 217 and / or p-Tau 181; and / or The sample diluent 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, 0.1%-1% (w / w) NP40 and / or Tween 20; and / or The sample in step (1) is a CSF, blood, or plasma sample; and / or Step (2) includes: adding 10-40 μL of CNBr-activated agarose gel antibody complex to solution A and incubating at room temperature by rotation for at least 2 hours; and / or The washing solution in step (3) includes: 10-50 mM Tris-HCl, 100-300 mM NaCl, and 0.5-2 mM EDTA. The immunoenriched complexes are washed with the washing solution at least three times; and / or Step 4) includes at least the following: washing the washed complex at least twice with 25-200 mM NH4HCO3 to replace the solvent; redissolving in 25-200 mM NH4HCO3; adding trypsin and shaking to digest; and adding formic acid aqueous solution to terminate the digestion.

4. The detection method according to claim 2, characterized in that, Step 6) includes adding isotopic peptides as shown in Seq No. 1 and / or isotopic peptides as shown in Seq No. 2 during mass spectrometry injection.

Images