Compositions, kits, and methods for detecting large cTnITCs, and their applications

Abstract

This application relates to a composition, kit, and method for detecting large cTnITCs, and its applications. Specifically, this application relates to a composition and kit comprising an antibody that specifically binds to any segment in the 67-222 amino acid sequence of cTnT and an antibody that specifically binds to any segment in the TnC amino acid sequence, a detection method, and its application in the in vitro diagnosis of myocardial injury.

Description

Technical Field

[0001] This application relates to the field of immunoassay, and specifically, to compositions and kits for detecting large-size cardiac troponin ternary complex (cTnITC), detection methods, and their applications in the in vitro diagnosis of myocardial injury.

Background Art

[0002] Cardiac troponin (cTn) is a specific marker for myocardial injury and can be used to detect myocardial injury after myocardial infarction or during the process of myocardial infarction. Troponin contains three subunits, namely cardiac troponin I (cTnI), cardiac troponin T (cTnT), and troponin C (TnC). The serum concentrations of cTnI and cTnT are highly correlated with the severity of myocardial injury and are markers with high specificity and sensitivity for myocardial infarction recommended by the American and European Societies of Cardiology. However, the causes of the increase in blood cardiac troponin concentration are diverse, including pulmonary embolism, acute or chronic heart failure, cardiac trauma, endocarditis, and myocarditis in addition to myocardial infarction. As a result, the clinical specificity of cTn for myocardial infarction decreases, and usually, other means are required to be used in combination for the diagnosis of myocardial infarction.

[0003] Cardiac troponin usually binds to actin filaments in the form of the ternary complex cTnITC. During myocardial injury, troponin is degraded from the myofilaments and released into the blood. In cells or in the blood circulation, full-length cTnITC is degraded by proteases and gradually forms low-molecular-weight ternary complexes (LMW-cTnITC), binary complexes (cTnIC), and free cTnT. Studies have shown that the forms of troponin present in the blood are related to the physiological and pathological states of individuals. Detecting different forms of cTnITC complexes helps physicians comprehensively understand the physiological and pathological states of patients, assist in rapid and accurate diagnosis, and guide the prognosis of patients.

[0004] Currently, conventional immunological methods for detecting cardiac troponin mainly include enzyme-linked immunosorbent assay (ELISA), gold colloid immunochromatography (GICA), electrochemiluminescence (ECL), and chemiluminescence immunoassay (CLIA). Of these, chemiluminescence immunoassay is the immunoanalytical method with the broadest potential for clinical application due to its high sensitivity, strong specificity, broad linear range, ease of operation, and high level of automation. Based on chemiluminescence immunoassay, conventional high-sensitivity cardiac troponin detection methods are typically used to detect cTnI and cTnT, and cannot distinguish between troponin complexes and free troponin. Existing literature reports indicate that the two-antibody sandwich immunoassay method for cTnITC complexes is achieved by having the capture antibody recognize the epitope of the binary TnIC complex and the detection antibody recognize the TnT antigen. Such detection methods have a low signal-to-noise ratio and low specificity, making it difficult to achieve rapid, highly sensitive, and specific detection of cTnITC complexes. [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of this application is to provide a technical solution that can detect a specific form of cTnITC in a sample with high sensitivity and rapid speed. This solution achieves specific detection by combining a capture antibody and a detection antibody, and in particular, adding an antibody that specifically binds to TnC to either the capture antibody or the detection antibody is highly advantageous in improving the signal-to-noise ratio of the detection system and increasing detection sensitivity. [Means for solving the problem]

[0006] According to a first aspect, the present application provides a composition for detecting large cTnITCs in a sample, the composition comprising a first group antibody and a second group antibody, wherein, The antibody of the first group comprises one or more antibodies 1, each antibody 1 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnT between positions 67 and 222. The second group of antibodies comprises one or more antibodies 2, each antibody 2 independently selected from antibodies that specifically bind to any segment in the TnC amino acid sequence.

[0007] According to a second aspect, the present application provides a kit for detecting large cTnITCs in a sample, the kit 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, The antibody of the first group comprises one or more antibodies 1, each antibody 1 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnT between positions 67 and 222. The second group of antibodies comprises one or more antibodies 2, each antibody 2 independently selected from antibodies that specifically bind to any segment in the TnC amino acid sequence.

[0008] According to a third aspect, the present application provides a method for detecting large cTnITCs in a sample in vitro, the method being: Steps include obtaining a sample for detection, The steps include: contacting the sample to be detected with a capture antibody to form an antibody-antigen complex; The steps include contacting the antibody-antigen complex with a detection antibody to which a detectable label is bound to form an antibody-antigen-antibody complex, The steps include detecting the signal generated by the detectable label to determine the presence and / or content of large cTnITCs in the sample to be detected, Here, the capture antibody is selected from either the antibody of group 1 or the antibody of group 2, and the detection antibody is selected from the other antibody of group 1 or group 2. The antibody of the first group comprises one or more antibodies 1, each antibody 1 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnT between positions 67 and 222. The second group of antibodies comprises one or more antibodies 2, each antibody 2 independently selected from antibodies that specifically bind to any segment in the TnC amino acid sequence.

[0009] According to a fourth aspect, the present application provides an application of the composition described in any one of the first aspects in the manufacture of a reagent for detecting large cTnITCs.

[0010] According to the fifth aspect, the present application provides an application for the composition described in any one of the first aspects, the kit described in any one of the second aspects, or the method described in any one of the third aspects in the in vitro diagnosis of myocardial injury.

[0011] According to a sixth aspect, the present application provides a method for diagnosing myocardial injury in vitro, the method comprising the step of detecting large cTnITCs in a sample from a subject using a composition described in any one of the first aspects, a kit described in any one of the second aspects, or the method described in any one of the third aspects. [Brief explanation of the drawing]

[0012] The drawings described herein are for further understanding of the present invention and constitute part of this application. Exemplary embodiments and descriptions of the present invention are for interpretation purposes only and do not improperly limit the present invention. In the drawings, [Figure 1] This figure shows the signal-to-noise ratio analysis of a large cTnITC detection kit. [Figure 2] This figure shows the validation of the specificity of the large cTnITC detection kit using serum samples. [Figure 3] This figure shows a linear analysis of the large-scale cTnITC detection kit 3. [Figure 4] This figure shows a linear analysis of the large-scale cTnITC detection kit 4. [Figure 5] This figure shows the signal-to-noise ratio analysis of the large cTnITC detection kit 3 and detection kit 3a. [Figure 6]Figure showing the signal-to-noise ratio analysis of the detection of clinical serum samples by the large cTnI TC detection kit 3 and the detection kit 3a. [Figure 7] Figure showing the verification of the specificity of the large cTnI TC detection kit 3a with serum samples. [Figure 8] Figure showing the analysis of the change trends of large cTnI detected by the large cTnI TC detection kit 3 and highly sensitive cTnI and cTnT detected by conventional kits. [Figure 9] Figure showing the analysis of the concentration change rates of large cTnI detected by the large cTnI TC detection kit 3 and highly sensitive cTnI and cTnT detected by conventional kits.

Mode for Carrying Out the Invention

[0013] [[ID=]16] Hereinafter, while referring to the drawings, the technical solutions in the embodiments will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. The following description of the embodiments is merely illustrative and should never be construed as any limitation to the present invention. All other embodiments obtained by those skilled in the art on the premise of not paying creative labor based on the embodiments belong to the protection scope of the present invention.

[0014] In this specification, unless otherwise specified, the scientific and technical terms used have the meanings generally understood by those skilled in the art. And the immunology laboratory operation steps used in this specification are all ordinary steps widely used in the corresponding fields. At the same time, in order to better understand the embodiments of the present invention, the definitions and interpretations of related terms are provided below.

[0015] As used herein, the technical terms "comprise," "comprising," or any other variant thereof cover non-exclusive inclusion, such that a method or apparatus that comprises a series of elements includes not only the elements explicitly recited, but also other elements not explicitly listed or further elements inherent to the method or apparatus. Without further limitation, an element limited by the phrase "comprising one" does not exclude the presence of other related elements in the method or apparatus that comprises the element.

[0016] As used herein, the term "at least one" means one or more under reasonable conditions, for example, two, three, four, five, or ten.

[0017] As used herein, the terms "first," "second," etc. are merely for distinguishing similar objects and do not represent a specific order of the objects. It can be understood that "first" and "second" can be interchanged in a specific order or sequence when permitted. It should be understood that the objects distinguished by "first" and "second" can be interchanged when appropriate. Therefore, the embodiments of the present application described herein can be implemented in an order different from that shown or described herein.

[0018] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), for example, the reaction between an antibody and the antigen to which it is directed. The binding affinity between two molecules can be described by the KD value. The KD value is the dissociation constant obtained from the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction, also called koff) and ka (the association rate of a specific binding molecule-target molecule interaction, also called kon), or refers to kd / ka expressed as molar concentration (M). The smaller the KD, the tighter the binding between the two molecules and the higher the affinity. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody having specificity for a certain antigen) has an antibody of about 10 -5Less than M, for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 This refers to binding to the antigen with a KD of less than or equal to M. Corresponding methods for analyzing antibody specificity are described, for example, in the following literature: 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 applicable studies include, for example, binding studies, blocking and competition studies performed using molecules that are structurally and / or functionally close. These studies can be performed by the following methods, for example, fluorescence-activated cell sorting (FACS) analysis, flow cytometry titration (FACS titration) analysis, surface plasmon resonance (SPR) technique, isothermal titration thermometry (ITC), fluorescence titration, or radiolabeled ligand binding assays. Other methods include, for example, Western blotting, ELISA (including competitive ELISA) tests, RIA tests, ECL tests, and IRMA tests.

[0019] In this specification, the terms "troponin" and "Tn" refer to proteins that regulate the calcium-mediated interaction between actin and myosin on myosilin within muscle cells, are present in cardiac and skeletal muscle, and consist of three subunits: troponin T (TnT), troponin I (TnI), and troponin C (TnC). Here, TnT is the tropomyosin-binding subunit, which interacts with actin and tropomyosin; TnI is the repressive subunit, which inhibits the activity of actinomyosin ATPase; and TnC is the calcium-binding subunit, which enables the contraction of skeletal or cardiac muscle. The terms "cardiac troponin" and "cTn" refer to all troponin isotypes expressed in cardiac cells, preferably subendocardial cells. These isotypes are well characterized in this field and are described, for example, by Anderson 1995, Circulation Research, vol. 76, no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493. The term “cardiac troponin” further includes variants of a particular cardiac troponin, such variants possessing at least the same basic biological and immunological properties as the particular cardiac troponin. In particular, they share the same basic biological and immunological properties if they are detected with the same specificity as referred herein. It is understood that troponin isotypes can be measured together (simultaneously or sequentially) or individually (i.e., without measuring any other isotypes).

[0020] As used herein, the terms “cardiac troponin T” and “cTnT” refer to the cardiac troponin T subunit, whose amino acid sequence is publicly available in the UniProt database and is numbered P45379.

[0021] As used herein, the terms “cardiac troponin I” and “cTnI” refer to the cardiac troponin I subunit, whose amino acid sequence is publicly available in the UniProt database and is numbered P19429.

[0022] As used herein, the terms “troponin C” and “TnC” refer to the troponin C subunit, whose amino acid sequence is publicly available in the UniProt database and is numbered P63316.

[0023] In this specification, the terms “large-size cardiac troponin ternary complex” or “large-size cTnITC” are used synonymously and are intended to include complexes formed by any full-length protein or fragment of TnC, a full-length protein or fragment of cTnI, one or more segments of cTnT at amino acid residues 223–287, and one or more segments of cTnT at amino acid residues 1–222.

[0024] In this specification, the term “antibody” has the meaning commonly understood in this field and refers to an immunoglobulin molecule, typically consisting of two polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified into kappa (κ) and lambda (λ) light chains. Heavy chains can be classified into μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a variable heavy chain region (VH) and a constant heavy chain region (CH). Each light chain consists of a variable light chain region (VL) and a constant light chain region (CL). The constant light chain region consists of a single domain CL. The constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, for example, it can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and host tissues or factors containing the first component of the classical complement system (C1q). The VH and VL regions may be further subdivided into highly variable regions (called complementarity-determining regions (CDRs)), with more conserved regions called framework regions (FRs) scattered between them. Each VH and VL consists of three CDRs and four FRs arranged from the amino group terminus to the carboxyl group terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The amino acid assignments in each region or domain follow the definitions in 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. In this specification, unless otherwise clearly indicated by the context, the term “antibody” includes not only complete antibodies but also antigen-binding fragments of antibodies.

[0025] This application is applicable to a variety of detection systems, including but not limited to fluorescent labeling reaction systems, enzyme-linked immune systems, and bioluminescent systems.

[0026] Detection reagents and methods One object of this application is to provide a detection reagent that can sensitively and specifically detect large cTnITC (containing the full-length cTnITC and a moderate amount of hydrolysis products, the hydrolysis products still containing cTnI, cTnT and TnC subunits or fragments thereof, and TnT fragments at positions 67-222), and furthermore, does not recognize hydrolyzed ternary complexes, binary complexes or free subunits and fragments thereof.

[0027] Specifically, according to the first aspect, the present application provides a composition for detecting large cTnITCs in a sample, the composition comprising a first group antibody and a second group antibody, where, The antibody of the first group comprises one or more antibodies 1, each antibody 1 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnT between positions 67 and 222. The second group of antibodies comprises one or more antibodies 2, each antibody 2 independently selected from antibodies that specifically bind to any segment in the TnC amino acid sequence.

[0028] Based on the first aspect, a second aspect of this application provides a kit for detecting large cTnITCs 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, The antibody of the first group comprises one or more antibodies 1, each antibody 1 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnT between positions 67 and 222. The second group of antibodies comprises one or more antibodies 2, each antibody 2 independently selected from antibodies that specifically bind to any segment in the TnC amino acid sequence.

[0029] In other words, the capture antibody contains at least antibody 1 and the detection antibody contains at least antibody 2, or the capture antibody contains at least antibody 2 and the detection antibody contains at least antibody 1.

[0030] In the embodiments of this application, the antibodies of the first group do not include antibodies that specifically bind to any segment in the amino acid sequence of cTnT between positions 223 and 287.

[0031] In some embodiments, capture antibodies are coated onto the surface of a solid support such as magnetic beads, latex particles, enzyme-labeled plates, plastic beads, plastic tubes, immunochromatography test strips, or detection cards to capture antigens in a sample, and then detection antibodies having a detectable label are bound to the capture antibodies coated on the solid support surface. The amount of the detectable label on the bound detection antibody can then be measured to determine the amount of antigen in the sample.

[0032] The detectable label may be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means. Particularly preferably, such a label is applicable to immunological detection (e.g., enzyme-linked immunoassay, 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 dot, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), chemiluminescent substances (e.g., acridinium ester compounds), and biotin for binding to avidin modified with the aforementioned labels (e.g., streptavidin). The labels encompassed in this invention can be detected by methods known to those skilled in the art. For example, radioactive labels can be detected with photographic film or a scintillation counter, and fluorescent labels can be detected with a photodetector to detect the emitted light. Enzyme labels are generally detected by supplying a substrate to an enzyme and detecting the reaction product produced when the enzyme acts on the substrate. Chemiluminescent substances (e.g., acridinium ester compounds) are generally detected by detecting the emission by supplying a trigger solution and / or a catalyst to the luminescent substance. Biotin is generally detected by supplying avidin modified with the label (e.g., streptavidin) to biotin and detecting the label carried by the avidin linked to the biotin. In some embodiments, the detectable labels can be linked to the antibody or its antigen-binding fragment of the present invention via linkers of different 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), radioisotopes, biotin, gold colloids, and magnetic particles.

[0033] In some embodiments, the kit may further include a reagent (substrate of the detectable label) for detecting the corresponding detectable label. For example, if the detectable label is an enzyme, the kit may further include 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, if the detectable label is a chemiluminescent reagent (e.g., an acridinium ester compound), the kit may further include a pre-trigger solution and / or trigger solution for chemiluminescence.

[0034] The kit described in this application is particularly applicable to immunoassays, such as ELISA, and especially to two-antibody sandwich chemiluminescence immunoassay methods.

[0035] In this application, the amount of large cTnITCs can be detected directly or indirectly. The amount or concentration of the detection antibody can be directly detected based on a detectable labeling signal of the detection antibody, the signal being directly related to the target antigen in the sample. Such a signal may also be referred to herein as an intensity signal and can be obtained, for example, by measuring the intensity of a specific physical or chemical property of the detection antibody. Indirect measurements include signals from a secondary component (i.e., the detection antibody itself) or a biological readout system, such as measuring a measurable cellular response, ligand, label, or enzymatic reaction product.

[0036] According to a third aspect, the present application provides a method for detecting large cTnITCs in a sample in vitro, the method being: The steps include obtaining the sample to be detected, The steps include: contacting the sample to be detected with a capture antibody to form an antibody-antigen complex; The steps include contacting the antibody-antigen complex with a detection antibody to which a detectable label is bound to form an antibody-antigen-antibody complex, The steps include detecting the signal generated by the detectable label to determine the presence and / or content of large cTnITCs in the sample to be detected, Here, the capture antibody is selected from either the antibody of group 1 or the antibody of group 2, and the detection antibody is selected from the other antibody of group 1 or group 2. The antibody of the first group comprises one or more antibodies 1, each antibody 1 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnT between positions 67 and 222. The second group of antibodies comprises one or more antibodies 2, each antibody 2 independently selected from antibodies that specifically bind to any segment in the TnC amino acid sequence.

[0037] In some embodiments of the first to third embodiments of this application, the antibodies of the second group are: One or more antibodies 3 independently selected from antibodies that specifically bind to cTnIC, and / or The present invention further comprises one or more antibodies 4 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnI between positions 18 and 210.

[0038] Here, by combining 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.

[0039] In the first to third embodiments of this application, as in some embodiments, the first group of antibodies comprises one or more antibodies 1, and the second group of antibodies comprises one or more antibodies 2 and one or more antibodies 3.

[0040] In some embodiments of the first to third embodiments of this application, the first group of antibodies comprises at least two antibodies 1. In some embodiments, the second group of antibodies comprises at least two antibodies 2. In some embodiments, the first group of antibodies comprises at least two antibodies 1, and the second group of antibodies comprises at least two antibodies 2 and at least two optionally selected antibodies 3. Combinations of multiple antibodies that bind to the same target protein are beneficial for the detection of large cTnITCs.

[0041] In this application, the antibody binding epitopes were screened. In some embodiments, each antibody 1 is independently selected from antibodies that specifically bind to the 67-86, 119-138, 132-151, 145-164, or 171-190 amino acids of cTnT. In some embodiments, each antibody 1 is independently selected from antibodies that specifically bind to the 119-138, 132-151, or 171-190 amino acids of cTnT. In some embodiments, each antibody 1 is independently selected from antibodies that specifically bind to the 119-138 or 132-151 amino acids of cTnT.

[0042] Regarding the binding epitope of antibody 4, in some embodiments, each antibody 4 is independently selected from antibodies that specifically bind to the amino acids at positions 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. In some embodiments, each antibody 4 is independently selected from antibodies that specifically bind to the 22-40 amino acids, 41-49 amino acids, or 83-89 amino acids of cTnI.

[0043] In some embodiments, the capture antibody is an antibody from group 1, and the detection antibody is an antibody from group 2. Studies have shown that when group 1 antibodies are used as the capture antibody and group 2 antibodies are used as the detection antibody, the detection of large cTnITCs has a higher signal-to-noise ratio.

[0044] In this application, a commercially available antibody against a specific epitope may be selected, or an antibody having specific binding activity to a specific epitope of large-size cTnITC in this application may be obtained by general technical means of the art. One exemplary method involves obtaining a monoclonal antibody with specific binding activity by hybridoma technology, specifically including steps such as selection and preparation of two parental cells, cell fusion, selective culture and cloning of hybridoma cells, preparation of the monoclonal antibody, identification of specificity and purification. Further steps of epitope identification and screening may be included, for example, by techniques such as ELISA or surface plasmon resonance (SPR) to identify the antibody-binding epitope.

[0045] application Conventional cTnITC detection kits suffer from low sensitivity, severely limiting their application in the diagnosis of myocardial injury. Clinically, cTnI and cTnT are typically selected as markers with high specificity and sensitivity to myocardial infarction. Based on the aforementioned composition, kit, and detection method, this application enables the rapid detection of large cTnITCs with high sensitivity and specificity, which is advantageous for a deeper understanding of the relationship between troponin and myocardial injury. Studies have shown that, compared to cTnI and cTnT, large cTnITCs are more sensitive to the context of myocardial injury and can be useful as a more sensitive myocardial injury marker for disease diagnosis and prognosis.

[0046] According to a fourth aspect, the present application provides an application of the composition described in any one of the first aspects in the manufacture of a reagent for detecting large cTnITCs.

[0047] According to the fifth aspect, the present application provides an application for the composition described in any one of the first aspects, the kit described in any one of the second aspects, or the method described in any one of the third aspects in the in vitro diagnosis of myocardial injury.

[0048] According to a sixth aspect, the present application provides a method for diagnosing myocardial injury in vitro, the method comprising the step of detecting large cTnITCs in a sample from a subject using a composition described in any one of the first aspects, a kit described in any one of the second aspects, or the method described in any one of the third aspects.

[0049] In some embodiments, the sample is a body fluid or tissue sample of the subject, such as whole blood, serum, plasma (including lithium heparin plasma and EDTA plasma), urine, saliva, biological tissue, or cells, and is preferably whole blood, serum, or plasma.

[0050] (Effects of the invention) 1) This application enables the rapid detection of large cTnITCs with high sensitivity and specificity. By selecting an antibody that specifically binds to TnC as the capture antibody or detection antibody, the signal-to-noise ratio of the system can be significantly improved, thereby increasing sensitivity.

[0051] 2) Because the form of troponin in the blood is related to the physiological and pathological state of an individual, the specific detection of large cTnITCs is advantageous for a deeper understanding of the relationship between troponin and disease. Experimental results show that the change trend of large cTnITCs is consistent with that of cTnT and cTnI, and has clinical value. Furthermore, the rate of change of large cTnITCs is faster, and they are more sensitive to the state of myocardial damage in patients. As a more sensitive myocardial damage marker, they can be useful for disease diagnosis and prognosis assessment.

[0052] 3) The composition or kit of this application can be used in combination with a fully automated chemiluminescence apparatus to enable automation, acceleration, and high-throughput detection of large cTnITCs.

[0053] 4) The kit described in this application has high sensitivity, good specificity, and good reproducibility. [Examples]

[0054] Example 1: Construction of a detection method and detection kit A capture antibody-detection antibody was applied to a two-antibody sandwich chemiluminescence immunoassay method to construct a detection kit, which was used to detect large cTnITCs in the sample.

[0055] (1) Large cTnITC detection kit 1 Capture antibody: Antibody 1: 329cc of antibody that specifically binds to cTnT amino acid fragments 119-138. Detected antibody: Antibody 3: 20C6cc of antibody that specifically binds to the cTnIC complex epitope.

[0056] (2) Large-scale cTnITC detection kit 2 Capture antibody: Antibody 1: 329cc of antibody that specifically binds to cTnT amino acid fragments 119-138. Detected antibody: Antibody 4: Antibody 19C7cc that specifically binds to cTnI amino acid fragments 41-49.

[0057] (3) Large-scale cTnITC detection kit 3 Capture antibody: Antibody 1: 329cc of antibody that specifically binds to cTnT amino acid fragments 119-138. Detected antibodies: Antibody 2: Antibody 7B9cc that specifically binds to TnC, and Antibody 3: Antibody 20C6cc that specifically binds to the cTnIC complex epitope.

[0058] (4) Large cTnITC detection kit 4 Capture antibody: Antibody 1: 329cc of antibody that specifically binds to cTnT amino acid fragments 119-138. Detected antibodies: Antibody 2: Antibody 7B9cc that specifically binds to TnC, and Antibody 4: Antibody 19C7cc that specifically binds to cTnI amino acid fragments 41-49.

[0059] (5) Large-scale cTnITC detection kit 5 Capture antibody: Antibody 1: 329cc of antibody that specifically binds to cTnT amino acid fragments 119-138. Detected antibody: Antibody 2: Antibody 7B9cc that specifically binds to TnC.

[0060] (6) Large cTnITC detection kit 6 Capture antibody: Antibody 1: Antibody 1C11cc that specifically binds to cTnT amino acid fragments 171-190. Detected antibodies: Antibody 2: Antibody 7B9cc, which specifically binds to TnC, and Antibody 3: Antibody Tcom8, which specifically binds to the cTnIC complex epitope.

[0061] (7) Large-scale cTnITC detection kit 7 Capture antibody: Antibody 1: 406cc of antibody that specifically binds to cTnT amino acid fragments 132-151. Detected antibodies: Antibody 2: Antibody 7B9cc that specifically binds to TnC, and Antibody 3: Antibody 20C6cc that specifically binds to the cTnIC complex epitope.

[0062] (8) Large-scale cTnITC detection kit 8 Capture antibody: Antibody 1: 300cc of antibody that specifically binds to cTnT amino acid fragments 119-138. Detected antibodies: Antibody 2: Antibody 7B9cc that specifically binds to TnC, and Antibody 3: Antibody 20C6cc that specifically binds to the cTnIC complex epitope.

[0063] In the experiment, kits obtained by replacing antibody 1 used in the aforementioned large cTnITC detection kits 3-8 with the following antibodies were also used: antibody 7F4 or 7G7 with a specific binding site on cTnT amino acid fragments 67-86, and antibody 2F3, 1A11, or 1F11cc with a specific binding site on cTnT amino acid fragments 145-164. In the experiment, kits obtained by replacing antibody 4 used in the aforementioned large cTnITC detection kit 4 with the following antibodies were also used: antibody M18cc with a specific binding site on cTnI amino acid fragments 18-28, antibody 16A11cc, 16A12cc, or 8E10cc with a specific binding site on cTnI amino acid fragments 86-90, antibody M46 with a specific binding site on cTnI amino acid fragments 130-145, and antibody MF4cc with a specific binding site on cTnI amino acid fragments 190-196. The antibodies described herein are purchased from Hi-Test Co., Ltd.

[0064] The large cTnITC detection kit includes the following:

[0065] A. A magnetic bead coating working solution for capturing large cTnITCs in a sample. The magnetic bead coating working solution contains a mixture of superparamagnetic microparticles that coat the capture antibody.

[0066] B. An enzyme-labeled working solution for detecting large cTnITC antigens captured by superparamagnetic microparticles. The enzyme-labeled working solution contains an alkaline phosphatase-labeled detection antibody.

[0067] The method for detecting large cTnITCs is as follows:

[0068] Step 1: The sample was added to a reaction tube along with the magnetic bead coating working solution and the enzyme labeling working solution, and incubated. Large cTnITCs in the sample bound to the antibody coated with magnetic beads, and the antibody-alkaline phosphatase label also bound to the large cTnITCs in the sample. After the reaction was complete, the solid phase was placed in a magnetic field, which adsorbed the magnetic beads, retaining the substances bound to the solid phase and washing away the unbound substances.

[0069] Step 2: The chemiluminescent substrate was added to the reaction tube. The luminescent substrate (3-(2-spiroadamantan)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxetane, AMPPD) was decomposed by alkaline phosphatase, removing one phosphate group. The unstable intermediate product was purified, and intramolecular electron transfer generated a methyl methoxobenzoate anion. Chemiluminescence occurred when the excited methyl methoxobenzoate anion returned from the excited state to the ground state. The number of photons generated in the reaction was then measured using a photomultiplier tube. The number of photons generated is directly proportional to the concentration of large cTnITC in the sample. The amount of analyte in the sample is determined by the calibration curve.

[0070] The above-mentioned large-capacity cTnITC detection kit can be used with Mairui fully automated chemiluminescence systems such as the CL2000i, CL6000i, and CL8000i.

[0071] Example 2: Signal-to-noise ratio analysis of a large cTnITC detection kit Samples containing antigens of different concentrations were prepared, including two high-concentration samples and two low-concentration samples, where the antigen was recombinant cardiac troponin ternary complex (Hytest, 8ITCR). Each sample was analyzed using detection kits 1-8. Simultaneously, the signal from a blank sample without the antigen was recorded, and the signal-to-noise ratio was calculated.

[0072] The test results are shown in Figure 1. Here, the signal-to-noise ratio of detection kit 3 was significantly higher than that of detection kits 1 and 5, and the signal-to-noise ratio of detection kit 4 was significantly higher than that of detection kits 2 and 5. This indicates that the combined use of antibody 2, which specifically binds to TnC, with antibody 3 or antibody 4 can significantly improve the signal-to-noise ratio. Detection kits 3, 7, and 8 also had high signal-to-noise ratios, indicating that antibody 1, whose specific binding sites are located at positions 119-138 and 132-151 of the cTnT amino acids, clearly contributes to improving the signal-to-noise ratio.

[0073] Furthermore, antibody 1 used in kit 3-8 can be replaced with antibody 7F4 or 7G7, whose specific binding site is on cTnT amino acid fragments 67-86, or antibody 2F3, 1A11, or 1F11cc, whose specific binding site is on cTnT amino acid fragments 145-164. Similarly, antibody 4 used in kit 4 can be replaced with antibody M18cc, whose specific binding site is on cTnI amino acid fragments 18-28, antibody 16A11cc, 16A12cc, or 8E10cc, whose specific binding site is on cTnI amino acid fragments 86-90, antibody M46, whose specific binding site is on cTnI amino acid fragments 130-145, or antibody MF4cc, whose specific binding site is on cTnI amino acid fragments 190-196. All of these can effectively reflect the differences in sample signals.

[0074] Example 3: Specificity analysis of a large cTnITC detection kit Equivalent concentrations of antigens were added to the serum of healthy individuals, and each was analyzed by chemiluminescence immunoassay using detection kits 1-8. The analyzed antigens included cTnT (Hytest, 8RTT5), cTnI (Hytest, 8RT17), cTnIC (Hytest, 8ICR3), and cTnITC (Hytest, 8ITCR).

[0075] The experimental results are shown in Figure 2. Detection kits 1-8 can recognize only the cTnITC antigen, but cannot recognize cTnT, cTnI, or binary cTnIC. Furthermore, antibody 1 used in kit 3-8 can be replaced with antibody 7F4 or 7G7, whose specific binding site is located at cTnT amino acid fragments 67-86, or antibody 2F3, 1A11, or 1F11cc, whose specific binding site is located at cTnT amino acid fragments 145-164. Similarly, antibody 4 used in kit 4 can be replaced with antibody M18cc, whose specific binding site is located at cTnI amino acid fragments 18-28, antibody 16A11cc, 16A12cc, or 8E10cc, whose specific binding site is located at cTnI amino acid fragments 86-90, antibody M46, whose specific binding site is located at cTnI amino acid fragments 130-145, or antibody MF4cc, whose specific binding site is located at cTnI amino acid fragments 190-196. All of these antibodies can effectively recognize the cTnITC antigen.

[0076] Example 4: Establishment of blank limit and detection limit for a large cTnITC detection kit. Based on the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (EP-17A2 Protocols for Determination of Limits of Detection and Limits of Quantitation), we established the blank limit (LoB) and the limit of detection (LoD).

[0077] LoB test results were derived from 5 blank samples, conducted over 4 days, and each test was repeated 4 times. The general formula is LoB = mean + 1.65 * SD.

[0078] The LoD test results were derived from five low-concentration samples, performed over four days, and each test was repeated four times. The general formula is LoD = LoB + 1.65 * SD.

[0079] Select kits 1-8, establish blank limits and detection limits, and the test results are shown in Table 1.

[0080] [Table 1]

[0081] Here, the blank limit and detection limit of detection kit 3 were significantly lower than those of detection kits 1 and 5, and the blank limit and detection limit of detection kit 4 were also significantly lower than those of detection kits 2 and 5. This indicates that adding antibody 2, which specifically binds to TnC, in combination with antibody 3 or antibody 4 can significantly improve the sensitivity of the kit.

[0082] Furthermore, antibody 1 used in kit 3-8 is replaced with antibody 7F4 or 7G7, whose specific binding site is located at cTnT amino acid fragments 67-86, or antibody 2F3, 1A11, or 1F11cc, whose specific binding site is located at cTnT amino acid fragments 145-164. Antibody 4 used in kit 4 is replaced with antibody M18cc, whose specific binding site is located at cTnI amino acid fragments 18-28, antibody 16A11cc, 16A12cc, or 8E10cc, whose specific binding site is located at cTnI amino acid fragments 86-90, antibody M46, whose specific binding site is located at cTnI amino acid fragments 130-145, or antibody MF4cc, whose specific binding site is located at cTnI amino acid fragments 190-196. All of these antibodies have low LoB and LoD values.

[0083] Example 5 Linear analysis of a large cTnITC detection kit Detection kits 3 and 4 had high signal-to-noise ratios and low detection limits, and were used for linear analysis.

[0084] A clinical serum sample is selected as a high-concentration sample, and this high-concentration sample is diluted at a certain ratio to obtain serially diluted samples, with a concentration range of 0 to 6000 ng / L.

[0085] Samples were analyzed using detection kit 3 by chemiluminescence immunoassay. Linear fitting was performed on the test concentration results and theoretical concentrations, and correlation coefficients within the linear range were calculated.

[0086] The experimental results are shown in Figure 3, where the test concentration and theoretical concentration of the diluted sample showed a linear relationship. The R² value within the linear range (0 to 6000 ng / L) is 0.9982.

[0087] Samples were analyzed using detection kit 4 by chemiluminescence immunoassay. Linear fitting was performed using the test concentration results and theoretical concentrations, and the correlation coefficient within the linear range was calculated. The experimental results are shown in Figure 4, and the test concentration and theoretical concentration of the diluted samples showed a linear relationship. The R² value within the linear range (0 to 6000 ng / L) is 0.9995.

[0088] Example 6: Construction and testing of a detection kit by exchanging the capture antibody and the detection antibody. Detection kit 3a was constructed by exchanging the capture antibody and detection antibody in detection kit 3.

[0089] Detection kit 3 includes the following: Capture antibody: Antibody 1: 329cc of antibody that specifically binds to cTnT amino acid fragments 119-138. Detected antibodies: Antibody 2: Antibody 7B9cc that specifically binds to TnC, and Antibody 3: Antibody 20C6cc that specifically binds to the cTnIC complex epitope.

[0090] Detection kit 3a includes the following: Capture antibodies: Antibody 2: Antibody 7B9cc that specifically binds to TnC, and Antibody 3: Antibody 20C6cc that specifically binds to the cTnIC complex epitope. Detected antibody: Antibody 1: 329cc of antibody that specifically binds to cTnT amino acid fragments 119-138.

[0091] Samples containing antigens of different concentrations were prepared, comprising three high-concentration samples and three low-concentration samples, where the antigen was recombinant cardiac troponin ternary complex (Hytest, 8ITCR). Each sample was analyzed using detection kits 3 and 3a. Simultaneously, the signal of a blank sample without the antigen was recorded, and the signal-to-noise ratio was calculated. The test results are shown in Figure 5.

[0092] Clinical serum samples from patients with cardiovascular disease were analyzed using detection kits 3 and 3a, respectively. Simultaneously, serum sample signals from healthy individuals were recorded, and the signal-to-noise ratio (SNO) was calculated. The test results are shown in Figure 6. Here, the SNO of detection kit 3 was significantly higher than that of detection kit 3a, indicating that the preferred combination of capture and detection antibodies is antibody 1 (specifically binding to cTnT amino acid fragments 119-138) as the capture antibody, and antibody 2 (specifically binding to TnC) and antibody 3 (specifically binding to the cTnIC complex epitope) as the detection antibodies, with detection kits having this combination of capture and detection antibodies exhibiting a higher SNO of detection. Detection kit 3a had a lower SNO of detection kit 3, and its signal difference change pattern across different samples was similar to that of detection kit 3.

[0093] The specificity of detection kit 3a was analyzed. Equivalent concentrations of antigens were added to the serum of healthy individuals, and each was analyzed using chemiluminescence immunoassay with detection kit 3a. The analyzed antigens included cTnT (Hytest, 8RTT5), cTnI (Hytest, 8RT17), cTnIC (Hytest, 8ICR3), and cTnITC (Hytest, 8ITCR). The experimental results are shown in Figure 7. Kit 3a can recognize the cTnITC antigen, but cannot recognize cTnT, cTnI, or binary cTnIC.

[0094] Overall, Kit 3a was suitable for detecting clinical serum samples, while Kit 3 was superior in terms of overall sensitivity and specificity.

[0095] Example 7: Clinical performance evaluation of specific immunoassay for large cTnITCs Detection kit 3 was used for clinical performance evaluation. To evaluate the clinical performance of large cTnITC detection using sandwich immunoassay, serum samples from patients with myocardial injury were dynamically monitored at different time points. Simultaneously, high-sensitivity cTnI and cTnT levels were detected in the patient serum.

[0096] Patients with myocardial injury were selected and included in the group, and the patients were 18 years of age or older. These patients had been diagnosed with myocardial infarction, heart failure, or had undergone cardiac surgery and showed elevated high-sensitivity cTnI or high-sensitivity cTnT test values. After inclusion in the group, heparin lithium plasma samples were collected from the patients for three consecutive days, and the concentration of large cTnITCs in the samples was tested using a Maiz chemiluminescence spectrometer and associated reagents.

[0097] The results are shown in Figure 8. The concentration values ​​of large cTnITCs were consistent with the trend of change in the test values ​​of high-sensitivity cTnI and cTnT. This indicates that the concentration values ​​of large cTnITCs have clinical value and can be used to assess the myocardial damage status of patients. Furthermore, analysis of the concentration changes of large cTnITCs, high-sensitivity cTnI, and cTnTs, as shown in Figure 9, revealed that the rate of change in the concentration value of large cTnITCs was faster compared to cTnI and cTnTs. This indicates that changes in the concentration value of large cTnITCs are more sensitive to the myocardial damage status of patients.

[0098] In addition to those described herein, various modifications of the invention will be apparent to those skilled in the art based on the foregoing description. Such modifications are also intended to be included within the claims. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A composition for detecting large cTnITCs in a sample, comprising a first group antibody and a second group antibody, wherein, The first group of antibodies comprises one or more antibodies 1, each antibody 1 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnT (cardiac troponin T) at positions 67 to 222. The second group of antibodies comprises one or more antibodies 2, each antibody 2 independently selected from antibodies that specifically bind to any segment in the TnC (troponin C) amino acid sequence.

2. The composition according to claim 1, wherein the antibodies of the first group do not include antibodies that specifically bind to any segment in the amino acid sequence of cTnT at positions 223 to 287.

3. The antibodies in the second group mentioned above are, One or more antibodies 3 independently selected from antibodies that specifically bind to cTnIC (cardiac troponin IC binary complex), and / or The composition according to claim 1 or 2, further comprising one or more antibodies 4 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnI (cardiac troponin I) between positions 18 and 210.

4. The composition according to any one of claims 1 to 3, wherein the antibody of the first group comprises one or more antibodies 1, and the antibody of the second group comprises one or more antibodies 2 and one or more antibodies 3.

5. Each of the aforementioned antibodies 1 is independently selected from antibodies that specifically bind to the amino acids at positions 67-86, 119-138, 132-151, 145-164, or 171-190 of cTnT. Preferably, each antibody 1 is independently selected from antibodies that specifically bind to the 119-138th amino acid, the 132-151st amino acid, or the 171-190th amino acid of cTnT, in the composition according to any one of claims 1 to 4.

6. Each of the aforementioned antibodies 4 is independently selected from antibodies that specifically bind to the amino acids at positions 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 antibody 4 is independently selected from antibodies that specifically bind to the 22-40 amino acids, 41-49 amino acids, or 83-89 amino acids of cTnI. More preferably, each antibody 4 is independently selected from antibodies that specifically bind to the amino acids at positions 41 to 49 of cTnI, the composition according to any one of claims 3 to 5.

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

8. The kit according to claim 7, wherein the antibodies in the first group do not include antibodies that specifically bind to any segment in the amino acid sequence of cTnT between positions 223 and 287.

9. The antibodies in the second group mentioned above are, One or more antibodies 3, independently selected from antibodies that specifically bind to cTnIC, and / or The kit according to claim 7 or 8, further comprising one or more antibodies 4 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnI between positions 18 and 210.

10. The kit according to any one of claims 7 to 9, wherein the antibody of the first group comprises one or more antibodies 1, and the antibody of the second group comprises one or more antibodies 2 and one or more antibodies 3.

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

12. Each of the aforementioned antibodies 1 is independently selected from antibodies that specifically bind to the amino acids at positions 67-86, 119-138, 132-151, 145-164, or 171-190 of cTnT, and preferably each of the aforementioned antibodies 1 is independently selected from antibodies that specifically bind to the amino acids at positions 119-138, 132-151, or 171-190 of cTnT, and / or Each antibody 4 independently controls the amino acids at positions 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 of cTnI. A kit according to any one of claims 7 to 11, wherein each antibody 4 is selected from antibodies that specifically bind to amino acids at positions 69 to 178, 186 to 192, 190 to 196, or 195 to 209, preferably each antibody 4 is independently selected from antibodies that specifically bind to amino acids at positions 22 to 40, 41 to 49, or 83 to 89 of cTnI, and more preferably each antibody 4 is independently selected from antibodies that specifically bind to amino acids at positions 41 to 49 of cTnI.

13. A method for in vitro detection of large cTnITCs in a sample, The steps include obtaining the sample to be detected, The steps include: contacting the sample to be detected with a capture antibody to form an antibody-antigen complex; The steps include: contacting the antibody-antigen complex with a detection antibody to which a detectable label is bound to form an antibody-antigen-antibody complex; The steps include detecting the signal generated by the detectable label to determine the presence and / or content of large cTnITCs in the sample to be detected, Here, the capture antibody is selected from either the antibody of group 1 or the antibody of group 2, and the detection antibody is selected from the other of the antibody of group 1 or the antibody of group 2. The first group of antibodies comprises one or more antibodies 1, each antibody 1 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnT between positions 67 and 222. The method wherein the second group of antibodies comprises one or more antibodies 2, each antibody 2 independently selected from antibodies that specifically bind to any segment in the TnC amino acid sequence.

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

15. The antibodies in the second group mentioned above are, One or more antibodies 3, independently selected from antibodies that specifically bind to cTnIC, and / or The method according to claim 13 or 14, further comprising one or more antibodies 4 independently selected from antibodies that specifically bind to any segment in the amino acid sequence of cTnI between positions 18 and 210.

16. The method according to claim 15, wherein the antibody of the first group comprises one or more antibodies 1, and the antibody of the second group comprises one or more antibodies 2 and one or more antibodies 3.

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

18. Uses of the composition according to any one of claims 1 to 6 in the production of a reagent for detecting large cTnITCs.

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

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

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