Composition, kit, and method for detecting large-size ctnitc, and use thereof

Abstract

The present application relates to a composition, kit, and method for detecting a large-size cTnITC, and a use thereof. Specifically, the present application relates to a composition and kit containing an antibody specifically binding to any segment of an amino acid sequence of cTnT from position 67 to position 222 and an antibody specifically binding to any segment of an amino acid sequence of TnC, a detection method, and a use in vitro diagnosis of myocardial injury.

Description

Compositions, kits, methods, and applications for detecting large-size cTnITC Technical Field

[0001] The present application relates to the field of immunoassays, and in particular to compositions and kits for detecting large-size cTnITC (large-size cardiac troponin ternary complex), detection methods, and their applications in in vitro diagnosis of myocardial injury. Background Art

[0002] Cardiac troponin (cTn) is a specific marker of myocardial injury and can be used to detect myocardial damage after or during myocardial infarction. Troponin consists of three subunits: cardiac troponin I (cTnI), cardiac troponin T (cTnT), and troponin C (TnC). Serum concentrations of cTnI and cTnT are highly correlated with the severity of myocardial injury and are highly specific and sensitive markers of myocardial infarction, recommended by the American and European Societies of Cardiology. However, elevated blood levels of cTn can be caused by a variety of factors, including pulmonary embolism, acute or chronic heart failure, cardiac trauma, endocarditis, and myocarditis, in addition to myocardial infarction. This reduces the clinical specificity of cTn for myocardial infarction, and diagnosis of myocardial infarction often requires additional testing.

[0003] Cardiac troponin typically binds to actin filaments in the form of a ternary complex, cTnITC. Upon myocardial injury, troponin is degraded from the myofilaments and released into the blood. Within cells or in the blood circulation, full-length cTnITC is degraded by proteases, gradually forming a low-molecular-weight ternary complex (LMW-cTnITC), a binary complex (cTnIC), and free cTnT. Studies have shown that the form of troponin in the blood is associated with an individual's physiological and pathological status. Detecting different forms of cTnITC complexes helps physicians gain a more comprehensive understanding of a patient's physiological and pathological status, facilitates rapid and accurate diagnosis, and guides prognosis.

[0004] Currently, traditional immunological methods for cardiac troponin detection primarily include enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography (GICA), electrochemiluminescence (ECL), and chemiluminescence immunoassay (CLIA). Chemiluminescence immunoassay technology offers the broadest clinical application prospects due to its high sensitivity, strong specificity, wide linear range, robust operability, and high degree of automation. Existing high-sensitivity cardiac troponin assays based on chemiluminescence immunoassays are typically used to detect cTnI and cTnT, but are unable to distinguish troponin complexes from free troponin. In existing literature, a double-antibody sandwich immunoassay for the cTnITC complex utilizes a capture antibody that recognizes the binary TnIC complex epitope and a detection antibody that recognizes the TnT antigen. However, this assay suffers from a low signal-to-noise ratio and weak specificity, making it difficult to achieve rapid, sensitive, and specific detection of the cTnITC complex.

[0005] Summary of the Invention

[0006] The task of this application is to provide a technical solution that can detect specific forms of cTnITC in samples with high sensitivity and rapidity. Specific detection is achieved through the combination of capture antibodies and detection antibodies. In particular, the addition of antibodies that specifically bind to TnC to the capture antibodies or detection antibodies is very beneficial for improving the signal-to-noise ratio of the detection system and enhancing the detection sensitivity.

[0007] In a first aspect, the present application provides a composition for detecting large-sized cTnITC in a sample, comprising a first group of antibodies and a second group of antibodies, wherein:

[0008] The first group of antibodies includes one or more antibodies 1, each of which is independently selected from antibodies that specifically bind to any section of the amino acid sequence of cTnT at positions 67-222;

[0009] The second group of antibodies includes one or more antibodies 2, and each antibody 2 is independently selected from an antibody that specifically binds to any segment of the TnC amino acid sequence.

[0010] In a second aspect, the present application provides a kit for detecting large-sized cTnITC in a sample, comprising a capture antibody and a detection antibody, wherein the capture antibody is selected from one of a first group of antibodies and a second group of antibodies, and the detection antibody is selected from the other of the first group of antibodies and the second group of antibodies; wherein,

[0011] The first group of antibodies includes one or more antibodies 1, each of which is independently selected from antibodies that specifically bind to any section of the amino acid sequence of cTnT at positions 67-222;

[0012] The second group of antibodies includes one or more antibodies 2, and each antibody 2 is independently selected from an antibody that specifically binds to any segment of the TnC amino acid sequence.

[0013] In a third aspect, the present application provides a method for detecting large-sized cTnITC in a sample in vitro, comprising the following steps:

[0014] Obtain test samples;

[0015] contacting the sample to be tested with a capture antibody to form an antibody-antigen complex;

[0016] contacting the antibody-antigen complex with a detection antibody conjugated to a detectable label to form an antibody-antigen-antibody complex; and

[0017] detecting the signal generated by the detectable label to determine the presence and / or amount of large-sized cTnITC in the test sample;

[0018] The capture antibody is selected from one of the first and second groups of antibodies, and the detection antibody is selected from the other of the first and second groups of antibodies:

[0019] The first group of antibodies includes one or more antibodies 1, each of which is independently selected from antibodies that specifically bind to any section of the amino acid sequence of cTnT at positions 67-222;

[0020] The second group of antibodies includes one or more antibodies 2, and each antibody 2 is independently selected from an antibody that specifically binds to any segment of the TnC amino acid sequence.

[0021] In a fourth aspect, the present application provides use of the composition according to any one of the first aspects in preparing a reagent for detecting large-sized cTnITC.

[0022] In a fifth aspect, the present application provides use of the composition described in any one of the first aspect, the kit described in any one of the second aspect, or the method described in any one of the third aspect in in vitro diagnosis of myocardial injury.

[0023] In a sixth aspect, the present application provides a method for diagnosing myocardial injury in vitro according to the first aspect, comprising the step of detecting large-sized cTnITC in a sample from a subject using the composition described in any one of the first aspect, the kit described in any one of the second aspect, or the method described in any one of the third aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0025] Figure 1 shows the signal-to-noise ratio analysis of the large-size cTnITC detection kit.

[0026] Figure 2 shows the specificity of the large-scale cTnITC detection kit validated using serum samples.

[0027] FIG3 shows the linearity analysis of the large-size cTnITC detection kit 3.

[0028] FIG4 shows the linearity analysis of the large-scale cTnITC detection kit 4.

[0029] FIG5 is a signal-to-noise ratio analysis of the large-size cTnITC detection kit 3 and the detection kit 3a.

[0030] FIG6 is an analysis of the signal-to-noise ratio of the large-size cTnITC detection kit 3 and the detection kit 3a for clinical serum samples.

[0031] FIG7 shows the specificity of the large-size cTnITC detection kit 3a validated using serum samples.

[0032] FIG8 is an analysis of the changing trends of large-size cTnITC detected by large-size cTnITC detection kit 3 and high-sensitivity cTnI and cTnT detected by existing kits.

[0033] FIG9 is an analysis of the concentration change rates of large-size cTnITC detected by the large-size cTnITC detection kit 3 and high-sensitivity cTnI and cTnT detected by the existing kits. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all. The following description of the embodiments is merely illustrative and in no way limits the present invention. All other embodiments obtained by ordinary technicians in this field based on the embodiments without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the immunology laboratory procedures used herein are conventional procedures widely used in the relevant fields. To better understand the embodiments of the present invention, definitions and explanations of relevant terms are provided below.

[0036] As used herein, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a method or apparatus comprising a series of elements includes not only the elements explicitly recited, but also other elements not explicitly listed, or elements inherent to the practice of the method or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other related elements in the method or apparatus comprising the element.

[0037] As used herein, the term "at least one" means 1 or more than 1 under reasonable conditions, such as 2, 3, 4, 5 or 10, etc.

[0038] As used herein, the terms "first" and "second" merely distinguish similar objects and do not represent a specific ordering of the objects. It is understood that "first" and "second" can be interchanged to indicate a specific order or precedence where appropriate. It should be understood that the objects distinguished by "first" and "second" can be interchanged where appropriate so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein.

[0039] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as a reaction between an antibody and the antigen to which it is directed. The binding affinity between two molecules can be described by a KD value. The KD value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as a molar concentration (M). The smaller the KD value, the tighter the binding of the two molecules and the higher the affinity. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) refers to an antibody that binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10The antibody binds to the antigen with a KD of M or less. Corresponding methods for analyzing antibody specificity are described, for example, in Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press and Harlow & Lane (1999) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Non-limiting examples of suitable studies are, for example, binding studies, blocking and competition studies using structurally and / or functionally closely related molecules. These studies can be performed using methods such as fluorescence activated cell sorting (FACS) analysis, flow cytometry titration (FACS titration) analysis, surface plasmon resonance (SPR, for example using), isothermal titration calorimetry (ITC), fluorescence titration or radiolabeled ligand binding assays. Further methods include, for example, immunoblotting (Western Blot), ELISA (including competitive ELISA) tests, RIA tests, ECL tests and IRMA tests.

[0040] As used herein, the terms "troponin" and "Tn" refer to proteins on myosin within myocytes that regulate the calcium-mediated interaction between actin and myosin. Found in cardiac and skeletal muscle, they are composed of three subunits: troponin T (TnT), troponin I (TnI), and troponin C (TnC). TnT is the tropomyosin-binding subunit, interacting with actin and tropomyosin; TnI is the inhibitory subunit, inhibiting the ATPase activity of actomyosin; and TnC is the only subunit that binds calcium, enabling skeletal or cardiac muscle contraction. The terms "cardiac troponin" and "cTn" refer to all troponin isoforms expressed in cardiac cells, preferably subendocardial cells. These isoforms have been well characterized in the art, for example as described in Anderson 1995, Circulation Research, vol. 76, no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493. The term "cardiac troponin" also includes variants of a particular cardiac troponin that have at least the same basic biological and immunological properties as that particular cardiac troponin. In particular, if they are detected by the same specificity as mentioned herein, they share the same basic biological and immunological properties. It will be understood that the isoforms of troponin can be assayed together (simultaneously or sequentially) or separately (i.e., without assaying the other isoforms at all).

[0041] As used herein, the terms "cardiac troponin T" and "cTnT" refer to the cardiac troponin T subunit, the amino acid sequence of which is disclosed in the UniProt database under accession number P45379.

[0042] As used herein, the terms "cardiac troponin I" and "cTnI" refer to the cardiac troponin I subunit, the amino acid sequence of which is disclosed in the UniProt database under accession number P19429.

[0043] As used herein, the terms "troponin C" and "TnC" refer to the troponin C subunit, the amino acid sequence of which is disclosed in the UniProt database under accession number P63316.

[0044] As used herein, the terms "large-size cardiac troponin ternary complex" or "large-size cTnITC" are used interchangeably herein and are intended to include a complex formed by any of the full-length protein or fragments of TnC, the full-length protein or fragments of cTnI, one or more stretches of amino acid residues 223-287 of cTnT, and one or more stretches of amino acid residues 1-222 of cTnT.

[0045] As used herein, the term "antibody" has the meaning commonly understood in the art and refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into regions of high variability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site, respectively. The allocation of amino acids within each region or domain can be based on the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0046] This application is applicable to different detection systems, including but not limited to fluorescent labeling reaction systems, enzyme-linked immunosorbent assay systems, bioluminescence systems, etc.

[0047] Detection reagents and methods

[0048] One object of the present application is to provide a detection reagent that can sensitively and specifically detect large-sized cTnITC (including full-length cTnITC and moderate hydrolysis products, wherein the hydrolysis products still include cTnI, cTnT and TnC subunits or fragments thereof, and TnT fragments at positions 67-222), without recognizing further hydrolyzed ternary complexes, binary complexes or free subunits and fragments thereof.

[0049] Specifically, in a first aspect, the present application provides a composition for detecting large-sized cTnITC in a sample, comprising a first group of antibodies and a second group of antibodies, wherein:

[0050] The first group of antibodies includes one or more antibodies 1, each of which is independently selected from antibodies that specifically bind to any section of the amino acid sequence of cTnT at positions 67-222;

[0051] The second group of antibodies includes one or more antibodies 2, and each antibody 2 is independently selected from an antibody that specifically binds to any segment of the TnC amino acid sequence.

[0052] Based on the first aspect, the second aspect of the present application provides a kit for detecting large-sized cTnITC in a sample, comprising a capture antibody and a detection antibody, wherein the capture antibody is selected from one of the first group of antibodies and the second group of antibodies, and the detection antibody is selected from the other of the first group of antibodies and the second group of antibodies; wherein,

[0053] The first group of antibodies includes one or more antibodies 1, each of which is independently selected from antibodies that specifically bind to any section of the amino acid sequence of cTnT at positions 67-222;

[0054] The second group of antibodies includes one or more antibodies 2, and each antibody 2 is independently selected from an antibody that specifically binds to any segment of the TnC amino acid sequence.

[0055] That is, the capture antibody includes at least antibody 1, and the detection antibody includes at least antibody 2; or, the capture antibody includes at least antibody 2, and the detection antibody includes at least antibody 1.

[0056] In the examples of the present application, the first group of antibodies does not include antibodies that specifically bind to any section of the amino acid sequence of cTnT from position 223 to position 287.

[0057] In some embodiments, the capture antibody can be coated on the surface of a solid phase carrier, such as magnetic beads, latex particles, ELISA plates, plastic beads, plastic tubes, immunochromatographic test strips, and test cards, to capture the antigen in the sample. Then, a detection antibody with a detectable label is used to bind to the capture antibody coated on the solid phase carrier surface, and the amount of the detectable label on the bound detection antibody is measured to determine the antigen content in the sample.

[0058] The detectable label can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Particularly preferably, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), chemiluminescent substances (e.g., acridinium ester compounds), and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. The labels encompassed in the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate. Chemiluminescent substances (such as acridinium ester compounds) are generally detected by providing an excitation solution and / or a catalyst to the luminescent substance to detect the emitted light. Biotin is generally detected by providing avidin (e.g., streptavidin) modified with the above-mentioned label to biotin and detecting the label carried by the avidin connected to biotin. In certain embodiments, the detectable label as described above can be connected to the antibody or antigen-binding fragment thereof of the present invention via linkers of varying lengths to reduce potential steric hindrance. In some embodiments, the detectable label is selected from fluorescein, chemiluminescent substances (e.g., acridinium ester compounds), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioactive isotopes, biotin, colloidal gold, and magnetic particles.

[0059] In some embodiments, the kit may further comprise a reagent for detecting the corresponding detectable label (a substrate for the detectable label). For example, when the detectable label is an enzyme, the kit may further comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS, or a luminol compound for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescent reagent (e.g., an acridinium ester compound), the kit may further comprise a pre-excitation solution and / or an excitation solution for chemiluminescence.

[0060] The kit of the present application is particularly suitable for immunoassay, such as ELISA, especially double antibody sandwich chemiluminescent immunoassay method.

[0061] In the present application, the amount of large-size cTnITC can be directly or indirectly detected. The amount or concentration of the detection antibody can be directly detected based on the detectable label signal of the detection antibody, and the signal is directly related to the target antigen in the sample. Such signals are sometimes also referred to as intensity signals herein - for example, they can be obtained by measuring the intensity value of the specific physical or chemical properties of the detection antibody. Indirect measurement includes measuring signals from secondary components (i.e., not the detection antibody itself) or biological readout systems, such as measurable cellular responses, ligands, markers, or enzymatic reaction products.

[0062] In a third aspect, the present application provides a method for detecting large-sized cTnITC in a sample in vitro, comprising the following steps:

[0063] Obtaining a sample to be tested;

[0064] contacting the sample to be tested with a capture antibody to form an antibody-antigen complex;

[0065] contacting the antibody-antigen complex with a detection antibody conjugated to a detectable label to form an antibody-antigen-antibody complex; and

[0066] detecting the signal generated by the detectable label to determine the presence and / or amount of large-sized cTnITC in the test sample;

[0067] The capture antibody is selected from one of the first and second groups of antibodies, and the detection antibody is selected from the other of the first and second groups of antibodies:

[0068] The first group of antibodies includes one or more antibodies 1, each of which is independently selected from antibodies that specifically bind to any section of the amino acid sequence of cTnT at positions 67-222;

[0069] The second group of antibodies includes one or more antibodies 2, and each antibody 2 is independently selected from an antibody that specifically binds to any segment of the TnC amino acid sequence.

[0070] In the first aspect to the third aspect of the present application, as some embodiments, the second group of antibodies further includes:

[0071] one or more antibodies 3, each of said antibodies 3 being independently selected from an antibody that specifically binds to cTnIC; and / or,

[0072] One or more antibodies 4, each of the antibodies 4 is independently selected from an antibody that specifically binds to any section of the amino acid sequence of cTnI from positions 18 to 210.

[0073] Here, by combining the antibody 2 with an antibody that specifically recognizes the cTnIC binary complex or an antibody that specifically recognizes cTnI, the signal-to-noise ratio of the detection reagent can be significantly improved.

[0074] In the first aspect to the third aspect of the present application, as some embodiments, in some embodiments, the first group of antibodies includes one or more of the antibody 1, and the second group of antibodies includes one or more of the antibody 2 and one or more of the antibody 3.

[0075] In the first to third aspects of the present application, as some embodiments, the first group of antibodies includes at least two antibodies 1. In some embodiments, the second group of antibodies includes at least two antibodies 2. In some embodiments, the first group of antibodies includes at least two antibodies 1; the second group of antibodies includes at least two antibodies 2 and optionally at least two antibodies 3. The combination of multiple antibodies that bind to the same target protein is beneficial for the detection of large-sized cTnITCs.

[0076] The present application screens for binding epitopes of antibodies. In some embodiments, each of the antibodies 1 is independently selected from an antibody that specifically binds to amino acids 67-86, amino acids 119-138, amino acids 132-151, amino acids 145-164, or amino acids 171-190 of cTnT. In some embodiments, each of the antibodies 1 is independently selected from an antibody that specifically binds to amino acids 119-138, amino acids 132-151, or amino acids 171-190 of cTnT. In some embodiments, each of the antibodies 1 is independently selected from an antibody that specifically binds to amino acids 119-138 or amino acids 132-151 of cTnT.

[0077] With respect to the binding epitope of antibody 4, in some embodiments, each of the antibodies 4 is independently selected from an antibody that specifically binds to amino acids 1-15, amino acids 13-22, amino acids 18-22, amino acids 18-28, amino acids 18-35, amino acids 22-31, amino acids 22-40, amino acids 23-29, amino acids 24-40, amino acids 25-40, amino acids 26-35, amino acids 34-37, amino acids 41-49, amino acids 83-89, amino acids 86-90, amino acids 87-90, amino acids 117-126, amino acids 130-145, amino acids 169-178, amino acids 186-192, amino acids 190-196, or amino acids 195-209 of cTnI. In some embodiments, each antibody 4 is independently selected from an antibody that specifically binds to amino acids 22-40, 41-49, or 83-89 of cTnI. In some embodiments, each antibody 4 is independently selected from an antibody that specifically binds to amino acids 41-49 of cTnI.

[0078] In some embodiments, the capture antibody is the first group of antibodies, and the detection antibody is the second group of antibodies. Studies have found that when the first group of antibodies are used as capture antibodies and the second group of antibodies are used as detection antibodies, the detection of large-sized cTnITCs has a higher signal-to-noise ratio.

[0079] In the present application, commercially available antibodies targeting specific epitopes can be selected, or antibodies with specific binding activity against specific epitopes of large-sized cTnITCs can be obtained using general techniques in the art. An exemplary approach involves obtaining a monoclonal antibody with specific binding activity through hybridoma technology, specifically including the selection and preparation of two parent cells, cell fusion, selective culture and cloning of hybridoma cells, preparation of monoclonal antibodies, specific identification, and purification. Furthermore, epitope identification and screening steps may be included, such as using ELISA or surface plasmon resonance (SPR) techniques to identify antibody binding epitopes.

[0080] application

[0081] Due to the low sensitivity of existing cTnITC detection kits, its application in the diagnosis of myocardial injury is seriously affected. In clinical practice, cTnI and cTnT are also commonly selected as highly specific and highly sensitive markers of myocardial infarction. Based on the aforementioned composition, kit and detection method, the present application achieves a highly sensitive and highly specific rapid detection of large-size cTnITC, which is conducive to a deeper understanding of the correlation between troponin and myocardial injury. Studies have shown that compared with cTnI and cTnT, large-size cTnITC is more sensitive to myocardial injury and can be used as a more sensitive myocardial injury marker to help diagnose the disease and make prognostic judgments.

[0082] In a fourth aspect, the present application provides use of the composition according to any one of the first aspects in preparing a reagent for detecting large-sized cTnITC.

[0083] In a fifth aspect, the present application provides use of the composition described in any one of the first aspect, the kit described in any one of the second aspect, or the method described in any one of the third aspect in in vitro diagnosis of myocardial injury.

[0084] In a sixth aspect, the present application provides a method for diagnosing myocardial injury in vitro, comprising the step of detecting large-sized cTnITC in a sample from a subject using the composition described in any one of the first aspect, the kit described in any one of the second aspect, or the method described in any one of the third aspect.

[0085] In some embodiments, the sample is a body fluid or tissue sample of a subject, such as whole blood, serum, plasma (including lithium heparin plasma, EDTA plasma), urine, saliva, biological tissue or cells, preferably whole blood, serum or plasma. Beneficial effects

[0086] 1) This application achieves highly sensitive and specific rapid detection of large-sized cTnITC. By selecting antibodies that specifically bind to TnC as capture or detection antibodies, the signal-to-noise ratio of the system can be significantly improved, thereby enhancing sensitivity.

[0087] 2) Because the form of troponin in the blood is associated with an individual's physiological and pathological state, the specific detection of large-size cTnITC facilitates a deeper understanding of the relationship between troponin and disease. Experimental results show that the changing trend of large-size cTnITC is consistent with that of cTnT and cTnI, which has clinical value. Furthermore, large-size cTnITC has a faster rate of concentration change and is more sensitive to myocardial injury in patients, making it a more sensitive marker of myocardial injury and aiding in disease diagnosis and prognosis.

[0088] 3) The composition or kit of the present application can be used in conjunction with a fully automatic chemiluminescence analyzer to achieve automated, rapid, and high-throughput detection of large-sized cTnITC.

[0089] 4) The kit of the present application has high sensitivity, good specificity and good repeatability.

[0090] Example

[0091] Example 1 Detection method and construction of detection kit

[0092] The capture antibody-detection antibody was applied to the double antibody sandwich chemiluminescence immunoassay method to construct a detection kit for detecting large-sized cTnITC in samples.

[0093] (1) Large size cTnITC detection kit 1

[0094] The capture antibodies were: Antibody 1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0095] The detection antibodies are: Antibody 3: Antibody 20C6cc that specifically binds to the cTnIC complex epitope.

[0096] (2) Large size cTnITC detection kit 2

[0097] The capture antibodies were: Antibody 1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0098] The detection antibodies are: Antibody 4: Antibody 19C7cc that specifically binds to amino acid fragment 41-49 of cTnI.

[0099] (3) Large size cTnITC detection kit 3

[0100] The capture antibodies were: Antibody 1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0101] The detection antibodies are: Antibody 2: 7B9cc, an antibody that specifically binds to TnC; and Antibody 3: 20C6cc, an antibody that specifically binds to an epitope of the cTnIC complex.

[0102] (4) Large size cTnITC detection kit 4

[0103] The capture antibodies were: Antibody 1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0104] The detection antibodies are: Antibody 2: antibody 7B9cc that specifically binds to TnC; and Antibody 4: antibody 19C7cc that specifically binds to amino acid fragment 41-49 of cTnI.

[0105] (5) Large size cTnITC detection kit 5

[0106] The capture antibodies were: Antibody 1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0107] The detection antibodies are: Antibody 2: Antibody 7B9cc that specifically binds to TnC.

[0108] (6) Large size cTnITC detection kit 6

[0109] The capture antibodies were: Antibody 1: Antibody 1C11cc that specifically binds to amino acid fragment 171-190 of cTnT;

[0110] The detection antibodies are: Antibody 2: 7B9cc, an antibody that specifically binds to TnC; and Antibody 3: Tcom8, an antibody that specifically binds to an epitope of the cTnIC complex.

[0111] (7) Large size cTnITC detection kit 7

[0112] The capture antibodies were: Antibody 1: Antibody 406cc that specifically binds to the amino acid fragment 132-151 of cTnT;

[0113] The detection antibodies are: Antibody 2: 7B9cc, an antibody that specifically binds to TnC; and Antibody 3: 20C6cc, an antibody that specifically binds to an epitope of the cTnIC complex.

[0114] (8) Large size cTnITC detection kit 8

[0115] The capture antibodies were: Antibody 1: 300cc of an antibody that specifically binds to amino acid fragment 119-138 of cTnT;

[0116] The detection antibodies are: Antibody 2: 7B9cc, an antibody that specifically binds to TnC; and Antibody 3: 20C6cc, an antibody that specifically binds to an epitope of the cTnIC complex.

[0117] The experiments also used kits obtained by replacing antibody 1 used in the large-size cTn ITC detection kits 3-8 with the following antibodies: 7F4 or 7G7, which specifically bind to the cTnT amino acid fragment 67-86; 2F3, 1A11, or 1F11cc, which specifically bind to the cTnT amino acid fragment 145-164. The experiments also used kits obtained by replacing antibody 4 used in the large-size cTn ITC detection kit 4 with the following antibodies: M18cc, which specifically binds to the cTnI amino acid fragment 18-28; 16A11cc, 16A12cc, or 8E10cc, which specifically bind to the cTnI amino acid fragment 86-90; M46, which specifically binds to the cTnI amino acid fragment 130-145; and MF4cc, which specifically binds to the cTnI amino acid fragment 190-196. These antibodies were purchased from Haitide Biotechnology Co., Ltd.

[0118] The large-format cTnITC assay kit includes:

[0119] A. Magnetic bead coating working solution, used to capture large-sized cTnITC in a sample. The magnetic bead coating working solution comprises: a mixture of superparamagnetic particles coated with capture antibodies.

[0120] B. Enzyme marker working solution, used to detect large-sized cTnITC antigens captured by superparamagnetic microparticles. The enzyme marker working solution includes: detection antibody labeled with alkaline phosphatase.

[0121] The detection method of large-size cTnITC is as follows:

[0122] Step 1: Add the sample, magnetic bead coating solution, and enzyme marker solution to a reaction tube. After incubation, the large cTnITC in the sample binds to the antibody coated on the magnetic beads, while the antibody-alkaline phosphatase marker also binds to the large cTnITC in the sample. After the reaction is complete, the solid phase is placed in a magnetic field, which attracts the magnetic beads, retaining the bound substances and washing away the unbound substances.

[0123] Step 2: A chemiluminescent substrate is added to the reaction tube. The luminescent substrate (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinoyl)-phenyl-1,2-dioxetane, AMPPD) is degraded by alkaline phosphatase, removing a phosphate group to form an unstable intermediate. This intermediate generates a methyl m-oxybenzoate anion through intramolecular electron transfer. When the excited methyl m-oxybenzoate anion returns to the ground state, it produces chemiluminescence. The number of photons generated in this reaction is then measured using a photomultiplier tube. The number of photons generated is proportional to the concentration of large-size cTnITC in the sample. The amount of analyte in the sample is determined using a calibration curve.

[0124] The above-mentioned large-size cTnITC detection kit can be used in conjunction with Mindray's fully automatic chemiluminescence analyzers CL2000i, CL6000i, CL8000i and other models.

[0125] Example 2 Signal-to-noise ratio analysis of large-size cTnITC detection kit

[0126] Prepare samples containing different antigen concentrations, including two high-concentration samples (high-value samples) and two low-concentration samples (low-value samples). The antigen is recombinant cardiac troponin ternary complex (Hytest, 8ITCR). Analyze these samples using assay kits 1-8. Simultaneously, record the signal from a blank sample without antigen and calculate the signal-to-noise ratio.

[0127] The test results are shown in Figure 1, where the signal-to-noise ratio of detection kit 3 was significantly higher than that of detection kit 1 and detection kit 5, and the signal-to-noise ratio of detection kit 4 was significantly higher than that of detection kit 2 and detection kit 5, indicating that the use of antibody 2 that specifically binds to TnC together with antibody 3 or antibody 4 can significantly improve the signal-to-noise ratio; detection kits 3, 7 and 8 have high signal-to-noise ratios, indicating that antibody 1 with specific binding sites at cTnT amino acids 119-138 and 132-151 is significantly helpful in improving the signal-to-noise ratio.

[0128] In addition, the antibody 1 used in kits 3-8 was replaced with the following antibodies: antibody 7F4 or 7G7 with a specific binding site at the cTnT amino acid 67-86 fragment, and antibody 2F3, 1A11, or 1F11cc with a specific binding site at the cTnT amino acid 145-164 fragment; the antibody 4 used in kit 4 was replaced with the following antibodies: antibody M18cc with a specific binding site at the cTnI amino acid 18-28 fragment, 16A11cc, 16A12cc, or 8E10cc with a specific binding site at the cTnI amino acid 86-90 fragment, antibody M46 with a specific binding site at the cTnI amino acid 130-145 fragment, and antibody MF4cc with a specific binding site at the cTnI amino acid 190-196 fragment, all of which can effectively reflect the signal differences of the samples.

[0129] Example 3 Specificity Analysis of Large-Size cTnITC Detection Kit

[0130] Equal concentrations of different antigens were added to the serum of healthy individuals and analyzed by chemiluminescent immunoassay using test kits 1-8. The antigens analyzed included: cTnT (Hytest, 8RTT5), cTnI (Hytest, 8RT17), cTnIC (Hytest, 8ICR3), and cTnITC (Hytest, 8ITCR).

[0131] The experimental results are shown in Figure 2. Test kits 1-8 only recognized the cTnITC antigen, but not cTnT, cTnI, or binary cTnIC. Furthermore, replacing Antibody 1 used in Kits 3-8 with the following antibodies: 7F4 or 7G7, whose specific binding site is at the cTnT amino acid 67-86 fragment; 2F3, 1A11, or 1F11cc, whose specific binding site is at the cTnT amino acid 145-164 fragment; and replacing Antibody 4 used in Kit 4 with the following antibodies: M18cc, whose specific binding site is at the cTnI amino acid 18-28 fragment; 16A11cc, 16A12cc, or 8E10cc, whose specific binding site is at the cTnI amino acid 86-90 fragment; M46, whose specific binding site is at the cTnI amino acid 130-145 fragment; and MF4cc, whose specific binding site is at the cTnI amino acid 190-196 fragment, all effectively recognized the cTnITC antigen.

[0132] Example 4 Establishment of Blank Limit and Detection Limit of Large-Size cTnITC Detection Kit

[0133] The limit of blank (LoB) and limit of detection (LoD) were established according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (EP-17A2 Protocols for Determination of Limits of Detection and Limits of Quantitation).

[0134] LoB test results are derived from five blank samples, run over four days, with four replicates per test. The general formula is LoB = mean + 1.65 * SD.

[0135] LoD test results are derived from five low-concentration samples, run over four days, with each test repeated four times. The general formula is LoD = LoB + 1.65 * SD.

[0136] Kits 1-8 were selected to establish blank limits and detection limits. The test results are shown in Table 1.

[0137] Table 1: Limit of Blank (LoB) and Limit of Detection (LoD) of the test kit

[0138] Among them, the blank limit and detection limit of detection kit 3 were significantly lower than those of detection kit 1 and detection kit 5, and the blank limit and detection limit of detection kit 4 were significantly lower than those of detection kit 2 and detection kit 5, indicating that the addition of antibody 2 that specifically binds to TnC and its use together with antibody 3 or antibody 4 can significantly improve the sensitivity of the kit.

[0139] In addition, the antibody 1 used in kits 3-8 was replaced with the following antibodies: antibody 7F4 or 7G7 with a specific binding site at the cTnT amino acid 67-86 fragment, antibody 2F3, 1A11 or 1F11cc with a specific binding site at the cTnT amino acid 145-164 fragment; the antibody 4 used in kit 4 was replaced with the following antibodies: antibody M18cc with a specific binding site at the cTnI amino acid 18-28 fragment, antibody 16A11cc, 16A12cc or 8E10cc with a specific binding site at the cTnI amino acid 86-90 fragment, antibody M46 with a specific binding site at the cTnI amino acid 130-145 fragment, and antibody MF4cc with a specific binding site at the cTnI amino acid 190-196 fragment, all of which had lower LoB and LoD values.

[0140] Example 5 Linearity Analysis of Large-Size cTnITC Detection Kit

[0141] Detection kits 3 and 4 had high signal-to-noise ratios and low limits of detection and were used for linear analysis.

[0142] A clinical serum sample was selected as a high-concentration sample, and the high-concentration sample was diluted according to a certain ratio to obtain a series of diluted samples, and the concentration range of the serial diluted samples was 0-6000 ng / L.

[0143] The samples were analyzed by chemiluminescent immunoassay using Detection Kit 3. A linear fit was performed between the mean value of the test concentration results and the theoretical concentration, and the correlation coefficient within the linear range was calculated.

[0144] The experimental results are shown in Figure 3. The test concentration of the diluted sample is linear with the theoretical concentration. The R2 value within the linear range (0-6000 ng / L) is 0.9982.

[0145] Samples were analyzed using chemiluminescent immunoassay using Detection Kit 4. A linear fit was performed between the average test concentration and the theoretical concentration, and the correlation coefficient was calculated within the linear range. The results are shown in Figure 4. The test concentrations of the diluted samples showed a linear relationship with the theoretical concentration. The R² value within the linear range (0-6000 ng / L) was 0.9995.

[0146] Example 6 Construction and testing of a detection kit for exchanging capture antibodies and detection antibodies

[0147] The capture antibody of detection kit 3 was exchanged with the detection antibody to construct detection kit 3a.

[0148] Detection Kit 3 includes:

[0149] The capture antibodies were: Antibody 1: Antibody 329cc that specifically binds to amino acid fragment 119-138 of cTnT;

[0150] The detection antibodies are: Antibody 2: 7B9cc, an antibody that specifically binds to TnC; and Antibody 3: 20C6cc, an antibody that specifically binds to an epitope of the cTnIC complex.

[0151] Detection Kit 3a includes:

[0152] The capture antibodies were: Antibody 2: antibody 7B9cc that specifically binds to TnC; and Antibody 3: antibody 20C6cc that specifically binds to an epitope of the cTnIC complex;

[0153] The detection antibodies are: Antibody 1: Antibody 329cc that specifically binds to the amino acid fragment 119-138 of cTnT.

[0154] Samples containing different antigen concentrations, including three high-concentration samples and three low-concentration samples, were prepared. The antigen was a recombinant cardiac troponin ternary complex (Hytest, 8ITCR). These samples were analyzed using assay kits 3 and 3a, respectively. The signal from a blank sample without antigen was also recorded, and the signal-to-noise ratio was calculated. The test results are shown in Figure 5.

[0155] Clinical serum samples from patients with cardiovascular disease were analyzed using test kits 3 and 3a, respectively. Simultaneously, signals from serum samples from healthy individuals were recorded, and the signal-to-noise ratio was calculated. The test results are shown in Figure 6. The signal-to-noise ratio of test kit 3 was significantly higher than that of test kit 3a, indicating that the preferred combination of capture and detection antibodies is: capture antibody 1 (specifically binding to cTnT amino acid fragment 119-138), and detection antibodies 2 (specifically binding to TnC) and 3 (specifically binding to the cTnIC complex epitope). The test kits with this combination of capture and detection antibodies exhibited a higher signal-to-noise ratio. The signal-to-noise ratio of test kit 3a was lower than that of test kit 3, and its signal difference variation pattern across different samples was similar to that of test kit 3.

[0156] The specificity of Detection Kit 3a was analyzed. Equal concentrations of different antigens were added to the serum of healthy individuals and analyzed using chemiluminescent immunoassay using Detection Kit 3a. The antigens analyzed included cTnT (Hytest, 8RTT5), cTnI (Hytest, 8RT17), cTnIC (Hytest, 8ICR3), and cTnITC (Hytest, 8ITCR). The results are shown in Figure 7. Kit 3a recognized the cTnITC antigen but not cTnT, cTnI, or the binary cTnIC.

[0157] Overall, Kit 3a can be used to detect clinical serum samples, and Kit 3 performs better in terms of overall sensitivity and specificity.

[0158] Example 7 Clinical Performance Evaluation of Large-Scale cTnITC-Specific Immunoassay

[0159] Assay Kit 3 was used for clinical performance evaluation. To assess the clinical performance of the sandwich immunoassay for detecting large-size cTnITC, serum samples from patients with myocardial injury were dynamically monitored at different time points. High-sensitivity cTnI and cTnT levels were also measured simultaneously in the patients' serum.

[0160] Patients aged 18 or older with myocardial injury were enrolled. These patients had been diagnosed with myocardial infarction, heart failure, or had undergone cardiac surgery and demonstrated elevated high-sensitivity cTnI or high-sensitivity cTnT test values. After enrollment, lithium heparin plasma samples were collected from the patients for three consecutive days and the concentration of large-size cTnITC in the samples was measured using a Mindray chemiluminescence analyzer and accompanying reagents.

[0161] The results, as shown in Figure 8, show that the concentration of large-size cTnITC showed a consistent trend with that of high-sensitivity cTnI and cTnT, indicating that the concentration of large-size cTnITC has clinical value and can be used to assess myocardial damage in patients. Further analysis of the concentration changes of large-size cTnITC, high-sensitivity cTnI, and cTnT, as shown in Figure 9, showed that the concentration of large-size cTnITC changed more rapidly than that of cTnI and cTnT, indicating that the concentration changes of large-size cTnITC are more sensitive to myocardial damage in patients.

[0162] In addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A composition for detecting large-sized cTnITC in a sample, comprising a first group of antibodies and a second group of antibodies, wherein: The first group of antibodies includes one or more antibodies 1, each of which is independently selected from an antibody that specifically binds to any section of the amino acid sequence of positions 67-222 of cTnT (cardiac troponin T); The second group of antibodies includes one or more antibodies 2, and each of the antibodies 2 is independently selected from antibodies that specifically bind to any segment of the TnC (troponin C) amino acid sequence.

2. The composition of claim 1, wherein the first group of antibodies does not include an antibody that specifically binds to any stretch of the amino acid sequence of positions 223-287 of cTnT.

3. The composition of claim 1 or 2, wherein the second set of antibodies further comprises: One or more antibodies 3, each of which is independently selected from antibodies that specifically bind to cTnIC (cardiac troponin IC binary complex); and / or, One or more antibodies 4, each of which is independently selected from an antibody that specifically binds to any section of the amino acid sequence at positions 18-210 of cTnI (cardiac troponin I). 4 . The composition of claim 1 , wherein the first group of antibodies comprises one or more of the antibody 1 , and the second group of antibodies comprises one or more of the antibody 2 and one or more of the antibody 3.

5. The composition of any one of claims 1 to 4, wherein each of the antibodies 1 is independently selected from an antibody that specifically binds to amino acids 67-86, amino acids 119-138, amino acids 132-151, amino acids 145-164, or amino acids 171-190 of cTnT; Preferably, each of the antibodies 1 is independently selected from an antibody that specifically binds to amino acids 119-138, amino acids 132-151, or amino acids 171-190 of cTnT.

6. The composition of any one of claims 3-5, wherein each of the antibodies 4 is independently selected from an antibody that specifically binds to amino acids 1-15, 13-22, 18-22, 18-28, 18-35, 22-31, 22-40, 23-29, 24-40, 25-40, 26-35, 34-37, 41-49, 83-89, 86-90, 87-90, 117-126, 130-145, 169-178, 186-192, 190-196, or 195-209 of cTnI; Preferably, each of the antibodies 4 is independently selected from an antibody that specifically binds to amino acids 22-40, amino acids 41-49, or amino acids 83-89 of cTnI; More preferably, each of the antibodies 4 is independently selected from antibodies that specifically bind to amino acids 41-49 of cTnI.

7. A kit for detecting large-size cTnITC in a sample, comprising a capture antibody and a detection antibody, wherein: The capture antibody is selected from one of the first group of antibodies and the second group of antibodies, and the detection antibody is selected from the other of the first group of antibodies and the second group of antibodies; wherein The first group of antibodies includes one or more of the antibodies 1, and each of the antibodies 1 is independently selected from antibodies that specifically bind to any segment of the amino acid sequence of positions 67-222 of cTnT; The second group of antibodies includes one or more antibodies 2, and each of the antibodies 2 is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence.

8. The kit of claim 7, wherein the first group of antibodies does not include an antibody that specifically binds to any section of the amino acid sequence of positions 223-287 of cTnT.

9. The kit of claim 7 or 8, wherein the second set of antibodies further comprises: One or more antibodies 3, each of which is independently selected from an antibody that specifically binds to cTnIC; and / or, One or more antibodies 4, each of which is independently selected from an antibody that specifically binds to any section of the amino acid sequence at positions 18-210 of cTnI. 10 . The kit according to claim 7 , wherein the first group of antibodies comprises one or more of the antibody 1 , and the second group of antibodies comprises one or more of the antibody 2 and one or more of the antibody 3 .

11. The kit of any one of claims 7 to 11, wherein the capture antibody is the first group of antibodies, and the detection antibody is the second group of antibodies.

12. The kit according to any one of claims 7 to 11, wherein each of the antibodies 1 is independently selected from an antibody that specifically binds to amino acids 67-86, amino acids 119-138, amino acids 132-151, amino acids 145-164, or amino acids 171-190 of cTnT; preferably, each of the antibodies 1 is independently selected from an antibody that specifically binds to amino acids 119-138, amino acids 132-151, or amino acids 171-190 of cTnT; and / or Each of the antibodies 4 is independently selected from the group consisting of antibodies that specifically bind to amino acids 1-15, 13-22, amino acids at positions 18-22, amino acids at positions 18-28, amino acids at positions 18-35, amino acids at positions 22-31, amino acids at positions 22-40, amino acids at positions 23-29, amino acids at positions 24-40, amino acids at positions 25-40, amino acids at positions 26-35, amino acids at positions 34-37, amino acids at positions 41-49, amino acids at positions 83-89, amino acids at positions 86-90, amino acids at positions 87-90, amino acids at positions 117-126, Preferably, each of the antibodies 4 is independently selected from an antibody that specifically binds to amino acids 130-145, amino acids 169-178, amino acids 186-192, amino acids 190-196 or amino acids 195-209 of cTnI; preferably, each of the antibodies 4 is independently selected from an antibody that specifically binds to amino acids 22-40, amino acids 41-49 or amino acids 83-89 of cTnI; more preferably, each of the antibodies 4 is independently selected from an antibody that specifically binds to amino acids 41-49 of cTnI.

13. A method for detecting large-sized cTnITC in a sample in vitro, comprising the following steps: Obtaining a sample to be tested; contacting the sample to be tested with a capture antibody to form an antibody-antigen complex; contacting the antibody-antigen complex with a detection antibody conjugated to a detectable label to form an antibody-antigen-antibody complex; and Detecting the signal generated by the detectable label to determine the presence and / or content of large-sized cTnITC in the sample to be tested; Wherein, the capture antibody is selected from one group of the first group of antibodies and the second group of antibodies, and the detection antibody is selected from the other group of the first group of antibodies and the second group of antibodies: The first group of antibodies includes one or more antibodies 1, each of which is independently selected from an antibody that specifically binds to any segment of the amino acid sequence of positions 67-222 of cTnT; The second group of antibodies includes one or more antibodies 2, and each of the antibodies 2 is independently selected from antibodies that specifically bind to any segment of the TnC amino acid sequence.

14. The method of claim 13, wherein the first group of antibodies does not include an antibody that specifically binds to any stretch of the amino acid sequence of positions 223-287 of cTnT.

15. The method of claim 13 or 14, wherein the second set of antibodies further comprises: One or more antibodies 3, each of which is independently selected from an antibody that specifically binds to cTnIC; and / or, One or more antibodies 4, each of which is independently selected from an antibody that specifically binds to any section of the amino acid sequence at positions 18-210 of cTnI. 16 . The method of claim 15 , wherein the first group of antibodies comprises one or more of the antibody 1 , and the second group of antibodies comprises one or more of the antibody 2 , and one or more of the antibody 3 .

17. The method of any one of claims 13-16, wherein the capture antibody is the first group of antibodies, and the detection antibody is the second group of antibodies.

18. Use of the composition according to any one of claims 1 to 6 in the preparation of a reagent for detecting large-sized cTnITC.

19. Use of the composition of any one of claims 1 to 6, the kit of any one of claims 7 to 12, or the method of any one of claims 13 to 17 in in vitro diagnosis of myocardial injury.

20. A method for diagnosing myocardial injury in vitro, comprising the step of detecting large-sized cTnITC in a sample from a subject using the composition of any one of claims 1 to 6, the kit of any one of claims 7 to 12, or the method of any one of claims 13 to 17.

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