Human phosphorylated Tau-217 protein detection method and kit based on spatial proximity chemiluminescence

Abstract

The invention relates to the technical field of biological detection, in particular to a human phosphorylated Tau-217 protein detection method and kit based on spatial proximity chemiluminescence. According to the method, a to-be-detected sample, an enzyme marker and a luminous marker are mixed and react to obtain a reactant, then a luminous auxiliary agent and a triggering agent are added, the luminous value of the reactant is detected to obtain the content of the human phosphorylated Tau-217 protein in the to-be-detected sample, the enzyme marker is a horse radish peroxidase-labeled first anti-p-tau-217 monoclonal antibody, and the luminous marker is 9, 10, 11, 12, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13, 13 and a second anti-p-tau-217 monoclonal antibody marked by the 1, 10-dihydroacridine. The method is high in sensitivity, good in specificity, good in stability and nontoxic and harmless to the environment and the human body; carrier and washing steps are not needed in the detection process, cost is low, efficiency is high, and accuracy is good; the kit has the advantages of few components, simple production process, low production cost and easiness in large-scale production and full-automatic detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method and a kit for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence. Background Art

[0002] Alzheimer's disease (AD) is the most common type of cognitive impairment, with an insidious onset and difficult early diagnosis. The disease is characterized by cognitive impairment, mental and behavioral abnormalities, and decreased social activities and living ability, which impose a heavy burden on patients' families and society. AD-derived mild cognitive impairment (mild cognitive impairment due to Alzheimer's disease), referred to as AD-derived MCI, is the early stage of the AD continuous disease spectrum. An epidemiological survey in 2020 pointed out that there are approximately 38.77 million MCI patients and 15.07 million dementia patients in the Chinese population over 60 years old, including approximately 9.83 million AD patients.

[0003] The core pathological manifestations of Alzheimer's disease are amyloid plaques and neurofibrillary tangles, which are formed by the accumulation of β-amyloid protein (Aβ) and hyperphosphorylated Tau protein, respectively. They gradually destroy the normal structure and function of the brain until memory, thinking and daily living ability decline completely.

[0004] In recent years, blood markers have emerged in the early screening, diagnosis, and staging of AD due to their advantages of minimally invasiveness, convenience, and adaptability to large-scale applications. Among many related protein markers, phosphorylated Tau protein 217 has shown unique potential. In July 2024, the National Institute on Aging and the Alzheimer's Association released the "Revised Criteria for Diagnosis and Staging of Alzheimer's Disease. Alzheimer's Association Workgroup", pointing out that the plasma marker P-tau-217 can be used for AD diagnosis, staging, prognosis, and treatment effect evaluation, while other plasma biomarkers can only be used for AD staging, prognosis, and treatment effect, not for diagnosis.

[0005] Phosphorylated Tau (p-tau) protein is a major candidate for blood biomarkers. Some validated blood marker candidates, such as the β-amyloid 42 / 40 (Aβ42 / 40) ratio, have limited applications due to their insufficient accuracy and stability for routine clinical testing. In contrast, p-tau protein, as the main blood marker for all stages of AD pathology, shows higher diagnostic accuracy and disease specificity. Among the proposed blood Tau biomarkers, p-tau-217 shows high performance in distinguishing AD from other neurodegenerative diseases. In addition, in patients with elevated Aβ pathology, an increase in p-tau-217 is associated with worsening brain atrophy and cognitive decline. Although plasma p-tau-217 has shown promise as a diagnostic tool for AD, there is still a lack of commercially validated usability assessments, so it is necessary to develop a kit for detecting p-tau-217. Currently, the common methods for detecting p-tau-217 are enzyme-linked immunosorbent assay and chemiluminescence.

[0006] The enzyme-linked immunosorbent assay method has complex operating steps, is time-consuming, requires professional personnel, and has many influencing factors during the operation, which can easily lead to false positive and false negative results. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method and a kit for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] The present invention provides a method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence. The method comprises the following steps: mixing and reacting a sample to be tested, an enzyme marker and a luminescent marker to obtain a reactant, adding a luminescent auxiliary agent and a triggering agent, detecting the luminescent value of the reactant, and obtaining the content of human phosphorylated Tau-217 protein in the sample to be tested. The enzyme marker is a first anti-p-tau-217 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-p-tau-217 monoclonal antibody labeled with 9,10-dihydroacridine.

[0010] Based on the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, the luminescence auxiliary agent includes hydrogen peroxide, and the triggering agent includes Tween 20.

[0012] Furthermore, the luminescence value of the reactant is positively correlated with the content of human phosphorylated Tau-217 protein in the sample to be tested.

[0013] Further, the following steps are included:

[0014] S1, obtaining the enzyme marker and the luminescent marker;

[0015] S2, obtaining the luminescence auxiliary agent and the triggering agent;

[0016] S3, obtaining a standard curve corresponding to the luminescence value and concentration of the human phosphorylated Tau-217 protein calibrator;

[0017] S4. Using the enzyme marker, the luminescent marker, the luminescent auxiliary agent and the trigger agent to detect the sample to be tested, the luminescent value of the sample to be tested is compared with the standard curve to obtain the content of human phosphorylated Tau-217 protein in the sample to be tested.

[0018] Further, in step S1, the method for obtaining the enzyme marker is: mixing the aqueous solution of horseradish peroxidase with sodium periodate and sodium acetate buffer and dialyzing; after adding the carbonate buffer, immediately adding the first anti-p-tau-217 monoclonal antibody, stirring at room temperature in the dark; adding sodium borohydride solution and mixing, and placing at 4°C for 2h; using PBS for dialyzing at 4°C to obtain the enzyme marker.

[0019] Furthermore, the method for obtaining the luminescent marker is: adding sodium borate buffer to the DMF solution of 9,10-dihydroacridine, then adding and diluting the second anti-p-tau-217 monoclonal antibody, mixing and standing at room temperature; shaking and mixing at 2-8°C overnight to obtain the luminescent marker.

[0020] Furthermore, the mass ratio of the horseradish peroxidase to the first anti-p-tau-217 monoclonal antibody is 1:0.5-1.3, and the mass ratio of the 9,10-dihydroacridine to the second anti-p-tau-217 monoclonal antibody is 4:0.5-1.5.

[0021] Further, in step S3, the standard curve is obtained by using the enzyme marker, the luminescent marker, the luminescent auxiliary agent and the trigger agent to respectively detect human phosphorylated Tau-217 protein calibrators with multiple concentration gradients, and drawing the standard curve according to the luminescent value and concentration of each human phosphorylated Tau-217 protein calibrator.

[0022] The present invention also provides a kit, which can be used to detect human phosphorylated Tau-217 protein. The kit includes an enzyme marker and a luminescent marker. The enzyme marker is a first anti-p-tau-217 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-p-tau-217 monoclonal antibody labeled with 9,10-dihydroacridine.

[0023] Furthermore, it also includes a luminescence auxiliary agent and a triggering agent.

[0024] The beneficial effects of the present invention are:

[0025] (1) The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence of the present invention comprises reacting a sample to be tested, an anti-p-tau-217 monoclonal antibody labeled with horseradish peroxidase, and an anti-p-tau-217 monoclonal antibody labeled with 9,10-dihydroacridine together to form an antigen-antibody sandwich complex, so that horseradish peroxidase and 9,10-dihydroacridine are spatially close to each other, and flash-type chemiluminescence is generated after adding a luminescence auxiliary agent and a triggering agent, thereby achieving detection;

[0026] (2) The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence of the present invention has high sensitivity, good specificity, good stability, and is non-toxic and harmless to the environment and human body;

[0027] (3) The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence of the present invention does not require a carrier and washing steps during the detection process, which not only reduces the cost of the detection method, but also improves the detection efficiency and has good accuracy;

[0028] (4) The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence of the present invention is a truly homogeneous reaction, and the detection results are more authentic and reliable;

[0029] (5) A kit for detecting human phosphorylated Tau-217 protein, which has few components, simple production process, low production cost, and is easy to scale up for production and fully automatic detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence of the present invention is a quantitative correlation curve of the detection results of the reagent to be evaluated and the comparison reagent in Example 3. DETAILED DESCRIPTION

[0031] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0032] The kit for detecting human phosphorylated Tau-217 protein of the present invention comprises an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger, wherein the enzyme marker is a first anti-p-tau-217 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-p-tau-217 monoclonal antibody labeled with 9,10-dihydroacridine.

[0033] The kit for detecting human phosphorylated Tau-217 protein of the present invention is based on the spatial proximity chemiluminescence human phosphorylated Tau-217 protein detection method, and adopts the spatial proximity chemiluminescence analysis method (SPARCL) to detect the concentration of p-tau-217. The sample to be tested, the anti-p-tau-217 monoclonal antibody labeled with horseradish peroxidase (HRP), and the anti-p-tau-217 monoclonal antibody labeled with 9,10-dihydroacridine (Acridan) are reacted together to form an antigen-antibody sandwich complex, so that the horseradish peroxidase and 9,10-dihydroacridine (Acridan) are spatially close to each other, and a solution containing hydrogen peroxide (H 2 O 2 ) luminescence auxiliary agent and triggering agent, produce flash chemiluminescence, the luminescence signal can be detected by chemiluminometer; while the unbound free HRP labeled antibody and Acridan labeled antibody do not emit light. The higher the p-tau-217 content in the sample to be tested, the greater the measured luminescence value (RLU). Therefore, within a certain concentration range, the luminescence value is positively correlated with the sample concentration. By drawing a standard curve with a known concentration of calibrator and its luminescence value, the p-tau-217 content in the sample can be calculated based on the luminescence value of the sample.

[0034] The kit of the present invention is based on spatial proximity chemiluminescence, has high sensitivity, good specificity, good stability, and is non-toxic and harmless to the environment and human body; the detection process does not require a carrier and a washing step, which not only reduces the cost of the detection method, but also improves the detection efficiency and has good accuracy. In addition, the method is a truly homogeneous reaction, and the detection result is more authentic and reliable.

[0035] Preferably, the first anti-p-tau-217 monoclonal antibody and the second p-tau-217 monoclonal antibody of the present invention are both purchased from Maichuan (Guangzhou) Biotechnology Co., Ltd., with clone numbers of P-tau-217-27 and t-tau-7, respectively. The clone number of the first anti-p-tau-217 monoclonal antibody is B-S43, and the clone number of the second anti-p-tau-217 monoclonal antibody is B-E53.

[0036] The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence of the present invention comprises the following steps:

[0037] S1. Prepare enzyme markers and the luminescent markers respectively.

[0038] Preferably, the preparation method of the enzyme marker is: mixing an aqueous solution of horseradish peroxidase with sodium periodate and sodium acetate buffer and dialyzing; after adding carbonate buffer, immediately adding the first anti-p-tau-217 monoclonal antibody, stirring at room temperature in the dark; adding sodium borohydride solution and mixing, and placing at 4°C for 2h; dialyzing with PBS at 4°C to obtain the enzyme marker.

[0039] Preferably, the mass ratio of horseradish peroxidase to the first anti-p-tau-217 monoclonal antibody is 1:1.

[0040] Preferably, the preparation method of the luminescent marker is: adding sodium borate buffer to a DMF solution of 9,10-dihydroacridine, then adding a second anti-p-tau-217 monoclonal antibody and diluting it, mixing it and standing it at room temperature; shaking and mixing it at 2-8°C overnight to obtain a luminescent marker.

[0041] Preferably, the mass ratio of 9,10-dihydroacridine to the second anti-p-tau-217 monoclonal antibody is 4:1.

[0042] S2. Prepare luminescence auxiliary agent and trigger agent respectively.

[0043] Preferably, the components of the luminescence auxiliary agent include citric acid, sodium citrate, hydrogen peroxide and water.

[0044] Preferably, the triggering agent comprises tris hydrochloride, NaCl, concentrated HCl, Tween 20 and water.

[0045] S3. Obtain a standard curve corresponding to the luminescence value and concentration of the human phosphorylated Tau-217 protein calibrator.

[0046] Preferably, the standard curve is obtained by using enzyme markers, luminescent markers, luminescent auxiliary agents and triggers to respectively detect multiple concentration gradients of human phosphorylated Tau-217 protein calibrators, and drawing a standard curve based on the luminescent value and concentration of each human phosphorylated Tau-217 protein calibrator.

[0047] Preferably, the calibrator is prepared using potassium dihydrogen phosphate, sodium dihydrogen phosphate dihydrate, Proclin 300, human phosphorylated Tau-217 protein and water.

[0048] Further preferably, the concentration gradient of the calibrator is 0 ng / mL, 5 ng / mL, 25 ng / mL, 150 ng / mL, 750 ng / mL, and 1500 ng / mL.

[0049] S4. Use enzyme markers, luminescent markers, luminescent auxiliary agents and triggering agents to detect the sample to be tested, compare the luminescent value of the sample to be tested with the standard curve, and obtain the content of human phosphorylated Tau-217 protein in the sample to be tested.

[0050] The present invention also provides a human phosphorylated Tau-217 protein detection kit based on spatial proximity chemiluminescence, the kit comprising an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger, the enzyme marker is an anti-p-tau-217 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is an anti-p-tau-217 monoclonal antibody labeled with 9,10-dihydroacridine.

[0051] The kit of the present invention has few components, simple production process, low production cost, and is easy to scale up for production and fully automatic detection.

[0052] The kit of the present invention can be used to detect human phosphorylated Tau-217 protein in a sample. It should be noted that the kit of the present invention can also include reagents for preparing the above-mentioned components and auxiliary reagents that may exist or may be used.

[0053] In the embodiments of the present invention, the sources of various materials are as follows:

[0054] Both anti-p-tau-217 monoclonal antibodies were purchased from Bioxinyi Biotechnology Co., Ltd. The catalog number of the first anti-p-tau-217 monoclonal antibody is A09N02-5D6, and the catalog number of the second anti-p-tau-217 monoclonal antibody is A09N02-B2. The antigen was purchased from Nanjing Huading Biotechnology Co., Ltd., catalog number: 60005.

[0055] Among other reagents, horseradish peroxidase, sodium periodate, sodium borohydride, sodium acetate, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, tris (hydroxymethyl)aminomethane hydrochloride (Tris), hydrochloric acid, citric acid, sodium citrate, glycerol, boric acid, sodium tetraborate, sucrose, aminopyrine, Tween 20, Triton 100, potassium dihydrogen phosphate, hydrogen peroxide, and dimethylformamide were purchased from Sinopharm Chemical Reagent Co., Ltd., bovine serum albumin, casein, Proclin 300, polyethylene glycol 2000, etc. were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., and 9, 10-dihydroacridine was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0056] Instrument: HomoG 100 fully automatic chemiluminescence analyzer produced by Guangzhou Jinde Biotechnology Co., Ltd.

[0057] Example 1 Preparation of various detection reagents for human phosphorylated Tau-217 protein

[0058] (1) Preparation of enzyme marker: Weigh 1 mg HRP and dissolve it in 200 μL distilled water, add 40 μL of freshly prepared 0.1 M sodium periodate, shake and mix at room temperature for 20 min, put the above liquid into a dialysis bag, dialyze it with 1 mM sodium acetate buffer, pH 4.4, at 4°C overnight, add 20 μL of 0.2 M carbonate buffer, pH 9.5, and then immediately add 1 mg of the first anti-p-tau-217 monoclonal antibody, gently stir for 2 h at room temperature in the dark, add 10 μL of freshly prepared 4 mg / mL sodium borohydride solution, mix well, and place at 4°C for 2 h. Put the above liquid into a dialysis bag, dialyze it with 0.15 M PBS, pH 7.4, at 4°C overnight, remove the dialysate, add 50% glycerol, and store it at -20°C for later use.

[0059] (2) Preparation of luminescent markers: Dissolve 1 mg of the luminescent substrate Acridan in 500 μL of DMF, take out 41.3 μL, add 708.7 μL of 0.05 M sodium borate buffer, then add 250 μL of anti-p-tau-217 (diluted to 1 mg / mL), invert and mix 5 times, let stand at room temperature for 30 min, place the labeled reaction tube on an oscillator and mix overnight at 2-8°C, take out, add 50% glycerol and store in a -20°C refrigerator.

[0060] (3) Luminescence auxiliary agent: Weigh 1.82 g of citric acid and 10.45 g of sodium citrate, dissolve in purified water and make up to 1000 mL, add 0.3 mL of 30% hydrogen peroxide to the buffer solution, mix well and place in a 4°C refrigerator for later use (pack into 10 mL / bottle).

[0061] (4) Trigger: Weigh 6.06 g of Tris and 9 g of NaCl, add appropriate amount of purified water to dissolve, add 2.1 mL of concentrated HCl and 2 mL of Tween 20, mix well and make up to 1000 mL, and place in a 4°C refrigerator for later use.

[0062] Example 2 Preparation of Calibrators

[0063] (1) Calibrator diluent: Weigh 14.1 g potassium dihydrogen phosphate and 3.0 g sodium dihydrogen phosphate dihydrate, add appropriate amount of purified water to dissolve, add 0.5 mL Proclin 300, mix well, add purified water to make up to 1000 mL, and place in a 4°C refrigerator for later use.

[0064] (2) Dilute recombinant P-tau-217 to 0 pg / mL, 2 pg / mL, 5 pg / mL, 15 pg / mL, 50 pg / mL, and 100 pg / mL using calibrator diluent and place in a 4°C refrigerator for later use.

[0065] Example 3 Detection of samples

[0066] In this embodiment, the kit is used to detect the calibrators and the samples to be tested in Example 2. The kit includes the detection reagent prepared in Example 1.

[0067] The specific detection process is carried out using a chemiluminescent immunoassay analyzer, and the system requires 10 μL of sample for each measurement. The detection process is as follows:

[0068] (1) Preparation before testing: Before testing, all materials required for the test should be prepared and the instruction manual of the chemiluminescent immunoassay analyzer should be carefully read to obtain information such as system operating procedures, sample management, precautions for use, maintenance and care.

[0069] (2) Calibration: Use the kit and calibrator to perform the calibration test. Before starting the kit calibration, the master curve in the 2D barcode of the kit should be scanned and imported into the system.

[0070] The chemiluminescent immunoassay analyzer adjusts the master curve based on the results of the calibration test to obtain the calibration curve of the current system. The instrument operation software has a calibration judgment function to automatically check the validity of the calibration curve. If the calibration fails, the instrument settings, status, location of the calibration (low-value calibration, high-value calibration), validity period, etc. should be considered.

[0071] A valid calibration curve is required before all tests. Recalibration is required in the following situations: when a new batch of reagent kit is used; when the same batch of reagent kit is used on the analyzer for more than 30 days; after instrument maintenance; or when the quality control value is out of the specified range.

[0072] (3) Quality Control: To ensure the reliability of the test results, it is recommended to test the high and low levels of quality control products once every 24 hours. In addition, it is also recommended to perform quality control tests after each calibration test, reagent batch change, maintenance and troubleshooting.

[0073] The test value of the quality control product should be within the specified range. If it exceeds the specified range, the user should check the detection system, such as the location, validity period, storage method, calibration process, instrument performance and status, etc. of the quality control product, and recalibrate if necessary. Sample testing can only be carried out after the quality control is qualified.

[0074] (4) Sample testing: Enter the sample application interface of the instrument and enter the sample number, sample rack number, position number, sample type, remarks and other information according to the test requirements. Select the test item, select the sample type in the item options, and enter the number of repetitions and other information. After preparing the test sample and placing it correctly, click the start button to start the test. The chemiluminescent immunoassay analyzer will perform the following operations in sequence:

[0075] 1. The sample processing system transfers the sample rack to the sample suction position;

[0076] 2. The cuvette loading and dispatching system loads the cuvette from the loading area to the sample loading position;

[0077] 3. The sample injection system completes the sample injection and sample needle cleaning;

[0078] 4. The reagent processing system provides the reagents required for the test, mixes each reagent and sends it to the reagent aspiration position to wait for aspiration;

[0079] 5. The sample adding system completes the dispensing of reagents and the cleaning of reagent needles;

[0080] 6. Mix the reaction mixture with the counter-liquid mixing system and incubate in the reaction plate;

[0081] 7. After the incubation is completed, inject the substrate solution into the substrate system, mix, incubate, and wait for light measurement;

[0082] 8. The light measurement reaction system sends each reaction cup to the signal collection position to collect signals for calculating the luminescence value;

[0083] 9. Calculate the amount of analyte in the sample.

[0084] (5) Calculation: Based on the saved calibration data, the system software uses a weighted four-parameter logarithmic curve (4PLC, Y-weighted) mathematical method to automatically determine the test results of the sample. The result unit is given in the form of pg / mL.

[0085] This example uses the above detection process to test samples from 40 Alzheimer's patients (reagents to be evaluated). For these blood samples, this example also uses the Hunan Novozyme Medical Technology Co., Ltd. phosphorylated tau-217 protein (p-tau-217) detection kit (chemiluminescence method, Hunan Medical Device Registration No. 20242400296) as a comparison reagent for detection, and the specific test results are shown in Table 1.

[0086] Table 1

[0087]

[0088]

[0089] The results of the comparative reagent are used as the horizontal axis, and the results of the reagent to be evaluated are used as the vertical axis. Figure 1 The quantitative correlation curve shown is used to examine the accuracy of the method of this embodiment compared with the existing method.

[0090] By comparison, the quantitative correlation coefficient R between the detection method of this embodiment and the detection result of the comparative reagent is 2 =0.9861, indicating that the method of the present invention has good accuracy.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence, characterized in that: The sample to be tested, an enzyme marker, and a luminescent marker are mixed and reacted to obtain a reactant, and then a luminescent auxiliary agent and a triggering agent are added to detect the luminescent value of the reactant to obtain the content of human phosphorylated Tau-217 protein in the sample to be tested. The enzyme marker is a first anti-p-tau-217 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-p-tau-217 monoclonal antibody labeled with 9,10-dihydroacridine.

2. A method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence according to claim 1, characterized in that: The luminescence auxiliary agent includes hydrogen peroxide, and the trigger agent includes Tween 20.

3. A method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence according to claim 1 or 2, characterized in that: The luminescence value of the reactant is positively correlated with the content of human phosphorylated Tau-217 protein in the sample to be tested.

4. The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence according to claim 3, characterized in that: The following steps are involved: S1, obtaining the enzyme marker and the luminescent marker; S2, obtaining the luminescence auxiliary agent and the triggering agent; S3, obtaining a standard curve corresponding to the luminescence value and concentration of the human phosphorylated Tau-217 protein calibrator; S4. Using the enzyme marker, the luminescent marker, the luminescent auxiliary agent and the trigger agent to detect the sample to be tested, the luminescent value of the sample to be tested is compared with the standard curve to obtain the content of human phosphorylated Tau-217 protein in the sample to be tested.

5. The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence according to claim 4, characterized in that: In step S1, the method for obtaining the enzyme marker is as follows: mixing the aqueous solution of horseradish peroxidase with sodium periodate and sodium acetate buffer and dialyzing; after adding the carbonate buffer, immediately adding the first anti-p-tau-217 monoclonal antibody, stirring at room temperature in the dark; adding the sodium borohydride solution and mixing, and placing at 4°C for 2h; and dialyzing with PBS at 4°C to obtain the enzyme marker.

6. The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence according to claim 5, characterized in that: The method for obtaining the luminescent marker is as follows: adding sodium borate buffer to a DMF solution of 9,10-dihydroacridine, then adding and diluting a second anti-p-tau-217 monoclonal antibody, mixing and standing at room temperature; shaking and mixing at 2-8°C overnight to obtain the luminescent marker.

7. A method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence according to claim 6, characterized in that: The mass ratio of the horseradish peroxidase to the first anti-p-tau-217 monoclonal antibody is 1:0.5-1.3, and the mass ratio of the 9,10-dihydroacridine to the second anti-p-tau-217 monoclonal antibody is 4:0.5-1.

5.

8. The method for detecting human phosphorylated Tau-217 protein based on spatial proximity chemiluminescence according to claim 4, characterized in that: In step S3, the standard curve is obtained by using the enzyme marker, the luminescent marker, the luminescent auxiliary agent and the trigger agent to respectively detect human phosphorylated Tau-217 protein calibrators with multiple concentration gradients, and drawing the standard curve according to the luminescent value and concentration of each human phosphorylated Tau-217 protein calibrator.

9. A kit, characterized in that: The kit can be used to detect human phosphorylated Tau-217 protein, and the kit includes an enzyme marker and a luminescent marker, wherein the enzyme marker is a first anti-p-tau-217 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-p-tau-217 monoclonal antibody labeled with 9,10-dihydroacridine.

10. The kit according to claim 9, characterized in that Also included are luminescence aids and triggers.

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