Human phosphorylated Tau-181 protein detection method based on spatial proximity chemiluminescence, kit and application

Abstract

The invention relates to the technical field of biological detection, in particular to a human phosphorylated Tau-181 protein detection method based on spatial proximity chemiluminescence, a kit and application. The kit comprises an enzyme marker, a luminous marker, a luminous auxiliary agent and a triggering agent, the enzyme marker is a first anti-p-tau-181 monoclonal antibody marked by horse radish peroxidase, the luminous marker is a second anti-p-tau-181 monoclonal antibody marked by 9, 10-dihydroacridine, the kit is adopted in the method, and the luminous auxiliary agent can be used for detecting the sensitivity of the kit. The human phosphorylated Tau-181 protein is detected based on spatial proximity chemiluminescence, the sensitivity is high, the specificity is good, the stability is good, and the kit is nontoxic and harmless to the environment and human bodies; 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, a kit and an application for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence. Background Art

[0002] Alzheimer's disease (AD) is a brain degenerative disease that causes irreversible degeneration of the patient's intelligence, memory, sensation, orientation, reasoning and judgment, seriously endangering human health.

[0003] my country has conducted a 20-year longitudinal follow-up investigation on the preclinical stage of AD and found that cerebrospinal fluid biomarkers, including Aβ42, Aβ42 / 40, phosphorylated tau (p-tau) 181 and neurofilament light chain (NfL), showed a specific pattern of change over time before the onset of symptoms. Paying attention to the etiology and pathological mechanism of AD-induced MCI, and early detection and management of AD-induced MCI will help maintain patients' existing cognitive function and delay disease progression.

[0004] In the diagnosis of AD, pathological diagnosis is often used as the gold standard for disease diagnosis in clinical medicine, but AD is a brain lesion. Performing brain tissue testing before the patient dies is an invasive examination, which is difficult for patients and their families to accept and has low feasibility. Neuroimaging is believed to be able to detect abnormalities related to AD, but there is no specific morphological change in the brain of early AD patients. Therefore, the detection of specific biomarkers is the key to early detection of AD. Currently, internationally recognized biological markers for early detection of AD include human amyloid β-protein 1-42 (Aβ1-42) and human phosphorylated tau-181 protein (p-tau-181).

[0005] In the preclinical stage of AD, the concentration levels of Aβ1-42 and p-tau-181 in the cerebrospinal fluid change. The excessive production of Aβ1-42 in the brain has strong aggregation and neurotoxicity. Its toxic effects will lead to increased permeability of the blood-cerebrospinal fluid barrier. Aβ1-42 and p-tau-181 proteins will enter the peripheral blood through the decreased function of the blood-cerebrospinal fluid barrier. Therefore, early screening and disease assessment of AD can be achieved by detecting the concentration levels of Aβ1-42 and p-tau-181 proteins in the peripheral blood, which greatly improves the convenience of operation, is safe and risk-free, and is convenient for dynamic clinical detection and efficacy evaluation.

[0006] Tau protein is a microtubule-stabilizing protein mainly expressed in neurons of the central nervous system. It is also expressed in a small amount in astrocytes and oligodendrocytes. Its hyperphosphorylation and aggregation lead to neurofibrillary tangles, which are the hallmark pathological changes of AD. There are abnormal hyperphosphorylation at many sites on the tau protein fragment, and threonine 181 (p-tau-181) is one of the many phosphorylation sites. Cerebrospinal fluid p-tau-181 has been recognized as one of the AD-specific biomarkers. In the "Guidelines for the Diagnosis and Treatment of Alzheimer's Disease in China", it is recommended that plasma p-tau-181 has a high accuracy in distinguishing AD dementia from non-AD dementia. In order to meet the clinical and social needs of early screening and prevention of Alzheimer's disease, it is very necessary to develop a kit for detecting p-tau-181 protein. At present, the common methods for detecting p-tau-181 protein are enzyme-linked immunosorbent assay and chemiluminescence method.

[0007] 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

[0008] The technical problem to be solved by the present invention is to provide a method, a kit and an application for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence.

[0009] The technical solution of the present invention to solve the above technical problems is as follows:

[0010] The invention provides a kit, which comprises an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger, wherein the enzyme marker is a first anti-p-tau-181 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-p-tau-181 monoclonal antibody labeled with 9,10-dihydroacridine.

[0011] Based on the above technical solution, the present invention can also be improved as follows.

[0012] Furthermore, the luminescence auxiliary agent includes hydrogen peroxide, and the triggering agent includes Tween 20.

[0013] The present invention also provides use of the above-mentioned kit in detecting human phosphorylated Tau-181 protein.

[0014] The present invention also provides a method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence, and the test sample is detected using the above-mentioned kit.

[0015] Furthermore, the sample to be tested, the enzyme marker, and the 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 luminescence value of the reactant to obtain the content of human phosphorylated Tau-181 protein in the sample to be tested.

[0016] Furthermore, the luminescence value of the reactant is positively correlated with the content of human phosphorylated Tau-181 protein in the sample to be tested.

[0017] Further, the following steps are included:

[0018] S1, obtaining the enzyme marker and the luminescent marker;

[0019] S2, obtaining the luminescence auxiliary agent and the triggering agent;

[0020] S3, obtaining a standard curve corresponding to the luminescence value and concentration of the human phosphorylated Tau-181 protein calibrator;

[0021] 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-181 protein in the sample to be tested.

[0022] 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-181 monoclonal antibody, stirring at room temperature in the dark; adding the sodium borohydride solution and mixing, and placing at 4°C for 2h; dialyzing with PBS at 4°C to obtain the enzyme marker;

[0023] 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-181 monoclonal antibody, mixing and standing at room temperature; shaking and mixing at 2-8°C overnight to obtain the luminescent marker.

[0024] Furthermore, the mass ratio of the horseradish peroxidase to the first anti-p-tau-181 monoclonal antibody is 1:0.8-1.2, and the mass ratio of the 9,10-dihydroacridine to the second anti-p-tau-181 monoclonal antibody is 4:0.8-2.

[0025] 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-181 protein calibrators with multiple concentration gradients, and drawing the standard curve according to the luminescence value and concentration of each human phosphorylated Tau-181 protein calibrator.

[0026] The beneficial effects of the present invention are:

[0027] (1) The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence of the present invention comprises reacting a sample to be tested, an anti-p-tau-181 monoclonal antibody labeled with horseradish peroxidase, and an anti-p-tau-181 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 realizing detection;

[0028] (2) The method for detecting human phosphorylated Tau-181 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;

[0029] (3) The method for detecting human phosphorylated Tau-181 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;

[0030] (4) The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence of the present invention is a truly homogeneous reaction, and the detection results are more authentic and reliable;

[0031] (5) A kit for detecting human phosphorylated Tau-181 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

[0032] Figure 1 The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence of the present invention is a quantitative correlation curve between the detection results of the reagent to be evaluated and the comparison reagent in Example 3. DETAILED DESCRIPTION

[0033] 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.

[0034] The kit for detecting human phosphorylated Tau-181 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-181 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-p-tau-181 monoclonal antibody labeled with 9,10-dihydroacridine.

[0035] The kit for detecting human phosphorylated Tau-181 protein of the present invention is based on the method for detecting human phosphorylated Tau-181 protein by spatial proximity chemiluminescence, and adopts spatial proximity chemiluminescence analysis (SPARCL) to detect the concentration of p-tau-181. The sample to be tested, the anti-p-tau-181 monoclonal antibody labeled with horseradish peroxidase (HRP), and the anti-p-tau-181 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 luminescent auxiliary agent and a triggering agent containing hydrogen peroxide (H2O2) are added to generate flash type chemiluminescence, and the luminescent signal can be detected by a chemiluminometer; while the unbound free HRP labeled antibody and Acridan labeled antibody do not emit light. The higher the p-tau-181 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 calibrant and its luminescence value, the p-tau-181 content in the sample can be calculated based on the luminescence value of the sample.

[0036] 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.

[0037] Preferably, the first anti-p-tau-181 monoclonal antibody and the second p-tau-181 monoclonal antibody of the present invention are both purchased from Maichuan (Guangzhou) Biotechnology Co., Ltd., with clone numbers of P-tau-181-27 and t-tau-7, respectively. The clone number of the first anti-p-tau-181 monoclonal antibody is B-S43, and the clone number of the second anti-p-tau-181 monoclonal antibody is B-E53.

[0038] The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence of the present invention comprises the following steps:

[0039] S1. Prepare enzyme markers and the luminescent markers respectively.

[0040] 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-181 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.

[0041] Preferably, the mass ratio of horseradish peroxidase to the first anti-p-tau-181 monoclonal antibody is 1:1.

[0042] Preferably, the preparation method of the luminescent marker is: adding sodium borate buffer to a DMF solution of 9,10-dihydroacridine, then adding and diluting a second anti-p-tau-181 monoclonal antibody, mixing and standing at room temperature; shaking and mixing at 2-8°C overnight to obtain a luminescent marker.

[0043] Preferably, the mass ratio of 9,10-dihydroacridine to the second anti-p-tau-181 monoclonal antibody is 4:1.

[0044] S2. Prepare luminescence auxiliary agent and trigger agent respectively.

[0045] Preferably, the components of the luminescence auxiliary agent include citric acid, sodium citrate, hydrogen peroxide and water.

[0046] Preferably, the triggering agent comprises tris hydrochloride, NaCl, concentrated HCl, Tween 20 and water.

[0047] S3. Obtain a standard curve corresponding to the luminescence value and concentration of the human phosphorylated Tau-181 protein calibrator.

[0048] 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-181 protein calibrators, and drawing a standard curve according to the luminescent value and concentration of each human phosphorylated Tau-181 protein calibrator.

[0049] Preferably, the calibrator is prepared using potassium dihydrogen phosphate, sodium dihydrogen phosphate dihydrate, Proclin 300, human phosphorylated Tau-181 protein and water.

[0050] 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.

[0051] 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-181 protein in the sample to be tested.

[0052] The present invention also provides a human phosphorylated Tau-181 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-181 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is an anti-p-tau-181 monoclonal antibody labeled with 9,10-dihydroacridine.

[0053] 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.

[0054] The kit of the present invention can be used to detect human phosphorylated Tau-181 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.

[0055] In the embodiments of the present invention, the sources of various materials are as follows:

[0056] Phosphorylated tubulin 181 (P-tau-181 antibody) was purchased from Maichuan (Guangzhou) Biotechnology Co., Ltd., catalog number AB1032, clone number: P-tau-181-27, t-tau-7, and the antigen was purchased from Nanjing Oukai Biotechnology Co., Ltd.

[0057] 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.

[0058] Instrument: HomoG 100 fully automatic chemiluminescence analyzer produced by Guangzhou Jinde Biotechnology Co., Ltd. (Guangdong Medical Device Registration No. 20212220196).

[0059] Example 1 Preparation of various detection reagents for human phosphorylated Tau-181 protein

[0060] (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 primary anti-p-tau-181 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.

[0061] (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-181 (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 refrigerator at -20°C.

[0062] (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).

[0063] (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.

[0064] Example 2 Preparation of Calibrators

[0065] (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.

[0066] (2) Dilute recombinant P-tau-181 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.

[0067] Example 3 Detection of samples

[0068] 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.

[0069] 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:

[0070] (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.

[0071] (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.

[0072] 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.

[0073] 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.

[0074] (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.

[0075] 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.

[0076] (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:

[0077] 1. The sample processing system transfers the sample rack to the sample suction position;

[0078] 2. The cuvette loading and dispatching system loads the cuvette from the loading area to the sample loading position;

[0079] 3. The sample injection system completes the sample injection and sample needle cleaning;

[0080] 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;

[0081] 5. The sample adding system completes the dispensing of reagents and the cleaning of reagent needles;

[0082] 6. Mix the reaction mixture with the counter-liquid mixing system and incubate in the reaction plate;

[0083] 7. After the incubation is completed, inject the substrate solution into the substrate system, mix, incubate, and wait for light measurement;

[0084] 8. The light measurement reaction system sends each reaction cup to the signal collection position to collect signals for calculating the luminescence value;

[0085] 9. Calculate the amount of analyte in the sample.

[0086] (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.

[0087] 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 human phosphorylated P-tau-181 assay kit (magnetic microparticle chemiluminescence immunoassay) of Tianjin Hongyutai Biotechnology Co., Ltd., Tianjin Medical Device No. 20232400121 (comparative reagent), and the specific test results are shown in Table 1.

[0088] Table 1

[0089]

[0090]

[0091] The results of the comparison 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 comparison method.

[0092] By comparison, the quantitative correlation coefficient R between the detection method of this embodiment and the detection result of the comparison reagent is 2 =0.9939, indicating that the method of the present invention has good accuracy.

[0093] 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 kit, characterized in that: The kit comprises an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger, wherein the enzyme marker is a first anti-p-tau-181 monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-p-tau-181 monoclonal antibody labeled with 9,10-dihydroacridine.

2. The kit according to claim 1, characterized in that The luminescence auxiliary agent includes hydrogen peroxide, and the trigger agent includes Tween 20.

3. Use of the kit according to claim 1 or 2 in detecting human phosphorylated Tau-181 protein.

4. A method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence, characterized in that: The test sample is tested using the kit as described in claim 1 or 2.

5. The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence according to claim 4, characterized in that: The sample to be tested, the enzyme marker, and the 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 luminescence value of the reactant to obtain the content of human phosphorylated Tau-181 protein in the sample to be tested.

6. The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence according to claim 5, characterized in that: The luminescence value of the reactant is positively correlated with the content of human phosphorylated Tau-181 protein in the sample to be tested.

7. The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence according to claim 5, 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-181 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-181 protein in the sample to be tested.

8. The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence according to claim 7, 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; immediately adding the first anti-p-tau-181 monoclonal antibody after adding the carbonate buffer, stirring at room temperature in the dark; adding the sodium borohydride solution and mixing, and placing at 4°C for 2h; dialyzing with PBS at 4°C to obtain the enzyme marker; 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-181 monoclonal antibody, mixing and standing at room temperature; shaking and mixing at 2-8°C overnight to obtain the luminescent marker.

9. The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence according to claim 8, characterized in that: The mass ratio of the horseradish peroxidase to the first anti-p-tau-181 monoclonal antibody is 1:0.8-1.2, and the mass ratio of the 9,10-dihydroacridine to the second anti-p-tau-181 monoclonal antibody is 4:0.8-2.

10. The method for detecting human phosphorylated Tau-181 protein based on spatial proximity chemiluminescence according to claim 7, 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-181 protein calibrators with multiple concentration gradients, and drawing the standard curve according to the luminescent value and concentration of each human phosphorylated Tau-181 protein calibrator.

Images