CTnI detection kit and preparation method thereof

Abstract

The invention discloses a cTnI detection kit and a preparation method thereof, and relates to the technical field of biochemical detection. The cTnI detection kit comprises a reagent R1 and a reagent R2, the reagent R1 comprises two cTnI specific mouse monoclonal antibodies which are connected with streptavidin magnetic particles and labeled by biotin; the reagent R2 comprises a cTnI specific mouse monoclonal antibody marked by acridinium ester; the two cTnI specific mouse monoclonal antibodies labeled by biotin are respectively and specifically combined with the 41st to 49th amino acid fragments of the cTnI and the joint between the cTnI and the cTnC in the cTnI-cTnC dimer compound; the cTnI specific mouse monoclonal antibody marked by acridinium ester is specifically combined with the 24 to 40 amino acid fragments of cTnI. The cTnI detection kit provided by the invention is simple, convenient and rapid to operate, high in sensitivity (hypersensitivity), good in specificity, wide in linear range and good in long-term stability and thermal stability.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection technology, and in particular to a cTnI detection kit and its preparation method. Background Technology

[0002] In striated muscle, the troponin complex binds to tropomyosin and is responsible for the precise regulation of the actin-myosin cross-bridge cycle. In the early stages, the most prevalent forms in peripheral circulation are free cardiac troponin I (cTnI), cardiac troponin T (cTnT), the cTnI-cTnC (cardiac troponin I-cardiac troponin C) dimer complex, and the cTnT-cTnI-cTnC (cardiac troponin T-cardiac troponin I-cardiac troponin C) trimer complex, with a molar ratio of approximately 1:1:1 for cTnI, cTnT, and cTnC. In the later stages (20-30 hours after symptom onset), cardiac troponin (cTn) exists primarily in the blood through the cTnI-cTnC dimer complex and free fragments of cTnT. Immunological detection of cTnI in the blood is an important method for diagnosing acute myocardial infarction (AMI). Conventional cTnI kits have low sensitivity and low detection rates, often delaying diagnosis. Although many manufacturers have introduced highly sensitive kits in recent years, these kits can improve detection sensitivity, effectively detecting cTnI changes at the onset of myocardial infarction and enabling early prevention. However, current detection reagents are still affected by various factors, including hydrolysis, phosphorylation, TnC complexes, heparin, heterophile antibodies, and TnI autoantibodies, thus affecting the test results. Among these, the most significant problem stems from cTnI degradation.

[0003] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a cTnI detection kit and its preparation method, which aims to provide a cTnI detection kit with high sensitivity, good specificity, wide linear range, good long-term stability and thermal stability.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a cTnI detection kit, wherein the cTnI detection kit includes reagent R1 and reagent R2; The reagent R1 comprises a complex comprising streptavidin magnetic particles and two biotin-labeled cTnI-specific mouse monoclonal antibodies linked to the streptavidin magnetic particles. The reagent R2 comprises an acridinium ester-labeled cTnI-specific mouse monoclonal antibody; Two biotin-labeled cTnI-specific mouse monoclonal antibodies specifically bind to amino acid fragments 41-49 of cTnI and the linker between cTnI and cTnC in the cTnI-cTnC dimer complex, respectively. The acridine ester-labeled cTnI-specific mouse monoclonal antibody specifically binds to amino acid fragments 24-40 of cTnI.

[0006] Optionally, the reagent R1 is prepared by mixing a solid-phase premixed reagent and a biotin premixed reagent; The solid-phase premixed reagent comprises the following components in varying amounts: A first buffer solution of 10–30 mmol / L, a first salt solution of 10–300 mmol / L, and streptavidin magnetic particles with a mass content of 0.07%–0.2%; The biotin premixed reagent comprises the following components in the following amounts: The mixture contained a second buffer solution of 20–400 mmol / L, a second salt solution of 10–300 mmol / L, a first protective agent of 0.02%–2% by mass, a first preservative of 0.02%–2% by mass, a first surfactant of 0.02%–2% by mass, water, and a biotin-labeled first cTnI-specific mouse monoclonal antibody and a biotin-labeled second cTnI-specific mouse monoclonal antibody; the concentration of the first cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL, and the concentration of the second cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL. In the first biotin-labeled cTnI-specific mouse monoclonal antibody, the molar ratio of the first cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:20. In the biotin-labeled second cTnI-specific mouse monoclonal antibody, the molar ratio of the second cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:20.

[0007] Optionally, the reagent R2 comprises the following components in varying amounts: The mixture contained a third buffer solution of 10–100 mmol / L, a third salt solution of 10–50 mmol / L, a second protective agent of 0.02%–2% by mass, a second preservative of 0.01%–0.1% by mass, a second surfactant of 0.05%–1% by mass, water, and an acridinium ester-labeled third cTnI-specific mouse monoclonal antibody; the concentration of the third cTnI-specific mouse monoclonal antibody was 0.1–0.8 μg / mL. In the acridine ester-labeled third cTnI-specific mouse monoclonal antibody, the molar ratio of the third cTnI-specific mouse monoclonal antibody to acridine ester is 1:1 to 1:10.

[0008] Optionally, the first, second, and third buffers each independently comprise at least one of HEPES buffer, PIPES buffer, MOPS buffer, disodium hydrogen phosphate-citrate buffer, phosphate buffer, BISTRIS buffer, TRIS buffer, and sodium barbital-hydrochloric acid buffer; and / or, The first salt, the second salt, and the third salt each independently include at least one of NaCl, KCl, and MgCl2; and / or, The magnetic particles have a particle size of 1~3μm.

[0009] Optionally, the first and second protective agents each independently comprise at least one of casein, bovine serum albumin, and human serum albumin; and / or, The first surfactant and the second surfactant each independently include at least one of polyoxyethylene lauryl ether, Triton X-100, Tween-20 and Tween-80.

[0010] Optionally, the cTnI detection kit further includes a chemiluminescent excitation solution, which includes an acidic excitation solution and an alkaline excitation solution; The acidic activating solution includes a mixed solution of nitric acid and hydrogen peroxide, and the alkaline activating solution includes a sodium hydroxide solution.

[0011] Optionally, the cTnI detection kit further includes cTnI calibrators and / or cTnI quality control samples, wherein the cTnI calibrators comprise cTnI antigen solutions with concentrations of 0 ng / L, 35 ng / L, and 15000 ng / L, respectively; and the cTnI quality control samples comprise cTnI antigen solutions with concentrations of 25 ng / L and 2000 ng / L, respectively.

[0012] Optionally, the cTnI detection kit further includes reagent R3, which comprises the following components in varying amounts: 20–400 mmol / L fourth buffer, 10–150 mmol / L fourth salt, 0.05%–2% by mass of third protective agent, and 0.01%–1% by mass of third surfactant.

[0013] A second aspect of the present invention provides a method for preparing the cTnI detection kit as described above, comprising the following steps: Reagents R1 and R2 were prepared separately to obtain the cTnI detection kit.

[0014] Optionally, the preparation method of reagent R1 includes the following steps: Solid-phase premixed reagents and biotin premixed reagents are provided in a volume ratio of 1:1; The solid-phase premixed reagent is placed on a magnetic separator for magnetic separation to obtain separated magnetic beads; Add the biotin premixed reagent to the separated magnetic beads, mix by inverting, and then premix in a mixer to obtain the reagent R1. The solid-phase premixed reagent comprises the following components in varying amounts: A first buffer solution of 10–30 mmol / L, a first salt solution of 10–300 mmol / L, and streptavidin magnetic particles with a mass content of 0.07%–0.2%; The biotin premixed reagent comprises the following components in the following amounts: The mixture contained a second buffer solution of 20–400 mmol / L, a second salt solution of 10–300 mmol / L, a first protective agent of 0.02%–2% by mass, a first preservative of 0.02%–2% by mass, a first surfactant of 0.02%–2% by mass, water, and a biotin-labeled first cTnI-specific mouse monoclonal antibody and a biotin-labeled second cTnI-specific mouse monoclonal antibody; the concentration of the first cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL, and the concentration of the second cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL. In the first biotin-labeled cTnI-specific mouse monoclonal antibody, the molar ratio of the first cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:20. In the biotin-labeled second cTnI-specific mouse monoclonal antibody, the molar ratio of the second cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:20.

[0015] Beneficial effects: The cTnI detection kit provided by this invention is simple and quick to operate, highly sensitive (ultrasensitive), specific, has a wide linear range, and exhibits good long-term and thermal stability. In this invention, the cTnI detection kit is based on chemiluminescent immunoassay, enabling the detection of cTnI using a fully automated chemiluminescent immunoassay analyzer. Therefore, when used with the instrument, this kit shortens the time required for clinical testing and provides high detection accuracy. Attached Figure Description

[0016] Figure 1 This is a hook diagram of the cTnI detection kit in Example 1. Detailed Implementation

[0017] This invention provides a cTnI detection kit and its preparation method. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0019] If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0020] Current detection reagents are affected by various factors, including hydrolysis, phosphorylation, TnC complexes, heparin, heterophilic antibodies, and TnI autoantibodies, thus affecting the detection results. Among these, the most significant problem stems from cTnI degradation. Therefore, the selection of antibody sites is crucial. Based on this, this invention provides a cTnI detection kit, comprising reagent R1 and reagent R2; The reagent R1 comprises a complex comprising streptavidin magnetic particles and two biotin-labeled cTnI-specific mouse monoclonal antibodies linked to the streptavidin magnetic particles. The reagent R2 comprises an acridinium ester-labeled cTnI-specific mouse monoclonal antibody; Two biotin-labeled cTnI-specific mouse monoclonal antibodies specifically bind to amino acid fragments 41-49 of cTnI and the linker between cTnI and cTnC in the cTnI-cTnC dimer complex, respectively. The acridine ester-labeled cTnI-specific mouse monoclonal antibody specifically binds to amino acid fragments 24-40 of cTnI.

[0021] In this invention, the antibody site selection avoids hydrolytic regions. Specifically, in vitro studies on necrotic myocardial tissue revealed several 14-28 kDa cTnI fragments (the molecular weight of complete cTnI is approximately 29 kDa). The 30-110 amino acid region in the middle of the cTnI molecule is considered the most stable region (this region contains two-thirds of the complete cTnI molecule). Meanwhile, most antibodies specifically recognizing amino acid fragments 50-80 and 90-130 of cTnI have difficulty recognizing cTnI-cTnC and cTnT-cTnI-cTnC complexes. Therefore, the recognition site can only be selected before amino acid 50 or between amino acids 80-90. However, studies have shown that antibodies specifically recognizing amino acid fragments 80-90 of cTnI are susceptible to interference from circulating antibodies, resulting in false negatives. Therefore, the antibody site in this invention selects amino acid fragments 41-49 and 24-40 of cTnI. Furthermore, in the cTnT-cTnI-cTnC complex, cTnI exists primarily as a full-length molecule of 29 kDa (amino acids 1-209) and fragment molecules of 27 kDa (amino acids 1-196) and 26 kDa (amino acids 23-209). In the cTnI-cTnC complex (molecular weights of 19 kDa and 14 kDa), cTnI exists primarily as fragment molecules of amino acids 23-145 and 23-126. The hydrolysis and phosphorylation sites of cTnI are mainly located at the two ends of the chain, primarily the N-terminus and C-terminus, while the middle region is relatively stable; therefore, there is no breakage at amino acids 24-40 and 41-49. The selection of antibody sites in this invention not only avoids hydrolysis regions and improves detection accuracy, but also, through the selection and combination of antibody sites, makes the cTnI detection kit highly sensitive, achieving hypersensitivity.

[0022] In this invention, the provided cTnI detection kit employs a multi-antibody sandwich method based on chemiluminescent immunoassay technology to detect different forms of cTnI in blood. Specifically, an acridil ester-labeled cTnI-specific mouse monoclonal antibody and two biotin-labeled cTnI-specific mouse monoclonal antibodies undergo an immune reaction with cTnI in the sample, forming an antibody-antigen-antibody complex, which is then bound to magnetic particles via biotin and streptavidin. The cTnI content in the sample is directly proportional to the relative optical units (RLU) detected by the system.

[0023] The cTnI detection kit provided by this invention is simple and quick to operate, highly sensitive (ultrasensitive), specific, has a wide linear range, and exhibits good long-term and thermal stability. In this invention, the cTnI detection kit is based on chemiluminescent immunoassay, enabling the detection of cTnI using a fully automated chemiluminescent immunoassay analyzer. Therefore, this kit, when used with the instrument, shortens the time required for clinical testing and provides high detection accuracy.

[0024] In addition, in this invention, a complex of two cTnI-specific mouse monoclonal antibodies, including streptavidin magnetic particles and biotin-labeled particles linked to the streptavidin magnetic particles, is used as the main component of reagent R1. This can reduce the interference of biotin in the sample on the detection, and solve the problems of high-concentration biotin interference and the complex, time-consuming, and difficult-to-control batch-to-batch processes caused by coating.

[0025] In some embodiments, the reagent R1 is prepared by mixing a solid-phase premixed reagent and a biotin premixed reagent; The solid-phase premixed reagent comprises the following components in varying amounts: A first buffer solution of 10–30 mmol / L, a first salt solution of 10–300 mmol / L, and streptavidin magnetic particles with a mass content of 0.07%–0.2%; For example, in the solid-phase premixed reagent, the concentration of the first buffer solution is 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, or 30 mmol / L, etc.; the concentration of the first salt is 10 mmol / L, 20 mmol / L, 50 mmol / L, 80 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, 250 mmol / L, or 300 mmol / L, etc.; and the mass content of the streptavidin magnetic particles is 0.07%, 0.08%, 0.09%, 0.1%, 0.12%, 0.15%, 0.18%, or 0.2%, etc.

[0026] Specifically, the first buffer includes, but is not limited to, at least one of HEPES buffer (where HEPES is 4-hydroxyethylpiperazine ethanesulfonic acid), PIPES buffer (where PIPES is piperazine-1,4-diethanesulfonic acid), MOPS buffer (where MOPS is sodium 3-(N-morpholino)propanesulfonate), disodium hydrogen phosphate-citrate buffer, phosphate buffer, BISTRIS buffer (where BISTRIS is bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane), TRIS buffer (where TRIS represents tris(hydroxymethyl)aminomethane), and sodium barbital-hydrochloric acid buffer.

[0027] Specifically, the first salt includes, but is not limited to, at least one of NaCl, KCl, and MgCl2.

[0028] Specifically, in the streptavidin magnetic particles, the particle size of the magnetic particles is 1~3µm, for example, it can be 1µm, 2µm or 3µm, etc.

[0029] The biotin premixed reagent comprises the following components in the following amounts: The mixture contained a second buffer solution of 20–400 mmol / L, a second salt solution of 10–300 mmol / L, a first protective agent of 0.02%–2% by mass, a first preservative of 0.02%–2% by mass, a first surfactant of 0.02%–2% by mass, water, and a biotin-labeled first cTnI-specific mouse monoclonal antibody and a biotin-labeled second cTnI-specific mouse monoclonal antibody; the concentration of the first cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL, and the concentration of the second cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL. In the first biotin-labeled cTnI-specific mouse monoclonal antibody, the molar ratio of the first cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:20. In the biotin-labeled second cTnI-specific mouse monoclonal antibody, the molar ratio of the second cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:20.

[0030] Specifically, the concentration of the second buffer solution is 20 mmol / L, 50 mmol / L, 80 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, 250 mmol / L, 300 mmol / L, 350 mmol / L, or 400 mmol / L; the concentration of the second salt is 10 mmol / L, 20 mmol / L, 50 mmol / L, 80 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, 250 mmol / L, or 300 mmol / L; and the mass content of the first protective agent is 0.02%, 0.05%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, or 1.2%. The first preservative has a mass content of 0.02%, 0.05%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2%, etc.; the first surfactant has a mass content of 0.02%, 0.05%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2%, etc.

[0031] The concentrations of the first cTnI-specific mouse monoclonal antibody were 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1.0 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5 μg / mL, 1.6 μg / mL, 1.7 μg / mL, 1.8 μg / mL, 1.9 μg / mL, or 2.0 μg / mL.

[0032] The concentrations of the second cTnI-specific mouse monoclonal antibody were 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1.0 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5 μg / mL, 1.6 μg / mL, 1.7 μg / mL, 1.8 μg / mL, 1.9 μg / mL, or 2.0 μg / mL.

[0033] In the first biotin-labeled cTnI-specific mouse monoclonal antibody, the molar ratio of the first cTnI-specific mouse monoclonal antibody to biotin is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20, etc.

[0034] In the biotin-labeled second cTnI-specific mouse monoclonal antibody, the molar ratio of the second cTnI-specific mouse monoclonal antibody to biotin is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20, etc.

[0035] Specifically, the second buffer includes, but is not limited to, at least one of HEPES buffer, PIPES buffer, MOPS buffer, disodium hydrogen phosphate-citrate buffer, phosphate buffer, BISTRIS buffer, TRIS buffer, and sodium barbital-hydrochloric acid buffer.

[0036] Specifically, the second salt includes, but is not limited to, at least one of NaCl, KCl, and MgCl2. These salts can balance the osmotic pressure of reagent R1.

[0037] Specifically, the first protective agent includes, but is not limited to, at least one of casein, bovine serum albumin, and human serum albumin. These protective agents can maintain the activity of cTnI-specific mouse monoclonal antibodies and maintain the spatial structural stability of cTnI-specific mouse monoclonal antibodies.

[0038] Specifically, the first preservative includes, but is not limited to, at least one of the ProClin series preservatives (whose active ingredients are 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT); specifically, it may be ProClin300, ProClin200, etc.), sodium azide, and BND-10 (whose main component is 5-bromo-5-nitro-1,3-dioxanepropanediol).

[0039] The first surfactant includes, but is not limited to, at least one of polyoxyethylene lauryl ether, Triton X-100, Tween-20 and Tween-80.

[0040] In some embodiments, the reagent R2 comprises the following components in varying amounts: The mixture contained a third buffer solution of 10–100 mmol / L, a third salt solution of 10–50 mmol / L, a second protective agent of 0.02%–2% by mass, a second preservative of 0.01%–0.1% by mass, a second surfactant of 0.05%–1% by mass, water, and an acridinium ester-labeled third cTnI-specific mouse monoclonal antibody; the concentration of the third cTnI-specific mouse monoclonal antibody was 0.1–0.8 μg / mL. In the acridine ester-labeled third cTnI-specific mouse monoclonal antibody, the molar ratio of the third cTnI-specific mouse monoclonal antibody to acridine ester is 1:1 to 1:10.

[0041] Specifically, in reagent R2, the concentration of the third buffer solution is 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, or 100 mmol / L; the concentration of the third salt is 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, or 50 mmol / L; and the mass content of the second protective agent is 0.02%, 0.05%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or 0. The concentrations of the first preservative are 6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2%, etc.; the mass content of the second preservative is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%, etc.; the mass content of the second surfactant is 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%, etc.; the concentration of the third cTnI-specific mouse monoclonal antibody can be 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, or 0.8 μg / mL, etc.

[0042] In the acridine ester-labeled third cTnI-specific mouse monoclonal antibody, the molar ratio of the third cTnI-specific mouse monoclonal antibody to acridine ester can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, etc.

[0043] Specifically, the third buffer includes, but is not limited to, at least one of HEPES buffer, PIPES buffer, MOPS buffer, disodium hydrogen phosphate-citrate buffer, phosphate buffer, BISTRIS buffer, TRIS buffer, and sodium barbital-hydrochloric acid buffer.

[0044] Specifically, the third salt includes, but is not limited to, at least one of NaCl, KCl, and MgCl2. These salts can balance the osmotic pressure of reagent R2.

[0045] Specifically, the second protective agent includes, but is not limited to, at least one of casein, bovine serum albumin, and human serum albumin. These protective agents can maintain the activity of cTnI-specific mouse monoclonal antibodies and maintain the spatial structural stability of cTnI-specific mouse monoclonal antibodies.

[0046] Specifically, the second preservative includes, but is not limited to, at least one of ProClin, sodium azide, and BND-10.

[0047] Specifically, the second surfactant includes, but is not limited to, at least one of polyoxyethylene lauryl ether, Triton X-100, Tween-20, and Tween-80.

[0048] In some embodiments, the cTnI detection kit further includes a chemiluminescent excitation solution, which includes an acidic excitation solution and an alkaline excitation solution; the acidic excitation solution includes a mixed solution of nitric acid and hydrogen peroxide (wherein, the concentration of HNO3 is 0.005~0.01mol / L (0.005mol / L, 0.006mol / L, 0.007mol / L, 0.008mol / L, 0.009mol / L or 0.01mol / L, etc.), and the concentration of H2O2 is 0.5%~2% (mass percentage), for example, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8% or 2%, etc.); the alkaline excitation solution includes a sodium hydroxide solution (wherein the concentration of sodium hydroxide is 0.1~0.5mol / L, for example, 0.1mol / L, 0.2mol / L, 0.3mol / L, 0.4mol / L or 0.5mol / L, etc.).

[0049] In some embodiments, the cTnI detection kit further includes cTnI calibrators and / or cTnI quality controls. The cTnI calibrators comprise cTnI antigen solutions with concentrations of 0 ng / L, 35 ng / L, and 15000 ng / L; the cTnI quality controls comprise cTnI antigen solutions with concentrations of 25 ng / L and 2000 ng / L.

[0050] In some embodiments, the cTnI detection kit further includes reagent R3, which comprises the following components in varying amounts: 20–400 mmol / L fourth buffer, 10–150 mmol / L fourth salt, 0.05%–2% by mass of third protective agent, and 0.01%–1% by mass of third surfactant.

[0051] Specifically, the fourth buffer includes, but is not limited to, at least one of HEPES buffer, PIPES buffer, MOPS buffer, disodium hydrogen phosphate-citrate buffer, phosphate buffer, BISTRIS buffer, TRIS buffer, and sodium barbital-hydrochloric acid buffer. The fourth salt includes, but is not limited to, at least one of NaCl, KCl, and MgCl2. The third protective agent includes, but is not limited to, at least one of casein, bovine serum albumin, and human serum albumin. The third surfactant includes, but is not limited to, at least one of polyoxyethylene lauryl ether, Triton X-100, Tween-20, and Tween-80.

[0052] This invention also provides a method for preparing the cTnI detection kit as described above, comprising the following steps: Reagents R1 and R2 were prepared separately to obtain the cTnI detection kit.

[0053] In some specific embodiments, reagents R1, R2, R3, chemiluminescent excitation solution, cTnI calibrator, and cTnI quality control are prepared respectively to obtain the cTnI detection kit.

[0054] Specifically, the components can be filled into specially treated reagent bottles to form a reagent kit. Before filling, the reagent bottles can be cleaned with a cleaning buffer.

[0055] In some embodiments, the preparation method of reagent R1 includes the following steps: A solid-phase premixed reagent and a biotin premixed reagent with a volume ratio of 1:1 are provided; the components of the solid-phase premixed reagent and the biotin premixed reagent are described above and will not be repeated here.

[0056] The solid-phase premixed reagent is placed on a magnetic separator for magnetic separation to obtain separated magnetic beads; Add the biotin premixed reagent to the separated magnetic beads, invert and mix, then place in a mixer (e.g., a 360-degree rotating mixer or a blood mixer) for premixing to obtain reagent R1.

[0057] In some embodiments, the preparation method of the solid-phase premixed reagent includes the following steps: (1) Preparation of streptavidin magnetic particles: Take 0.5-1 mL of streptavidin magnetic particle solution with a concentration of 50-100 mg / mL, add 5-10 mL of TBST buffer (Tris-Buffered Saline with Tween 20, mainly composed of Tris-HCl, NaCl and Tween 20), mix thoroughly for 10-15 min, place on a magnetic separator until the supernatant is clear, discard the supernatant and keep the magnetic particles. Repeat the washing 3 times to obtain streptavidin magnetic particles; (2) Prepare a solid-phase premixed reagent with a mass concentration (i.e. mass content) of 0.07% to 0.2% by using a buffer solution containing 25 mmol / L TRIS, 150 mmol / L NaCl, and pH 7.2 for the above streptavidin magnetic particles, and store it at 2℃ to 8℃.

[0058] In some embodiments, the preparation method of the biotin-labeled first cTnI-specific mouse monoclonal antibody includes the following steps: The first cTnI-specific mouse monoclonal antibody was placed in a centrifuge tube, carbonate buffer was added, and biotin was added after mixing. The tube was centrifuged and vortexed in the dark (e.g., in an air bath constant temperature shaker). Blocking buffer was added and vortexed (e.g., in an air bath constant temperature shaker). The blocked biotin-labeled first cTnI-specific mouse monoclonal antibody was purified and collected.

[0059] In some embodiments, the preparation method of the first biotin-labeled cTnI-specific mouse monoclonal antibody specifically includes the following steps: (1) Replacement: After equilibrating the first cTnI-specific mouse monoclonal antibody to room temperature, mix it thoroughly and add it to an ultrafiltration centrifuge tube. Centrifuge at 1500 rpm for 5-10 min (e.g., 5 min, 6 min, 7 min, 8 min, 9 min or 10 min, etc.) and discard the liquid in the lower tube. Add replacement buffer to the upper tube of the ultrafiltration centrifuge tube, dilute the first cTnI-specific mouse monoclonal antibody in the upper tube to 2 mg / mL, centrifuge at 1500 rpm for 5-10 min, and discard the liquid in the lower tube after centrifugation; add replacement buffer to the upper tube again, dilute the first cTnI-specific mouse monoclonal antibody in the upper tube to 2 mg / mL, centrifuge at 1500 rpm for 5-10 min, and discard the liquid in the lower tube after centrifugation; add replacement buffer to the upper tube again, dilute the first cTnI-specific mouse monoclonal antibody in the upper tube to 2 mg / mL, centrifuge at 1500 rpm for 5-10 min, and discard the liquid in the lower tube after centrifugation (i.e., repeat the steps of adding replacement buffer, diluting, centrifuging, and discarding the liquid in the lower tube three times), and collect the first cTnI-specific mouse monoclonal antibody in the upper tube.

[0060] (2) Labeling: Place the first cTnI-specific mouse monoclonal antibody into a centrifuge tube, add carbonate buffer, mix thoroughly, and then add 0.75~20µL of 2mg / mL biotin in N,N-dimethylformamide (DMF) solution (so that the molar ratio of the first cTnI-specific mouse monoclonal antibody to biotin is 1:5~1:20). Centrifuge at room temperature for 0.5~1min. Seal the centrifuge tube with sealing film and place it in a light-proof dark box. Then place the dark box in an air bath constant temperature shaker (23~25℃) and mix for 3~4h. Add 0.5~1mL of 20%~30% lysine blocking solution, place in an air bath constant temperature shaker (23~25℃), mix at medium speed, and block for 1~2h.

[0061] (3) Purification: The first biotin-labeled cTnI-specific mouse monoclonal antibody, after blocking, was purified by loading onto a Superdex 200 column, eluted with phosphate (PB) buffer, and collected stepwise. The collected concentrated solution of the first biotin-labeled cTnI-specific mouse monoclonal antibody was stored at 2℃~8℃.

[0062] When using (e.g., when preparing biotin premixed reagent), dilute the purified biotin-labeled first cTnI-specific mouse monoclonal antibody concentrate with a buffer solution containing 50 mM BISTRIS, 10 mmol / L NaCl, 1% (w / v) bovine serum albumin, 0.05% (w / v) Tween, 0.05% (w / v) Proclin 300, and 0.05% (w / v) BND-10 at pH 6.5 to a final concentration of 0.1 µg / mL, and store at 2–8 °C.

[0063] The preparation method of the second biotin-labeled cTnI-specific mouse monoclonal antibody is basically the same as that of the first biotin-labeled cTnI-specific mouse monoclonal antibody, except that the "first cTnI-specific mouse monoclonal antibody" is replaced with the "second cTnI-specific mouse monoclonal antibody".

[0064] In some embodiments, the preparation method of the acridinium ester-labeled third cTnI-specific mouse monoclonal antibody in reagent R2 includes the following steps: The third cTnI-specific mouse monoclonal antibody was placed in a centrifuge tube, carbonate buffer was added, and after mixing, acrid ester was added. The tube was centrifuged and vortexed in the dark (e.g., in an air bath constant temperature shaker). Blocking buffer was added and vortexed (e.g., in an air bath constant temperature shaker). The blocked acrid ester-labeled cTnI-specific mouse monoclonal antibody was purified, collected, and then diluted in buffer.

[0065] In some specific embodiments, the preparation method of the acridinium ester-labeled third cTnI-specific mouse monoclonal antibody includes the following steps: (1) Replacement: The specific steps are the same as those in the preparation method of the first cTnI-specific mouse monoclonal antibody labeled with biotin mentioned above.

[0066] (2) Labeling: Place the third cTnI-specific mouse monoclonal antibody into a centrifuge tube, add carbonate buffer, mix thoroughly, and then add 0.75~15µL of 2mg / mL acridine ester DMF solution (so that the molar ratio of the third cTnI-specific mouse monoclonal antibody to acridine ester is 1:1~1:10). Centrifuge at room temperature for 0.5~1min. Seal the centrifuge tube with sealing film and place it in a light-proof dark box. Then place the dark box in an air bath constant temperature shaker (23~25℃) and mix for 3~4h. Add 0.5~1mL of 20%~30% lysine blocking solution, place in an air bath constant temperature shaker (23~25℃), mix at medium speed, and block for 1~2h.

[0067] (3) Purification: The blocked acridinium ester-labeled third cTnI-specific mouse monoclonal antibody was purified on a Superdex 200 column, eluted with PB buffer, and collected stepwise. The collected concentrated solution of acridinium ester-labeled third cTnI-specific mouse monoclonal antibody was stored at 2℃~8℃.

[0068] When using (e.g., when preparing reagent R2), dilute the purified acrid ester-labeled third cTnI-specific mouse monoclonal antibody concentrate with a buffer solution of 50 mM BISTRIS, 10 mmol / L NaCl, 1% (w / v) bovine serum albumin, 0.05% (w / v) Tween, 0.05% (w / v) Proclin 300, and 0.05% (w / v) BND-10 at pH 6.0 to a final concentration of 0.1 µg / mL, and store at 2°C to 8°C.

[0069] The present invention will be further described below through specific embodiments.

[0070] In the following examples, unless otherwise specified, all materials and equipment used are commercially available products. Specifically, cTnI-specific mouse monoclonal antibody 1, which specifically binds to amino acid fragments 41-49 of cTnI, was purchased from HaiTai Biotechnology (Shanghai) Co., Ltd.

[0071] cTnI-specific mouse monoclonal antibody 2, specifically binds to the linker between cTnI and cTnC in the cTnI-cTnC dimer complex; purchased from HaiTai Biotechnology (Shanghai) Co., Ltd.

[0072] cTnI-specific mouse monoclonal antibody 3, specifically binds to amino acid fragments 24-40 of cTnI; purchased from HaiTai Biotechnology (Shanghai) Co., Ltd.

[0073] Example 1 This embodiment provides a cTnI detection kit, including reagents R1, R2 and R3, as well as cTnI calibrators, cTnI quality control samples and chemiluminescent excitation solution.

[0074] The preparation method of reagent R1 includes the following steps: The solid-phase premixed reagent and biotin premixed reagent were measured in a 1:1 volume ratio. The solid-phase premixed reagent was placed on a magnetic separator for magnetic separation. After visually inspecting the supernatant for clarity and transparency, the supernatant was aspirated and discarded to obtain the separated magnetic beads. The biotin premixed reagent was added to the separated magnetic beads at once, and after inverting and mixing, it was placed in a 360-degree rotating mixer for premixing to obtain reagent R1.

[0075] The solid-phase premixed reagent includes the following components in the following amounts: 25 mmol / L TRIS buffer, 150 mmol / L NaCl, 0.072% (w / w) streptavidin magnetic particles; The magnetic particles have a diameter of 3µm.

[0076] Biotin premixed reagents contain the following components in varying amounts: The diet consisted of 50 mmol / L BISTRIS buffer, 10 mmol / L NaCl, 1% (w / v) bovine serum albumin, 0.05% (w / v) Tween-20, 0.05% (w / v) ProClin300, 0.05% (w / v) BND-10, 1 L of purified water, and biotin-labeled cTnI-specific mouse monoclonal antibody 1 and biotin-labeled cTnI-specific mouse monoclonal antibody 2. The molar ratio of cTnI-specific mouse monoclonal antibody 1 to biotin was 1:10, and the concentration of cTnI-specific mouse monoclonal antibody 1 was 0.5 μg / mL. The molar ratio of cTnI-specific mouse monoclonal antibody 2 to biotin was 1:10, and the concentration of cTnI-specific mouse monoclonal antibody 2 was 1.0 μg / mL.

[0077] The preparation method of streptavidin magnetic particles used in solid-phase premixed reagents includes the following steps: Take 1 mL of a 100 mg / mL streptavidin magnetic particle solution (available commercially), add 10 mL of TBST solution and mix thoroughly (mixing time is 15 min). Place the solution on a magnetic separator until the supernatant is clear. Discard the supernatant and keep the magnetic particles. Repeat the washing process 3 times to obtain the streptavidin magnetic particles.

[0078] The preparation method of biotin-labeled cTnI-specific mouse monoclonal antibody 1 includes the following steps: (1) Replacement: After equilibrating the extracted cTnI-specific mouse monoclonal antibody 1 to room temperature, mix thoroughly. Add the mixed antibody to an ultrafiltration centrifuge tube, centrifuge at 1500 rpm for 10 min, and discard the liquid in the lower tube. Add replacement buffer to the upper tube of the ultrafiltration centrifuge tube, dilute the antibody in the upper tube to 2 mg / mL, centrifuge at 1500 rpm for 10 min, discard the liquid in the lower tube, add replacement buffer to the upper tube again, dilute the antibody in the upper tube to 2 mg / mL, centrifuge at 1500 rpm for 10 min, discard the liquid in the lower tube, add replacement buffer to the upper tube again, dilute the antibody in the upper tube to 2 mg / mL, centrifuge at 1500 rpm for 10 min, discard the liquid in the lower tube, and collect the antibody in the upper tube.

[0079] (2) Labeling: Place cTnI-specific mouse monoclonal antibody 1 into a centrifuge tube, add carbonate buffer, mix thoroughly, and then add 18.92 µL of 2 mg / mL biotin DMF solution (so that the molar ratio of cTnI-specific mouse monoclonal antibody 1 to biotin is 1:10). Centrifuge at room temperature for 1 min. Seal the centrifuge tube with sealing film and place it in a light-proof dark box. Then place the dark box in an air bath constant temperature shaker (23~25℃) and mix for 4 h. Add 1 mL of 25% (w / w) lysine blocking solution, place in an air bath constant temperature shaker (23~25℃), mix at medium speed, and block for 2 h.

[0080] (3) Purification: The product obtained in step (2) was purified on a Superdex 200 column, eluted with PB buffer, and collected stepwise to obtain biotin-labeled cTnI-specific mouse monoclonal antibody 1.

[0081] Following the method described above, prepare biotin-labeled cTnI-specific mouse monoclonal antibody 2.

[0082] Reagent R2 comprises the following components in the following amounts: The mixture consisted of 50 mmol / L BISTRIS buffer, 10 mmol / L NaCl, 1% (w / v) bovine serum albumin, 0.05% (w / v) Tween-20, 0.05% (w / v) ProClin300, 0.05% (w / v) BND-10, 1 L of purified water, and acridine ester-labeled cTnI-specific mouse monoclonal antibody 3; wherein the molar ratio of cTnI-specific mouse monoclonal antibody 3 to acridine ester was 1:5, and the concentration of cTnI-specific mouse monoclonal antibody 3 was 0.8 μg / mL.

[0083] The preparation method of acridine ester-labeled cTnI-specific mouse monoclonal antibody 3 includes the following steps: (1) Replacement: Same as the replacement step in the preparation of biotin-labeled cTnI-specific mouse monoclonal antibody 1 in Example 1.

[0084] (2) Labeling: Place cTnI-specific mouse monoclonal antibody 3 into a centrifuge tube, add carbonate buffer, mix thoroughly, and then add 11.36 µL of 2 mg / mL acridine ester DMF solution (so that the molar ratio of cTnI-specific mouse monoclonal antibody 3 to acridine ester is 1:5). Centrifuge at room temperature for 1 min. Seal the centrifuge tube with sealing film and place it in a light-proof dark box. Then place the dark box in an air bath constant temperature shaker (23~25℃) and mix for 4 h. Add 1 mL of 25% lysine blocking solution, place in an air bath constant temperature shaker (23~25℃), mix at medium speed, and block for 2 h.

[0085] (3) Purification: The product obtained in step (2) was purified on a Superdex 200 column, eluted with PB buffer, and collected stepwise to obtain acridinium ester-labeled cTnI-specific mouse monoclonal antibody 3.

[0086] Reagent R3 contains the following components in varying amounts: 50 mmol / L BISTRIS buffer, 10 mmol / L NaCl, 1% (w / w) bovine serum albumin, 0.05% (w / w) Tween-20 and 1 L of purified water.

[0087] cTnI calibrators are cTnI antigen solutions with concentrations of 0 ng / L, 35 ng / L, and 15000 ng / L.

[0088] cTnI quality control products are cTnI antigen solutions with concentrations of 25 ng / L and 20000 ng / L, respectively.

[0089] Chemiluminescence excitation solutions include acidic and alkaline excitation solutions. The acidic excitation solution is a mixed solution of nitric acid and hydrogen peroxide (with a HNO3 concentration of 0.008 mol / L and a H2O2 mass percentage of 1%), and the alkaline excitation solution is a sodium hydroxide solution (with a NaOH concentration of 0.3 mol / L).

[0090] test: (1) The method of using the cTnI detection kit in Example 1 above includes the following steps: The detection instrument was a fully automated chemiluminescence immunoassay analyzer (CM320). The reagent kit's detection principle is a polyclonal sandwich method. Specifically, 75 µL of the sample to be tested is added sequentially to the instrument, followed by 40 µL of reagent R1 and 50 µL of reagent R2. After reacting for 5 min, 50 µL of reagent R3 is added, and after another 5 min of reaction, magnetic separation is performed. The instrument then transfers the reactants to a dark chamber, where acidic and alkaline excitation solutions are added sequentially for further reaction. Finally, the relative light units are recorded.

[0091] (2) Based on the usage method of the cTnI detection kit in each of the above Examples 1, the performance indicators of the cTnI detection kit in Example 1 are evaluated.

[0092] Linearity evaluation: High-value samples close to the upper limit of the linear range were diluted to at least five concentrations according to a certain ratio, while low-value concentration samples must be close to the lower limit of the linear range. Each concentration of sample was tested three times using the cTnI detection kit in Example 1, and the average value was calculated. The average value of the measured concentration was then fitted to the theoretical concentration using the least squares method to calculate the linear correlation coefficient r. The results are shown in Table 1, where the linear correlation coefficient r is 1.0000.

[0093] Table 1. Evaluation results of linearity

[0094] Blank limit evaluation: The photon number of the zero-value calibrator was measured 20 times, and the average value was added to twice the standard deviation. The result obtained by substituting the average value into the standard curve is the blank limit. The results are shown in Table 2. The blank limit of the cTnI detection kit in Example 1 is 0.31 ng / L.

[0095] Table 2. Evaluation results of blank limits

[0096] Repeatability evaluation: Using the same batch of myocardial cTnI detection kits from Example 1, three samples with repeatability low (25 ng / L), repeatability medium (15000 ng / L), and repeatability high (20000 ng / L) values ​​were each tested 10 times. The mean M and standard deviation SD of the 10 concentration measurements were calculated. The results are shown in Table 3. The CV (coefficient of variation, i.e., the ratio of standard deviation to mean) of the cTnI detection kits in Example 1 for the low concentration samples was 3.30%, the CV for the medium concentration samples was 2.04%, and the CV for the high concentration samples was 1.41%.

[0097] Table 3. Results of Repeatability Evaluation

[0098] Evaluation of Biotin Interference: Samples with different concentrations of biotin interference were tested using the cTnI detection kit in Example 1. The biotin concentrations were 70 ng / mL, 100 ng / mL, 1000 ng / mL, and 3500 ng / mL. The results are shown in Table 4 (wherein, low-value samples: biotin was added at a low cTnI concentration (25 ng / L) as the test sample; low-value control: the same volume of buffer solution was added at a low cTnI concentration as the control test sample; high-value samples: the interferon was added at a high cTnI concentration (10000 ng / L) as the test sample; high-value control: the same volume of buffer solution was added at a high cTnI concentration as the control test sample)). The cTnI detection kit provided in Example 1 showed good anti-interference effect, and biotin interference at various concentrations did not significantly interfere with either low-value or high-value cTnI samples.

[0099] Table 4. Results of Biotin Interference

[0100] HOOK evaluation: Ultra-high concentration samples (concentration range 1.5–2 times the hook point concentration) were taken and diluted proportionally with the calibrator matrix (i.e., buffer solution) to obtain 7 samples (H1–H7). The highest concentration at the HOOK point was 3138022 ng / L. These 7 concentrations were then used for validation, and the results are shown in Table 5. Figure 1 As shown, no decanting occurred at a concentration of 2,000,000 ng / L, and a slight decrease began at 3,138,022 ng / L. Therefore, the decanting point of the cTnI detection kit in Example 1 is 2,000,000 ng / L.

[0101] Table 5. HOOK Validation Results

[0102] Drug interference evaluation: Common interfering substances, including endogenous and drug-induced interfering substances, were added to cTnI samples at different concentrations. The results are shown in Table 6 (where L sample: test sample with interfering substance added at low cTnI concentration; L-pair: test sample with the same volume of buffer added at low cTnI concentration as a control; H sample: test sample with interfering substance added at high cTnI concentration; H-pair: test sample with the same volume of buffer added at high cTnI concentration as a control). The interference rate was within ±10%.

[0103] Table 6. Results of Endogenous and Drug Interference

[0104] Specificity evaluation: 1000 ng / mL of cardiac troponin C (cTnC), cardiac troponin T (cTnT), and skeletal troponin (skcTnI) were added to a sample containing a certain amount of the analyte cTnI. The cTnI detection kits from Example 1 and Comparative Examples 1-4 were used for testing, and the cross-reactivity rate of each group of test results was calculated. The results are shown in Table 7. The cross-reactivity rates of cardiac troponin C (cTnC), cardiac troponin T (cTnT), and skeletal troponin (skcTnI) calculated according to Equation (1) were all less than 1.0%.

[0105] R 交叉 =(XT) 对照 ) / C 干扰 ×100% (1) In equation (1): R 交叉 — Cross-reactivity rate; X — Mean value of measurements after addition; T 对照 —Mean measurement of the control sample; C 干扰 —The concentration of added interfering substances.

[0106] The results are shown in Table 7, where H 对 H 样 L 对 and L 样 See above for the meaning of .

[0107] Table 7. Specificity Validation Results

[0108] Long-term stability evaluation: The cTnI detection kit in Example 1 was stored at 2℃~8℃, and the appearance, blank limit, linearity, and repeatability were evaluated at 3 months, 6 months, 9 months, 12 months, 18 months, and 19 months. The results are shown in Table 8. The blank limit, linearity, and repeatability of the reagents at different shelf lives all met the detection requirements, indicating that the reagents in the cTnI detection kit have good shelf-life stability and are easy to store for a long time.

[0109] Table 8. Long-term stability evaluation

[0110] Thermal stability evaluation: The cTnI detection kit from Example 1 within its expiration date was placed at 37°C for 3, 7, and 10 days, and then placed in a CM-320 fully automated chemiluminescence immunoassay analyzer to test the appearance blank limit, linearity, and repeatability. The results are shown in Table 9. After high-temperature storage, the blank limit, linearity, and repeatability of the kit all met the detection requirements, indicating that the reagents in the cTnI detection kit have good thermal stability and are convenient for transportation.

[0111] Table 9. Thermal stability evaluation

[0112] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A cTnI detection kit, characterized in that, The cTnI detection kit includes reagent R1 and reagent R2; The reagent R1 comprises a complex comprising streptavidin magnetic particles and two biotin-labeled cTnI-specific mouse monoclonal antibodies linked to the streptavidin magnetic particles. The reagent R2 comprises an acridinium ester-labeled cTnI-specific mouse monoclonal antibody; Two biotin-labeled cTnI-specific mouse monoclonal antibodies specifically bind to amino acid fragments 41-49 of cTnI and the linker between cTnI and cTnC in the cTnI-cTnC dimer complex, respectively. The acridine ester-labeled cTnI-specific mouse monoclonal antibody specifically binds to amino acid fragments 24-40 of cTnI.

2. The cTnI detection kit according to claim 1, characterized in that, The reagent R1 is prepared by mixing a solid-phase premixed reagent and a biotin premixed reagent; The solid-phase premixed reagent comprises the following components in varying amounts: A first buffer solution of 10–30 mmol / L, a first salt solution of 10–300 mmol / L, and streptavidin magnetic particles with a mass content of 0.07%–0.2%; The biotin premixed reagent comprises the following components in the following amounts: The mixture contained a second buffer solution of 20–400 mmol / L, a second salt of 10–300 mmol / L, a first protective agent of 0.02%–2% by mass, a first preservative of 0.02%–2% by mass, a first surfactant of 0.02%–2% by mass, water, and a biotin-labeled first cTnI-specific mouse monoclonal antibody and a biotin-labeled second cTnI-specific mouse monoclonal antibody; the concentration of the first cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL, and the concentration of the second cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL. In the first biotin-labeled cTnI-specific mouse monoclonal antibody, the molar ratio of the first cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:

20. In the biotin-labeled second cTnI-specific mouse monoclonal antibody, the molar ratio of the second cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:

20.

3. The cTnI detection kit according to claim 2, characterized in that, The reagent R2 comprises the following components in varying amounts: The mixture contained a third buffer solution of 10–100 mmol / L, a third salt solution of 10–50 mmol / L, a second protective agent of 0.02%–2% by mass, a second preservative of 0.01%–0.1% by mass, a second surfactant of 0.05%–1% by mass, water, and an acridinium ester-labeled third cTnI-specific mouse monoclonal antibody; the concentration of the third cTnI-specific mouse monoclonal antibody was 0.1–0.8 μg / mL. In the acridine ester-labeled third cTnI-specific mouse monoclonal antibody, the molar ratio of the third cTnI-specific mouse monoclonal antibody to acridine ester is 1:1 to 1:

10.

4. The cTnI detection kit according to claim 3, characterized in that, The first, second, and third buffers each independently comprise at least one of HEPES buffer, PIPES buffer, MOPS buffer, disodium hydrogen phosphate-citrate buffer, phosphate buffer, BISTRIS buffer, TRIS buffer, and sodium barbital-hydrochloric acid buffer; and / or, The first salt, the second salt, and the third salt each independently include at least one of NaCl, KCl, and MgCl2; and / or, The magnetic particles have a particle size of 1~3μm.

5. The cTnI detection kit according to claim 3, characterized in that, The first and second protective agents each independently comprise at least one of casein, bovine serum albumin, and human serum albumin; and / or, The first surfactant and the second surfactant each independently include at least one of polyoxyethylene lauryl ether, Triton X-100, Tween-20 and Tween-80.

6. The cTnI detection kit according to any one of claims 1-5, characterized in that, The cTnI detection kit also includes a chemiluminescent excitation solution, which includes an acidic excitation solution and an alkaline excitation solution; The acidic activating solution includes a mixed solution of nitric acid and hydrogen peroxide, and the alkaline activating solution includes a sodium hydroxide solution.

7. The cTnI detection kit according to any one of claims 1-5, characterized in that, The cTnI detection kit further includes cTnI calibrators and / or cTnI quality control products. The cTnI calibrators include cTnI antigen solutions with concentrations of 0 ng / L, 35 ng / L, and 15000 ng / L, respectively. The cTnI quality control products include cTnI antigen solutions with concentrations of 25 ng / L and 2000 ng / L, respectively.

8. The cTnI detection kit according to any one of claims 1-5, characterized in that, The cTnI detection kit also includes reagent R3, which comprises the following components in varying amounts: 20–400 mmol / L fourth buffer, 10–150 mmol / L fourth salt, 0.05%–2% by mass of third protective agent, and 0.01%–1% by mass of third surfactant.

9. A method for preparing a cTnI detection kit according to any one of claims 1-8, characterized in that, Includes the following steps: Reagents R1 and R2 were prepared separately to obtain the cTnI detection kit.

10. The preparation method according to claim 9, characterized in that, The preparation method of the reagent R1 includes the following steps: Solid-phase premixed reagents and biotin premixed reagents are provided in a volume ratio of 1:1; The solid-phase premixed reagent is placed on a magnetic separator for magnetic separation to obtain separated magnetic beads; Add the biotin premixed reagent to the separated magnetic beads, mix by inverting, and then premix in a mixer to obtain the reagent R1. The solid-phase premixed reagent comprises the following components in varying amounts: A first buffer solution of 10–30 mmol / L, a first salt solution of 10–300 mmol / L, and streptavidin magnetic particles with a mass content of 0.07%–0.2%; The biotin premixed reagent comprises the following components in the following amounts: The mixture contained a second buffer solution of 20–400 mmol / L, a second salt of 10–300 mmol / L, a first protective agent of 0.02%–2% by mass, a first preservative of 0.02%–2% by mass, a first surfactant of 0.02%–2% by mass, water, and a biotin-labeled first cTnI-specific mouse monoclonal antibody and a biotin-labeled second cTnI-specific mouse monoclonal antibody; the concentration of the first cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL, and the concentration of the second cTnI-specific mouse monoclonal antibody was 0.5–2.0 μg / mL. In the first biotin-labeled cTnI-specific mouse monoclonal antibody, the molar ratio of the first cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:

20. In the biotin-labeled second cTnI-specific mouse monoclonal antibody, the molar ratio of the second cTnI-specific mouse monoclonal antibody to biotin is 1:5 to 1:20.

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