Hypersensitive troponin T detection method and kit based on spatial proximity chemiluminescence

Abstract

The invention relates to the technical field of biological detection, in particular to a hypersensitive troponin T detection method and kit based on spatial proximity chemiluminescence. The method comprises the following steps: mixing and reacting a to-be-detected sample, an enzyme marker and a luminous marker to obtain a reactant, adding a luminous auxiliary agent and a triggering agent, and detecting the luminous value of the reactant to obtain the content of the hypersensitive troponin T in the to-be-detected sample, the enzyme marker is a first anti-cTnT monoclonal antibody marked by horse radish peroxidase, and the luminous marker is a second anti-cTnT monoclonal antibody marked by 9, 10-dihydroacridine. The detection method disclosed by the invention is high in sensitivity, good in specificity and good in stability, and is nontoxic and harmless to the environment and a 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 ultrasensitive troponin T based on spatial proximity chemiluminescence. Background Art

[0002] Troponin (Tn) is a heterotrimeric complex containing three subunits, TnC, TnT, and Tn I. It is expressed in both skeletal and cardiac muscles and is an important structural protein in muscle excitation-contraction coupling. Cardiac troponin I (cTnI) and cardiac troponin T (cTnT) in cardiomyocytes are encoded by separate genes, which are completely different from sTn I and sTnT in skeletal muscle cells. Slow skeletal muscle Tn I and cardiac muscle Tn I are co-expressed in the heart within 0 to 9 months after birth, and then cardiomyocytes only express cTn I. Most cTn is bound to thin filaments to form the myocardial cell skeleton, and only 2% to 8% exists in the cytoplasm in free form.

[0003] When myocardial cells undergo necrosis, apoptosis, renewal, changes in cell membrane permeability, and bleb formation, troponin is released into the blood and detected by sufficiently sensitive methods. After myocardial infarction, the main forms in the peripheral circulation are free cTn I, cTnT, cTn I-cTnC complex, and full molecular weight and low molecular weight cTnT-cTn I-cTnC trimers. After release, cTn in the circulation is degraded, fragmented, and cleared by the kidneys. Renal damage can affect its clearance rate in the blood, especially cTnT with a slightly larger molecular weight. cTnT will peak quickly after myocardial infarction and then slowly decrease. During the decline (4-5 days), a slight secondary increase occurs due to further enzymatic hydrolysis of related fragments containing cTnT, which is called the "double peak phenomenon."

[0004] In patients with myocardial infarction, cardiac troponin levels rise rapidly after the onset of symptoms (usually within 1 hour after the onset of symptoms, if using high-sensitivity troponin testing), and remain elevated for a period of time (usually several days). It should be noted that in the era of high-sensitivity testing, troponin is no longer a marker for myocardial infarction, but a marker for myocardial injury. Many reasons can cause myocardial injury, so the concept of false positives should be correctly understood. High-sensitivity troponin does not increase the false positive rate, but rather discovers more subtle myocardial damage. In addition, healthy people sometimes have positive troponin results. Therefore, clinicians need to clarify how to distinguish myocardial injury from myocardial infarction, and it is necessary to develop a rapid detection kit for cTnT. Currently, the common methods for detecting cTnT are enzyme-linked immunosorbent assay and traditional chemiluminescence.

[0005] The enzyme-linked immunosorbent assay method has complex operation steps, is time-consuming, requires professionals, and has many influencing factors in the operation process, which can easily lead to false positive and false negative results. The traditional magnetic microparticle chemiluminescence method has high sensitivity, good specificity, good stability, and is non-toxic and harmless to the environment and human body. However, it requires the use of magnetic microspheres and combined with magnetic separation technology for testing, which is not only costly but also may cause distortion of some test results. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method and a kit for detecting ultrasensitive troponin T based on spatial proximity chemiluminescence.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The present invention provides a method for detecting high-sensitive troponin T based on spatial proximity chemiluminescence, wherein a 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 high-sensitive troponin T in the sample to be tested;

[0009] Wherein, the enzyme marker is a first anti-cTnT monoclonal antibody labeled with horseradish peroxidase, and the mass ratio of the horseradish peroxidase to the first anti-cTnT monoclonal antibody is 1:0.5-2; the luminescent marker is a second anti-cTnT monoclonal antibody labeled with 9,10-dihydroacridine, and the mass ratio of the 9,10-dihydroacridine to the second anti-cTnT monoclonal antibody is 4:0.5-4.5.

[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.

[0012] Furthermore, the triggering agent includes Tween 20.

[0013] Furthermore, the luminescence value of the reactant is positively correlated with the content of high-sensitive troponin T in the sample to be tested.

[0014] Further, the following steps are included:

[0015] S1, preparing the enzyme marker and the luminescent marker respectively;

[0016] S2, preparing the luminescence auxiliary agent and the triggering agent respectively;

[0017] S3, obtaining a standard curve corresponding to the luminescence value and concentration of the high-sensitive troponin T calibrator;

[0018] 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 high-sensitive troponin T in the sample to be tested.

[0019] Further, in step S1, the preparation method of 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-cTnT 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.

[0020] Further, in step S1, the preparation method of the luminescent marker is: adding sodium borate buffer to the DMF solution of 9,10-dihydroacridine, then adding and diluting the second anti-cTnT monoclonal antibody, mixing and standing at room temperature; shaking and mixing at 2-8°C overnight to obtain the luminescent marker.

[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 multiple concentration gradients of high-sensitive troponin T calibrators, and drawing the standard curve according to the luminescent value and concentration of each high-sensitive troponin T calibrator.

[0022] The present invention also provides a kit for detecting high-sensitive troponin T, comprising an enzyme marker and a luminescent marker, wherein the enzyme marker is a first anti-cTnT monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-cTnT 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 present invention is a method for detecting ultrasensitive troponin T based on spatial proximity chemiluminescence, wherein a sample to be tested, an anti-cTnT monoclonal antibody labeled with horseradish peroxidase, and an anti-cTnT monoclonal antibody labeled with 9,10-dihydroacridine are reacted together to form an antigen-antibody sandwich complex, so that the horseradish peroxidase and the 9,10-dihydroacridine are spatially close to each other, and after adding a luminescence auxiliary agent and a triggering agent, flash-type chemiluminescence is generated, thereby achieving detection;

[0026] (2) The ultrasensitive troponin T detection method 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 ultrasensitive troponin T detection method 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 ultrasensitive troponin T detection method 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) The kit for detecting high-sensitive troponin T 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 This is the ultra-sensitive troponin T detection method based on spatial proximity chemiluminescence of the present invention, in Example 3, the quantitative correlation curve of the detection results of the reagent to be evaluated and the comparison reagent. 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 invention discloses a method for detecting supersensitive troponin T 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 supersensitive troponin T in the sample to be tested. The enzyme marker is a first anti-cTnT monoclonal antibody labeled with horseradish peroxidase, the mass ratio of horseradish peroxidase to the first anti-cTnT monoclonal antibody is 1:0.5-2, and the luminescent marker is a second anti-cTnT monoclonal antibody labeled with 9,10-dihydroacridine, the mass ratio of 9,10-dihydroacridine to the second anti-cTnT monoclonal antibody is 4:0.5-4.5.

[0033] The present invention discloses a method for detecting cTnT based on spatial proximity chemiluminescence, and adopts spatial proximity chemiluminescence analysis (SPARCL) to detect the concentration of cTnT. The sample to be tested, the anti-cTnT monoclonal antibody labeled with horseradish peroxidase (HRP), and the anti-cTnT 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 the Acridan-labeled antibody do not emit light. The higher the cTnT 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 calibrators of known concentrations and their luminescence values, the cTnT content in the sample can be calculated based on the luminescence value of the sample.

[0034] The detection method 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 results are more authentic and reliable.

[0035] Preferably, the first anti-cTnT monoclonal antibody and the second cTnT monoclonal antibody of the present invention are purchased from Feipeng Biotechnology Co., Ltd. The catalog number of the first anti-cTnT monoclonal antibody is CTNT-McAb-1#, and the catalog number of the second anti-cTnT monoclonal antibody is CTNT-McAb-2#.

[0036] The method for detecting ultrasensitive troponin T 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-cTnT 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-cTnT 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 and diluting a second anti-cTnT monoclonal antibody, mixing and standing at room temperature; shaking and mixing at 2-8°C overnight to obtain a luminescent marker.

[0041] Preferably, the mass ratio of 9,10-dihydroacridine to the second anti-cTnT 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 high-sensitive troponin T calibrator.

[0046] Preferably, the standard curve is obtained by using an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger agent to respectively detect multiple concentration gradients of high-sensitive troponin T calibrators, and drawing a standard curve according to the luminescent value and concentration of each high-sensitive troponin T calibrator.

[0047] Preferably, the calibrator is prepared with potassium dihydrogen phosphate, sodium dihydrogen phosphate dihydrate, Proc lin 300, high-sensitivity troponin T and water.

[0048] Further preferably, the concentration gradient of the calibrator is 0 pg / mL, 9 pg / mL, 45 pg / mL, 180 pg / mL, 450 pg / mL, 1800 pg / mL, 10000 pg / mL, and 21600 pg / 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 high-sensitive troponin T in the sample to be tested.

[0050] The present invention also provides a supersensitive troponin T detection kit based on spatial proximity chemiluminescence, which includes an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger, wherein the enzyme marker is a first anti-cTnT monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-cTnT 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 production and fully automatic detection.

[0052] The kit of the present invention can be used to detect high-sensitive troponin T 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] Anti-cTnT monoclonal antibodies were purchased from Feipeng Biotechnology Co., Ltd. The clone number of the first anti-cTnT monoclonal antibody was CTNT-McAb-1#, and the clone number of the second anti-cTnT monoclonal antibody was CTNT-McAb-2#.

[0055] The cTnT antigen in the examples was purchased from Nanjing Ainuodi Biotechnology Co., Ltd., with the catalog number: AEP0076.

[0056] 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(hydroxymethylaminomethane) 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, Procline 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.

[0057] The instrument is the HomoG 100 fully automatic chemiluminescence analyzer produced by Guangzhou Jinde Biotechnology Co., Ltd.

[0058] Example 1 Preparation of various detection reagents for high-sensitive troponin T

[0059] (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-cTnT monoclonal antibody, gently stir at room temperature in the dark for 2 h, 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.

[0060] (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 the secondary anti-cTnT (diluted to 1 mg / mL), invert and mix five 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.

[0061] (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).

[0062] (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.

[0063] Example 2 Preparation of Calibrators

[0064] (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 Proc lin 300, mix well, add purified water to make up to 1000 mL, and place in a 4°C refrigerator for later use.

[0065] (2) Use the calibrator diluent to dilute the cTnT antigen to 0 pg / mL, 9 pg / mL, 45 pg / mL, 180 pg / mL, 450 pg / mL, 1800 pg / mL, 10000 pg / mL, and 21600 pg / mL and place in a 4°C refrigerator for later use.

[0066] Example 3 Detection of samples

[0067] 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.

[0068] 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:

[0069] (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.

[0070] (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.

[0071] 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.

[0072] 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.

[0073] (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.

[0074] 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.

[0075] (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:

[0076] 1. The sample processing system transfers the sample rack to the sample suction position;

[0077] 2. The cuvette loading and dispatching system loads the cuvette from the loading area to the sample loading position;

[0078] 3. The sample injection system completes the sample injection and sample needle cleaning;

[0079] 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;

[0080] 5. The sample adding system completes the dispensing of reagents and the cleaning of reagent needles;

[0081] 6. Mix the reaction mixture with the counter-liquid mixing system and incubate in the reaction plate;

[0082] 7. After the incubation is completed, inject the substrate solution into the substrate system, mix, incubate, and wait for light measurement;

[0083] 8. The light measurement reaction system sends each reaction cup to the signal collection position to collect signals for calculating the luminescence value;

[0084] 9. Calculate the amount of analyte in the sample.

[0085] (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.

[0086] This example uses the above detection process to test samples from 74 patients with myocardial injury (reagents to be evaluated). For these samples, this example also uses Roche Diagnostics' high-sensitivity troponin T detection kit (electrochemiluminescence method, National Medical Device Registration No. 20152400203) for comparative testing (Roche), and the specific test results are shown in Table 1.

[0087] Table 1

[0088]

[0089]

[0090] The results of Roche reagent are plotted as the horizontal axis and the results of the reagent to be evaluated are plotted as the vertical axis. Figure 1 The quantitative correlation curve shown is used to investigate the accuracy of the method of this example compared with the Roche method.

[0091] After comparison, the 74 samples of patients with myocardial injury tested had a consistency rate of more than 94% with the results of Roche high-sensitivity troponin T test, and the quantitative correlation coefficient R 2 =0.9958. This shows that the method of the present invention has good accuracy.

[0092] 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 high-sensitive troponin T based on spatial proximity chemiluminescence, 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 high-sensitive troponin T in the sample to be tested; Wherein, the enzyme marker is a first anti-cTnT monoclonal antibody labeled with horseradish peroxidase, and the mass ratio of the horseradish peroxidase to the first anti-cTnT monoclonal antibody is 1:0.5-2; the luminescent marker is a second anti-cTnT monoclonal antibody labeled with 9,10-dihydroacridine, and the mass ratio of the 9,10-dihydroacridine to the second anti-cTnT monoclonal antibody is 4:0.5-4.

5.

2. The method for detecting high-sensitive troponin T based on spatial proximity chemiluminescence according to claim 1, characterized in that: The luminescence auxiliary agent includes hydrogen peroxide.

3. The method for detecting high-sensitive troponin T based on spatial proximity chemiluminescence according to claim 1, characterized in that: The triggering agent includes Tween 20.

4. A method for detecting high-sensitive troponin T based on spatial proximity chemiluminescence according to any one of claims 1 to 3, characterized in that: The luminescence value of the reactant is positively correlated with the content of high-sensitive troponin T in the sample to be tested.

5. A method for detecting high-sensitive troponin T based on spatial proximity chemiluminescence according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, preparing the enzyme marker and the luminescent marker respectively; S2, preparing the luminescence auxiliary agent and the triggering agent respectively; S3, obtaining a standard curve corresponding to the luminescence value and concentration of the high-sensitive troponin T 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 high-sensitive troponin T in the sample to be tested.

6. The method for detecting high-sensitive troponin T based on spatial proximity chemiluminescence according to claim 5, characterized in that: In step S1, the preparation method of 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-cTnT monoclonal antibody, stirring at room temperature in the dark; adding the sodium borohydride solution and mixing, and placing at 4°C for 2h; using PBS for dialyzing at 4°C to obtain the enzyme marker.

7. The method for detecting high-sensitive troponin T based on spatial proximity chemiluminescence according to claim 5, characterized in that: In step S1, the preparation method of the luminescent marker is: adding sodium borate buffer to the DMF solution of 9,10-dihydroacridine, then adding and diluting the second anti-cTnT monoclonal antibody, mixing and standing at room temperature; shaking and mixing at 2-8°C overnight to obtain the luminescent marker.

8. The method for detecting high-sensitive troponin T based on spatial proximity chemiluminescence according to claim 5, 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 multiple concentration gradients of high-sensitive troponin T calibrators, and drawing the standard curve according to the luminescent value and concentration of each high-sensitive troponin T calibrator.

9. A kit for detecting high-sensitive troponin T, characterized in that: The kit comprises an enzyme marker and a luminescent marker, wherein the enzyme marker is a first anti-cTnT monoclonal antibody labeled with horseradish peroxidase, and the luminescent marker is a second anti-cTnT monoclonal antibody labeled with 9,10-dihydroacridine.

10. A kit for detecting high-sensitive troponin T according to claim 9, characterized in that: Also included are luminescence aids and triggers.

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