Kit for detecting N-terminal B-type natriuretic peptide precursor as well as preparation method and application thereof

Abstract

The invention relates to a kit for detecting an N-terminal B-type natriuretic peptide precursor and a preparation method and application thereof, and relates to the technical field of biology, the kit comprises an enzyme marker, a luminous marker, a luminous auxiliary agent and a triggering agent; the enzyme marker comprises an anti-NT-proBNP monoclonal antibody marked by horse radish peroxidase, and the anti-NT-proBNP monoclonal antibody is a monoclonal antibody. The luminous marker comprises an anti-NT-proBNP monoclonal antibody marked by 9, 10-dihydroacridine, and the anti-NT-proBNP monoclonal antibody is a monoclonal antibody marked by 9, 10- The luminous auxiliary agent comprises citric acid, sodium citrate and hydrogen peroxide; the triggering agent is prepared from tris (hydroxymethyl) aminomethane hydrochloride, chlorine salt, HCl and a surfactant. The kit adopts a space adjacent chemical double antibody sandwich method, is simple to operate, can realize full-automatic detection, does not need magnetic beads and washing, not only reduces the reagent cost, but also shortens the detection time, and adopts homogeneous reaction to avoid the influence brought by the traditional chemiluminescence magnetic separation technology, so that the detection result is more accurate and stable.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kit for detecting N-terminal B-type natriuretic peptide precursor, and a preparation method and application thereof. Background Art

[0002] Heart failure (HF) is a syndrome caused by various structural or functional cardiac diseases that impair ventricular filling and ejection capacity. It is a severe stage of various heart diseases and carries a poor prognosis. With improvements in living standards, the prevalence of unhealthy lifestyles, and an aging population, the prevalence of cardiovascular disease and heart failure has increased annually, seriously endangering human health. Among the neuroendocrine changes in HF, brain natriuretic peptide (BNP) is the primary protective factor. BNP counteracts sodium and water retention by directly inhibiting sodium transport in the proximal convoluted tubule and intramedullary collecting duct, increasing glomerular filtration rate, and inhibiting angiotensin-converting enzyme activity and the release of renin and aldosterone. By inhibiting adrenaline production, BNP reduces pulmonary artery wedge pressure and vascular resistance, increasing stroke volume, and thereby reducing cardiac preload and afterload, slowing the progression of myocardial hypertrophy and cardiomegaly. Therefore, BNP is a natural antagonist of the RAAS system, and its primary physiological effects can be summarized as natriuretic, diuretic, and dilation.

[0003] When myocardial cells are stimulated by pressure or stretch, they first synthesize the precursor polypeptide of BNP, namely pro-B-type natriuretic peptide (pre-proBNP). Pre-proBNP contains 134 amino acids and is subsequently cleaved by proteases into pro-B-type natriuretic peptide (proBNP) containing 108 amino acids and a 26-amino acid signal peptide. During the secretion process, proBNP is further cleaved by endonucleases into N-terminal pro-B-type natriuretic peptide (NT-proBNP) containing 76 amino acids and BNP containing 32 amino acids. Both are then secreted into the blood. Although both BNP and NT-proBNP are members of the natriuretic peptide family, their biological effects differ. BNP has a half-life of 22 minutes, poor in vitro stability, and low concentrations in peripheral blood. NT-proBNP, on the other hand, has a half-life of 2 hours and is stable at 4°C for 5 days ex vivo. Its peripheral blood concentration is over 10 times that of BNP, making it easier to detect. Furthermore, its concentration reflects the transient release of newly synthesized BNP rather than existing BNP, thus better reflecting BNP pathway activation. It also better reflects cardiac function than levels of other endocrine hormones, making it more clinically relevant for patients with heart failure. To meet the clinical and societal needs for the prevention and treatment of heart failure, the development of a test kit for measuring NT-proBNP concentrations is crucial.

[0004] Currently, common methods for detecting NT-proBNP concentrations include enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assays. ELISA has complex and time-consuming procedures, requires specialized personnel, and is subject to numerous operational factors, which can easily lead to false-positive and false-negative results. Chemiluminescence assays offer high sensitivity, good specificity, and stability, and are non-toxic to the environment and the human body. In light of this, the present invention provides a kit for detecting N-terminal pro-B-type natriuretic peptide (NT-proBNP), as well as its preparation and use. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a kit for detecting N-terminal pro-B-type natriuretic peptide, as well as its preparation method and application. The purpose is to utilize spatial proximity chemiluminescence analysis, eliminating the need for a carrier and washing process, reducing kit costs, shortening detection time, and making the test results more reliable.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, a kit for detecting N-terminal B-type natriuretic peptide precursor comprises an enzyme label, a luminescent label, a luminescent auxiliary agent and a triggering agent;

[0008] The enzyme marker includes horseradish peroxidase-labeled anti-NT-proBNP monoclonal antibody;

[0009] The luminescent marker includes an anti-NT-proBNP monoclonal antibody labeled with 9,10-dihydroacridine;

[0010] The luminescence auxiliary agent includes citric acid, sodium citrate and hydrogen peroxide;

[0011] The triggering agent includes tris(hydroxymethyl)aminomethane hydrochloride, chloride salt, HCl and surfactant.

[0012] On the basis of the above technical solution, the present invention can also be improved as follows.

[0013] Furthermore, the chloride salt includes NaCl; and the surfactant includes Tween 20.

[0014] Furthermore, the enzyme marker also includes bovine serum albumin, sucrose and trehalose;

[0015] The concentration of the horseradish peroxidase-labeled anti-NT-proBNP monoclonal antibody in the enzyme label is 1 mg / ml;

[0016] The mass concentration of bovine serum albumin in the enzyme marker is 0.5% to 2%;

[0017] The mass concentration of sucrose in the enzyme marker is 2% to 5%;

[0018] The mass concentration of trehalose in the enzyme marker is 1% to 3%.

[0019] Furthermore, the luminescent marker also includes casein, sucrose and trehalose;

[0020] The concentration of the 9,10-dihydroacridine-labeled anti-NT-proBNP monoclonal antibody in the luminescent marker is 0.5 mg / ml;

[0021] The mass concentration of casein in the luminescent marker is 0.1% to 1%;

[0022] The mass concentration of sucrose in the luminescent marker is 2% to 5%;

[0023] The mass concentration of trehalose in the luminescent marker is 1% to 3%.

[0024] Furthermore, the luminescence auxiliary agent also includes citric acid and sodium citrate;

[0025] Each 1L of the luminescence auxiliary agent includes the following components in the following amounts: 1.5g to 2g of citric acid, 5g to 11g of sodium citrate, and 0.15mL to 0.45mL of 30% hydrogen peroxide;

[0026] Each 1L of the triggering agent includes the following components: 3g to 9g of tris (hydroxymethyl)aminomethane hydrochloride, 6g to 12g of NaCl, 1mL to 4mL of concentrated HCl, and 1mL to 4mL of Tween 20;

[0027] The mass content of the concentrated HCl is 36% to 38%.

[0028] In a second aspect, a kit for detecting N-terminal B-type natriuretic peptide precursor is prepared, comprising the following steps:

[0029] The enzyme label was prepared using horseradish peroxidase and anti-NT-proBNP monoclonal antibody;

[0030] Luminescent markers were prepared using 9,10-dihydroacridine and anti-NT-proBNP monoclonal antibodies;

[0031] A luminescence auxiliary agent is prepared by using citric acid, sodium citrate and hydrogen peroxide;

[0032] The trigger is prepared by using tris(hydroxymethylaminomethane) hydrochloride, chloride salt, HCl and surfactant.

[0033] Furthermore, the mass ratio of the horseradish peroxidase to the anti-NT-proBNP monoclonal antibody is 1:1.

[0034] The preparation of the enzyme marker comprises the following specific steps: dissolving horseradish peroxidase in distilled water, adding sodium periodate solution, mixing, and dialyzing in sodium acetate buffer, then adding carbonate buffer, anti-NT-proBNP monoclonal antibody, and sodium borohydride solution, mixing, and dialyzing again in PBS, removing the dialyzate, adding 50% glycerol, and storing at -20°C for future use.

[0035] Furthermore, the mass ratio of the 9,10-dihydroacridine to the anti-NT-proBNP monoclonal antibody is 1:3.

[0036] The preparation of the luminescent marker includes the following specific steps: dissolving 9,10-dihydroacridine, adding sodium borate buffer and anti-NT-proBNP monoclonal antibody, shaking and mixing overnight, taking out, adding 50% glycerol and storing in a -20°C refrigerator.

[0037] Furthermore, the preparation of the luminescence auxiliary agent includes the following specific steps: taking citric acid, sodium citrate and hydrogen peroxide solution, dissolving them in purified water and fixing the volume to obtain the luminescence auxiliary agent;

[0038] The preparation of the trigger comprises the following specific steps: weighing tris(hydroxymethyl)aminomethane hydrochloride), NaCl, concentrated HCl and a surfactant, dissolving them and then fixing the volume to obtain the trigger.

[0039] In a third aspect, a kit for detecting N-terminal B-type natriuretic peptide precursor is used to prepare a detection reagent for preventing and treating heart failure.

[0040] The present invention uses a spatial proximity chemiluminescence analysis method to detect the concentration of NT-proBNP. A sample, a horseradish peroxidase (HRP)-labeled anti-NT-proBNP monoclonal antibody, and a 9,10-dihydroacridine (Acridan)-labeled anti-NT-proBNP monoclonal antibody are reacted together to form an antigen-antibody sandwich complex, so that the horseradish peroxidase and the 9,10-dihydroacridine (Acridan) are spatially close to each other. A luminescence auxiliary agent and a triggering agent are added to generate flash chemiluminescence. Unbound, free HRP-labeled and Acridan-labeled antibodies do not emit light. The higher the NT-proBNP content in the sample, the greater the measured luminescence value (RLU). Therefore, within a certain concentration range, the luminescence value is positively correlated with the sample concentration. A working curve is plotted using calibrators of known concentrations and their luminescence values, and the NT-proBNP content in the sample can be calculated based on the sample luminescence value.

[0041] The beneficial effects of the present invention are as follows: the spatial proximity chemical double antibody sandwich method adopted by the kit of the present invention is simple to operate and can be fully automatically detected compared with other methods such as enzyme-linked immunosorbent assay and fluorescent immunochromatography, without the need for magnetic beads and washing, which not only reduces the reagent cost but also shortens the detection time. In addition, the kit adopts a homogeneous reaction, avoiding the influence of traditional chemiluminescence magnetic separation technology, making the detection results more accurate and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the standard curve diagram of the present invention. DETAILED DESCRIPTION

[0043] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0044] Example

[0045] (1) Materials and reagents

[0046] (1) Antigen and Antibody:

[0047] NT-proBNP-REAB-c1-019 and NT-proBNP-REAB-c1-016 were purchased from Feipeng Biotechnology Co., Ltd., and the antigen was purchased from Nanjing Ainuodi Biotechnology Co., Ltd.

[0048] (2) Other reagents:

[0049] 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, and polyethylene glycol 2000 were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. 9,10-Dihydroacridine was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0050] (3) Instruments:

[0051] Guangzhou Jinde Biotechnology Co., Ltd. HomoG 100 fully automatic chemiluminescence analyzer (Guangdong Medical Device Registration No. 20212220196)

[0052] 2. Kit and Preparation Method

[0053] 2.1 Kit for detecting N-terminal pro-B-type natriuretic peptide

[0054] A kit for detecting N-terminal B-type natriuretic peptide precursor, comprising an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger;

[0055] The enzyme marker includes horseradish peroxidase-labeled anti-NT-proBNP monoclonal antibody;

[0056] The luminescent marker includes an anti-NT-proBNP monoclonal antibody labeled with 9,10-dihydroacridine;

[0057] The luminescence auxiliary agent includes citric acid, sodium citrate and hydrogen peroxide;

[0058] The triggering agent includes tris(hydroxymethyl)aminomethane hydrochloride, chloride salt, HCl and surfactant.

[0059] In this embodiment, the chloride salt includes NaCl; the surfactant includes Tween 20.

[0060] Preferably, the enzyme marker further comprises bovine serum albumin, sucrose and trehalose;

[0061] The concentration of the horseradish peroxidase-labeled anti-NT-proBNP monoclonal antibody in the enzyme label is 1 mg / ml;

[0062] The mass concentration of bovine serum albumin in the enzyme marker is 0.5% to 2%;

[0063] The mass concentration of sucrose in the enzyme marker is 2% to 5%;

[0064] The mass concentration of trehalose in the enzyme marker is 1% to 3%.

[0065] Preferably, in this embodiment, the luminescent marker further comprises casein, sucrose and trehalose;

[0066] The concentration of the 9,10-dihydroacridine-labeled anti-NT-proBNP monoclonal antibody in the luminescent marker is 0.5 mg / ml;

[0067] The mass concentration of casein in the luminescent marker is 0.1% to 1%;

[0068] The mass concentration of sucrose in the luminescent marker is 2% to 5%;

[0069] The mass concentration of trehalose in the luminescent marker is 1% to 3%.

[0070] In this embodiment, preferably, each 1 L of the luminescence auxiliary agent includes the following components: 1.5 g to 2 g of citric acid, 5 g to 11 g of sodium citrate, and 0.15 mL to 0.45 mL of 30% hydrogen peroxide.

[0071] Furthermore, each 1L of the triggering agent includes the following components: 3g to 9g of tris(hydroxymethyl)aminomethane hydrochloride, 6g to 12g of NaCl, 1mL to 4mL of concentrated HCl, and 1mL to 4mL of Tween 20;

[0072] The mass content of the concentrated HCl is 36% to 38%.

[0073] The following is a detailed description of the preparation process of this kit, and the prepared kit is used for experimental detection.

[0074] 2.2 Specific preparation method of the kit

[0075] (1) Preparation of enzyme markers:

[0076] Weigh 1 mg of HRP and dissolve it in 200 μL of distilled water. Add 40 μL of freshly prepared 0.1 M sodium periodate and shake at room temperature for 20 min. Pour the above liquid into a dialysis bag and dialyze it against 1 mM sodium acetate buffer (pH 4.4) at 4°C overnight. Add 20 μL of 0.2 M carbonate buffer (pH 9.5). Immediately add 1 mg of anti-NT-proBNP monoclonal antibody and 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 let it stand at 4°C for 2 h. Pour the above liquid into a dialysis bag and dialyze it against 0.15 M PBS (pH 7.4) at 4°C overnight. Remove the dialysate, add 50% glycerol, and store it at -20°C until use.

[0077] (2) Preparation of luminescent markers:

[0078] 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-NT-proBNP (diluted to 1 mg / mL), invert and mix five times, let stand at room temperature for 30 minutes, place the labeled reaction tube on a shaker at 2-8°C and mix overnight, take out and add 50% glycerol and store in a refrigerator at -20°C.

[0079] (3) Luminescence auxiliary agent:

[0080] Dissolve 1.82 g of citric acid and 10.45 g of sodium citrate in purified water and dilute 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 (divide into 10 mL / bottle).

[0081] (4) Trigger:

[0082] Weigh 6.06 g of Tris and 9 g of NaCl, dissolve them in appropriate amount of purified water, add 2.1 mL of concentrated HCl and 2 mL of Tween 20, mix well, and make up to 1000 mL. Place in a 4°C refrigerator until ready for use.

[0083] (5) Calibrators:

[0084] ① Calibrator diluent: Weigh 14.1 g of potassium dihydrogen phosphate and 3.0 g of sodium dihydrogen phosphate dihydrate, dissolve them in an appropriate amount of purified water, add 0.5 mL of Proclin 300, mix well, and dilute to 1000 mL with purified water. Place in a 4°C refrigerator until ready for use.

[0085] ②Dilute recombinant NT-proBNP to 29.3 pg / mL, 58.59 pg / mL, 117.19 pg / mL, 234.38 pg / mL, 468.75 pg / mL, 937.5 pg / mL, 1875 pg / mL, 3750 pg / mL, 7500 pg / mL, 15000 pg / mL, and 30000 pg / mL with calibrator diluent and place in a 4°C refrigerator until used.

[0086] 3. Detection method:

[0087] The system requires a sample volume of 10 μL per measurement. Before applying for a test, you should prepare all the materials required for the test and carefully read the chemiluminescence immunoassay user manual to obtain information on system operating procedures, sample management, usage precautions, maintenance, and other information. The main steps for applying for a test are as follows:

[0088] 3.1 Calibration:

[0089] Perform a calibration test using the included kit and calibrator. Before starting the kit calibration, scan the master curve in the kit's 2D barcode and import it into the system.

[0090] The chemiluminescence immunoassay analyzer uses the results of calibrator tests to adjust the master curve and generate the calibration curve for the current system. The instrument operating software includes a calibration check function that automatically verifies the validity of the calibration curve. If calibration fails, consider the instrument settings, status, location of the calibrators (place low-value calibrators first, then high-value calibrators), and expiration dates.

[0091] A valid calibration curve is required before all tests. Recalibration is required in the following situations:

[0092] ⑴Use a new batch of test kit;

[0093] (2) The same batch of test kits has been used on the analyzer for more than 30 days;

[0094] ⑶After instrument maintenance;

[0095] (4) The quality control value exceeds the specified range;

[0096] 3.2 Quality Control:

[0097] To ensure the reliability of test results, it is recommended that both high and low level quality control products be tested once every 24 hours. In addition, quality control testing is also recommended after each calibration test, reagent batch change, maintenance and troubleshooting.

[0098] The test values of quality control products should be within the specified range. If they are outside the specified range, the user should check the detection system, such as the location, expiration date, storage method, calibration process, instrument performance and status, and recalibrate if necessary. Sample testing can only be carried out after the quality control is qualified.

[0099] 3.3 Sample testing:

[0100] ⑴ Enter the sample application interface and enter the sample number, sample rack number, position number, sample type, remarks and other information according to the test requirements;

[0101] ⑵Select the test item, select the sample type in the item options, enter the number of repetitions and other information;

[0102] ⑶ After preparing the test sample and placing it correctly, click the start button to start the test. The chemiluminescence immunoassay analyzer will perform the following operations in sequence:

[0103] ①The sample processing system transfers the sample rack to the sample suction position;

[0104] ② The cuvette loading and scheduling system loads the cuvette from the loading area to the sample loading position;

[0105] ③The sample injection system completes the sample injection and cleaning of the sample needle;

[0106] (4) The reagent processing system provides the reagents required for the test, mixes each reagent, and sends it to the reagent aspiration position for aspiration;

[0107] (5) The sample injection system completes the reagent injection and cleaning of the reagent needle;

[0108] ④Mix the reaction mixture with the counter-liquid mixing system and incubate in the reaction plate;

[0109] ⑤After the incubation is completed, inject the substrate solution into the substrate system, mix, incubate, and wait for light detection;

[0110] ⑥The light measurement reaction system sends each reaction cup to the signal collection position to collect signals for calculating the luminescence value;

[0111] ⑦Calculate the content of the analyte in the sample.

[0112] 3.4 Calculation

[0113] Using stored calibration data, the system software automatically determines the sample test results using a weighted four-parameter logarithmic curve (4PLC, Y-weighted) mathematical method. Results are given in pg / mL.

[0114] 3.5 Comparison reagent: N-terminal atrial natriuretic peptide (NT-proBNP) detection kit (chemiluminescence method) produced by Ruibo Biotechnology Co., Ltd. (Guangzhou), Guangdong Medical Device Registration No. 20212400971.

[0115] 4. Results

[0116] Detection of 50 samples from patients with heart failure, and the quantitative correlation coefficient R with the control reagent 2 =0.9993( Figure 1 and Table 1 ).

[0117] Table 1

[0118]

[0119]

[0120] according to Figure 1 As shown in Table 1, the two are well correlated, and the yin-yang coincidence rate is high.

[0121] (2) Minimum detection limit

[0122] Using PBS buffer as a blank sample, the detection and comparison reagents prepared in the present invention were simultaneously measured 25 times. The test strip test and comparison values prepared in the present invention, plus twice the standard deviation, were substituted into the fitted curve for regression. The results showed a minimum detection limit of 19.6 pg / mL for the detection kit prepared in the present invention, and a minimum detection limit of 21.3 for the comparison reagent. The results are shown in Table 2.

[0123] Table 2

[0124]

[0125]

[0126] (3) Accuracy

[0127] A high-concentration reference substance (Solution A) was added to a low-concentration reference substance (Solution B), with the volume ratio between the high-concentration reference substance (Solution A) and the low-concentration reference substance (Solution B) being no greater than 1:9. Each sample was measured three times and the average value was obtained. Calculated according to formula (1), the recovery rate of the detection preparation method prepared by the present invention was 99.3%, indicating good accuracy. The results are shown in Table 3.

[0128]

[0129] Where: R is the recovery rate; V is the volume of solution A added; V0 is the volume of serum sample B; c is the detection concentration of serum sample after adding solution A; c0 is the detection concentration of serum sample B; c s is the concentration of solution A.

[0130] Table 3

[0131]

[0132] In summary, the spatial proximity chemical double antibody sandwich method used in this kit is simple to operate and can be fully automatically detected compared to other methods such as enzyme-linked immunosorbent assay and fluorescent immunochromatography. It does not require magnetic beads and washing, which not only reduces reagent costs but also shortens detection time. In addition, it adopts a homogeneous reaction, avoiding the influence of traditional chemiluminescence magnetic separation technology, making the detection results more accurate and stable.

[0133] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A kit for detecting N-terminal B-type natriuretic peptide precursor, characterized in that: The kit includes an enzyme marker, a luminescent marker, a luminescent auxiliary agent and a trigger; The enzyme marker includes horseradish peroxidase-labeled anti-NT-proBNP monoclonal antibody; The luminescent marker includes an anti-NT-proBNP monoclonal antibody labeled with 9,10-dihydroacridine; The luminescence auxiliary agent includes citric acid, sodium citrate and hydrogen peroxide; The triggering agent includes tris(hydroxymethyl)aminomethane hydrochloride, chloride salt, HCl and surfactant.

2. A kit for detecting N-terminal B-type natriuretic peptide precursor according to claim 1, characterized in that: The chloride salt includes NaCl; the surfactant includes Tween 20.

3. A kit for detecting N-terminal B-type natriuretic peptide precursor according to claim 1, characterized in that: The enzyme marker also includes bovine serum albumin, sucrose and trehalose; The concentration of the horseradish peroxidase-labeled anti-NT-proBNP monoclonal antibody in the enzyme label is 1 mg / ml; The mass concentration of bovine serum albumin in the enzyme marker is 0.5% to 2%; The mass concentration of sucrose in the enzyme marker is 2% to 5%; The mass concentration of trehalose in the enzyme marker is 1% to 3%.

4. The kit for detecting N-terminal B-type natriuretic peptide precursor according to claim 1, characterized in that: The luminescent marker also includes casein, sucrose and trehalose; The concentration of the 9,10-dihydroacridine-labeled anti-NT-proBNP monoclonal antibody in the luminescent marker is 0.5 mg / ml; The mass concentration of casein in the luminescent marker is 0.1% to 1%; The mass concentration of sucrose in the luminescent marker is 2% to 5%; The mass concentration of trehalose in the luminescent marker is 1% to 3%.

5. The kit for detecting N-terminal B-type natriuretic peptide precursor according to claim 1, characterized in that: Each 1L of the luminescence auxiliary agent includes the following components in the following amounts: 1.5g to 2g of citric acid, 5g to 11g of sodium citrate, and 0.15mL to 0.45mL of 30% hydrogen peroxide; Each 1L of the triggering agent includes the following components: 3g to 9g of tris (hydroxymethyl)aminomethane hydrochloride, 6g to 12g of NaCl, 1mL to 4mL of concentrated HCl, and 1mL to 4mL of Tween 20; The mass content of the concentrated HCl is 36% to 38%.

6. Preparation of a kit for detecting N-terminal B-type natriuretic peptide precursor according to any one of claims 1 to 5, characterized in that: The steps include: The enzyme label was prepared using horseradish peroxidase and anti-NT-proBNP monoclonal antibody; Luminescent markers were prepared using 9,10-dihydroacridine and anti-NT-proBNP monoclonal antibodies; A luminescence auxiliary agent is prepared by using citric acid, sodium citrate and hydrogen peroxide; The trigger is prepared by using tris(hydroxymethylaminomethane) hydrochloride, chloride salt, HCl and surfactant.

7. The preparation of a kit for detecting N-terminal B-type natriuretic peptide precursor according to claim 6, characterized in that: The mass ratio of the horseradish peroxidase to the anti-NT-proBNP monoclonal antibody is 1:

1.

8. The preparation of a kit for detecting N-terminal B-type natriuretic peptide precursor according to claim 6, characterized in that: The mass ratio of the 9,10-dihydroacridine to the anti-NT-proBNP monoclonal antibody is 1:

3.

9. The preparation of a kit for detecting N-terminal B-type natriuretic peptide precursor according to claim 6, characterized in that: The preparation of the luminescence auxiliary agent includes the following specific steps: taking citric acid, sodium citrate and hydrogen peroxide solution, dissolving them in purified water and adjusting the volume to obtain the luminescence auxiliary agent; The preparation of the trigger comprises the following specific steps: weighing tris(hydroxymethyl)aminomethane hydrochloride), NaCl, concentrated HCl and a surfactant, dissolving them and then fixing the volume to obtain the trigger.

10. Use of a kit for detecting N-terminal B-type natriuretic peptide precursor, characterized in that: The kit for detecting N-terminal B-type natriuretic peptide precursor according to any one of claims 1 to 5 is used to prepare a detection reagent for preventing and treating heart failure.

Images