Anti-ctni antibody and use thereof

Abstract

The present disclosure relates to the field of antibodies. Disclosed are an anti-cTnI antibody and a use thereof. The anti-cTnI antibody of the present disclosure comprises a heavy chain complementarity determining region and a light chain complementarity determining region; the antibody provides an important raw material source for cTnI detection, and has good affinity or activity, high detection sensitivity and specificity, and good clinical relevance.

Description

An anti-cTnI antibody and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure claims priority to Chinese patent application number 202410155057.2 filed with the Chinese Patent Office on February 3, 2024, entitled “An anti-cTnI antibody and its application,” the entire contents of which are incorporated by reference into the present disclosure. Technical Field

[0003] The present disclosure relates to the field of antibody technology, and in particular, to an anti-cTnI antibody and applications thereof. Background Art

[0004] Before the 1980s, the World Health Organization (WHO) used myocardial enzyme activity as one of the diagnostic criteria for acute myocardial infarction (AMI). In the late 1980s, researchers discovered that troponin (Tn) was more sensitive and specific than biomarkers such as creatine phosphokinase (CK), creatine phosphokinase isoenzyme (CK-MB), lactate dehydrogenase, and aspartate aminotransferase. Cardiac troponin I (cTnI), present only in the myocardium, is a marker of cardiomyocytes. Abnormal changes in cTnI can affect cardiac systolic and diastolic function and can be used to diagnose myocardial necrosis and myocardial damage. It has become one of the most sensitive and specific markers of myocardial cell injury and is recognized as a key biochemical marker for the rapid diagnosis of AMI and acute coronary syndromes (ACS), as well as for assisting in ACS risk stratification and reflecting ACS prognosis.

[0005] The normal level of cTnI in human blood is generally less than 0.3 μg / L. When the integrity of myocardial cell membranes is compromised by ischemia or hypoxia, free cTnI can rapidly penetrate the cell membrane and enter the bloodstream. Therefore, rapid, sensitive, and accurate measurement of cTnI in human blood and its changing trends in the early stages of disease development are of great clinical significance for the diagnosis of acute myocardial infarction, risk stratification of acute coronary syndromes, and monitoring of myocardial damage caused by various factors. Clinically, methods used to measure cTnI levels include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, and colloidal gold. Each method has its own advantages and disadvantages, but all require antibodies specific to cTnI.

[0006] Therefore, there is a strong need in the art for anti-cTnI antibodies with good performance. Summary of the Invention

[0007] The present application provides an anti-cTnI antibody, which provides an important source of raw materials for the detection of cTnI, has good activity or affinity, high detection sensitivity and specificity, and high clinical relevance.

[0008] To achieve the above objectives, according to one aspect of the present disclosure, an anti-cTnI antibody is provided, comprising three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 22, 23, 24, and 25, and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 30, 31, 32, and 33.

[0009] To achieve the above object, according to a second aspect of the present disclosure, an anti-cTnI antibody is provided, wherein the antibody comprises the following complementarity determining regions:

[0010] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0011] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0012] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0013] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0014] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 5;

[0015] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0016] To achieve the above objectives, according to the third aspect of the present disclosure, an anti-cTnI antibody is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 22, 23, 24, and 25; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 30, 31, 32, and 33.

[0017] To achieve the above objectives, according to a fourth aspect of the present disclosure, an anti-cTnI antibody is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 26, 27, 28, and 29; and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 34, 35, 36, and 37.

[0018] In order to achieve the above object, according to a fifth aspect of the present disclosure, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.

[0019] In order to achieve the above-mentioned object, according to the sixth aspect of the present disclosure, a reagent or a kit is provided, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0020] To achieve the above objectives, according to a seventh aspect of the present disclosure, there is provided a use of the above-mentioned antibody, antibody conjugate, reagent or kit in detecting cTnI, or diagnosing cTnI-related diseases, or preparing a product for detecting cTnI.

[0021] To achieve the above objectives, according to an eighth aspect of the present disclosure, a method for diagnosing whether a subject suffers from a cTnI-related disease is provided, comprising: a) contacting the above-mentioned antibody, antibody conjugate, or reagent or kit with cTnI in a test sample from the subject under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample; and c) if the antigen is present in the test sample, or the antigen is present in the test sample and the level of the antigen is higher than that in a control sample, determining that the subject suffers from the cTnI-related disease; if the antigen is not present in the test sample, or the antigen is present in the test sample and the level of the antigen is lower than that in the control sample, determining that the subject does not suffer from the cTnI-related disease.

[0022] To achieve the above objectives, according to a ninth aspect of the present disclosure, a method for detecting cTnI is provided, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with cTnI in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0023] To achieve the above objectives, the present disclosure also provides a nucleic acid, a vector, a cell and a method for preparing the above antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 is a standard curve of the clinical relevance of the detection of Beckman-defined cTnI samples by the luminescence platform Anti-CTNI 8H5RMb6. DETAILED DESCRIPTION

[0026] In a first aspect, the presently disclosed embodiments provide an anti-cTnI antibody, comprising three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 22, 23, 24, and 25, and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 30, 31, 32, and 33.

[0027] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody described in the first aspect.

[0028] For example, the HCDR1, HCDR2, and HCDR3 have amino acid sequences consistent with those of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in SEQ ID NO: 22; and the LCDR1, LCDR2, and LCDR3 have amino acid sequences consistent with those of LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in SEQ ID NO: 31.

[0029] In the present disclosure, the term "antibody" is used in the broadest sense, and may include full-length monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity. Antigen-binding fragments include any one of F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv, and generally have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art based on the contents of the present disclosure that the above-mentioned antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present disclosure, it is easy for those skilled in the art to obtain the antigen-binding fragments of the above-mentioned antibodies.

[0030] Antigen-binding fragments can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0031] In the present disclosure, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more, or even all, of the primary amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of the antibodies.

[0032] In the present disclosure, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0033] Methods for defining CDRs are well known in the art, and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200,255,854,554, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes, but may still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literature vary slightly. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0034] Table 1: CDR Definition 1 1The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+number" and amino acid numbers on the light chain represented by "L+number." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software. 3 If both H35A and H35B are absent, HCDR1 ends at position 35; if only H35A is present, HCDR1 ends at position 35A; if both H35A and H35B are present, HCDR1 ends at position 35B. 4 If both H35A and H35B are absent, HCDR1 ends at position 32; if only H35A is present, HCDR1 ends at position 33; if both H35A and H35B are present, HCDR1 ends at position 34. 5 If both H35A and H35B are absent, HCDR1 ends at position 33; if only H35A is present, HCDR1 ends at position 34; if both H35A and H35B are present, HCDR1 ends at position 35.

[0035] According to an embodiment of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0036] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0037] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0038] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

[0039] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0040] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0041] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0042] According to an embodiment of the present disclosure, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, Chothia, AbM or IMGT systems are as follows:

[0043] In a second aspect, the present disclosure provides an anti-cTnI antibody, comprising the following complementarity determining regions:

[0044] HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0045] HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0046] HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0047] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4.

[0048] LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5.

[0049] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0050] According to an embodiment of the present disclosure, the HCDRs and LCDRs are defined by the Kabat system.

[0051] In the present disclosure, "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0052] In the present disclosure, the heavy chain variable region is obtained by arranging and connecting the following CDRs and FRs with the following numbers in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following CDRs and FRs with the following numbers in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0053] In an alternative embodiment, the antibody of the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0054] In an alternative embodiment, the HFR1 comprises / is SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0055] The HFR2 comprises / is as SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0056] The HFR3 comprises / is SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0057] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0058] The LFR1 comprises / is SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0059] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0060] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and

[0061] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0062] It should be noted that, in other embodiments, the amino acid sequence of each framework region of the anti-cTnI antibody provided herein may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0063] In an optional embodiment, the HFR1 includes / is represented by an amino acid sequence as shown in any one of SEQ ID NOs: 17, 18, and 19.

[0064] In an optional embodiment, the LFR1 comprises / is represented by the amino acid sequence shown in SEQ ID NO:20.

[0065] In an optional embodiment, the HFR2 comprises / is represented by the amino acid sequence shown in SEQ ID NO:21.

[0066] In an alternative embodiment, the antibody has a KD < 4.04×10 -8 M's affinity binds to cTnI.

[0067] In an alternative embodiment, the antibody has a KD ≤ 10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M or KD≤10 -12 M binds cTnI with high affinity.

[0068] In an alternative embodiment, the antibody has a KD ≤ 4.70×10 -9 M binds cTnI with high affinity.

[0069] There are many methods for determining antibody affinity (KD). Based on the detection principle, they can be divided into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods. Among them, thermodynamic detection methods such as isothermal titration calorimetry (ITC) are common; kinetic detection methods such as surface plasmon resonance (SPR) and biofilm interferometry (BLI) are common; and dynamic equilibrium detection methods such as enzyme-linked immunosorbent assay (ELISA) are common.

[0070] In alternative embodiments, KD is determined using a kinetic assay; optionally, surface plasmon resonance, for example, by using a kinetic assay such as System of biosensor systems.

[0071] In a third aspect, the presently disclosed embodiments provide an anti-cTnI antibody comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NOs: 22, 23, 24, and 25, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NOs: 30, 31, 32, and 33.

[0072] In an optional embodiment, the heavy chain variable region and the light chain variable region of the first aspect or the third aspect are selected from any one of the following combinations:

[0073] In an optional embodiment, the antibody described in the first, second, and third aspects above further comprises a constant region.

[0074] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0075] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0076] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0077] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0078] In an alternative embodiment, the light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0079] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0080] In an alternative embodiment, the species origin of the constant region is sheep.

[0081] In this article, the division of variable and constant region sequences refers to the IMGT division method, see Lefranc, M.-P. and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGHC,IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. Variable regions divided by different methods may differ from the variable region C-terminus or constant region N-terminus divided by IMGT. Variable regions or constant regions divided by other methods known in the art are also within the scope of protection of this disclosure.

[0082] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO:15, and the light chain constant region (CL) sequence is shown as SEQ ID NO:16.

[0083] It should be noted that, in other embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0084] In an alternative embodiment, the antibody comprises any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv.

[0085] In a fourth aspect, the present disclosure provides an anti-cTnI antibody, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 26, 27, 28, and 29, and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 34, 35, 36, and 37.

[0086] In an optional embodiment, the antibody of the first, second, third or fourth aspect comprises any combination of the following heavy chains and light chains:

[0087] In a fifth aspect, the present disclosure provides an antibody conjugate comprising the above-mentioned antibody.

[0088] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0089] In an optional embodiment, the antibody conjugate further comprises a label or purification tag coupled to the antibody.

[0090] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0091] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0092] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present disclosure.

[0093] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0094] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0095] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0096] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0097] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0098] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0099] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0100] In an optional embodiment, the colloidal metal is colloidal gold.

[0101] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody.

[0102] In an alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0103] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0104] In a sixth aspect, the present disclosure provides a reagent or a kit, which comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0105] As previously mentioned, the antibodies disclosed in some embodiments or examples of the present disclosure are capable of effectively binding to cTnI. Therefore, reagents or kits comprising such cTnI antibodies are capable of effectively performing qualitative or quantitative detection of cTnI. The reagents or kits provided herein can be used, for example, in immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of cTnI and its antibodies. As previously mentioned, the antibodies disclosed in some embodiments or examples of the present disclosure have higher binding activity or affinity for cTnI. Therefore, reagents or kits comprising such antibodies have higher detection sensitivity or specificity.

[0106] In a seventh aspect, the present disclosure provides a method for detecting cTnI, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with cTnI in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0107] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody.

[0108] In an alternative embodiment, the immune complex further comprises a second antibody, which binds to cTnI.

[0109] In an eighth aspect, the present disclosure provides uses of the aforementioned anti-cTnI antibodies, antibody conjugates, or reagents or kits for detecting cTnI, or diagnosing cTnI-related diseases, or preparing products for detecting cTnI.

[0110] In an optional embodiment, the cTnI-related diseases include but are not limited to myocardial injury, myocardial failure, myocardial infarction, acute coronary syndrome, left ventricular hypertrophy in essential hypertension, myocardial lysis in non-ischemic heart failure, skeletal muscle injury with myocardial injury, or chronic renal failure with myocardial injury.

[0111] It should be noted that the products disclosed herein include but are not limited to reagents, test kits, test strips or reagent plates.

[0112] In a ninth aspect, the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody.

[0113] In a tenth aspect, the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.

[0114] In an eleventh aspect, the present disclosure provides cells containing the above-mentioned vector.

[0115] In a twelfth aspect, the present disclosure provides a method for preparing an anti-cTnI antibody, comprising: culturing the cells as described above.

[0116] In the present application, term " vector " refers to a kind of vehicle that can operatively insert genetic element (for example aforementioned nucleic acid molecule) and make this genetic element obtain expression, for example, produce the protein, RNA or DNA encoded by this genetic element, or copy described genetic element.Carrier can be used for transforming, transducing or transfecting host cell, so that the genetic element that it carries is expressed in host cell.Described carrier can comprise the carrier that is mainly used for DNA or RNA is inserted into cell, the carrier that is mainly used for copying DNA or RNA, and the carrier of expression that is mainly used for transcribing and / or translating DNA or RNA.Described carrier also comprises the carrier with multiple above-mentioned function.Described carrier can be the polynucleotide that can be transcribed and translated into polypeptide when introducing suitable cell or host.Usually, by cultivating suitable cell or host that comprise described carrier, described carrier can produce desired expression product.

[0117] As used herein, the term "cell" generally refers to cells obtained by modifying or recombining the genetic material of host cells using genetic engineering techniques or cell fusion techniques to obtain cells with unique traits that are stably inherited. The term "cell" refers to prokaryotic or eukaryotic cells into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art, followed by the use of these cells to express the antibodies provided by the present invention, and the cells are cultured to obtain the corresponding antibodies.

[0118] In a thirteenth aspect, the present disclosure provides use of the above-mentioned antibody, antibody conjugate, or the above-mentioned reagent or kit in detecting cTnI or indicating a cTnI-related disease.

[0119] In a fourteenth aspect, the present invention provides a method for diagnosing whether a subject has a cTnI-related disease, comprising:

[0120] a) contacting the above-mentioned antibody, or antibody conjugate, or the above-mentioned reagent or kit with cTnI in a test sample from a subject under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen or the presence or status of the cTnI-related disease in the subject; and c) if the antigen is present in the test sample, or the antigen is present in the test sample and the level of the antigen is higher than that in a control sample, determining that the subject has the cTnI-related disease; if the antigen is not present in the test sample, or the antigen is present in the test sample and the level of the antigen is lower than that in a control sample, determining that the subject does not have the cTnI-related disease.

[0121] In an optional embodiment, the immune complex further comprises a second antibody, which binds to the above-mentioned antibody;

[0122] In an alternative embodiment, the immune complex further comprises a second antibody, which binds to cTnI.

[0123] In an optional embodiment, the cTnI-related disease of the thirteenth aspect or the fourteenth aspect is selected from myocardial infarction.

[0124] Based on the amino acid sequence of the anti-cTnI antibody disclosed in the present disclosure, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-cTnI antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells that can recombinantly express the antibody as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the anti-cTnI antibody of the present disclosure, it falls within the scope of protection of the present disclosure.

[0125] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0126] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0127] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0128] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0129] Example 1 Antibody Discovery of Monoclonal Antibodies

[0130] 1. Sheep Immunity

[0131] An emulsified preparation of CTNI recombinant antigen (from Feipeng Bio) prepared with incomplete Freund's adjuvant was injected subcutaneously to stimulate the immune response of sheep, and pre- and post-immunization sera were collected on days 0, 14, 28, 42, and 69, respectively; peripheral blood was drawn from the sheep to prepare cell suspensions.

[0132] 2. Construction of phage library

[0133] RNA was extracted from sheep peripheral blood cells and reverse-transcribed into cDNA. Using specifically designed sheep antibody gene amplification primers and cDNA as templates, the heavy-chain variable region (VH) and light-chain variable region (VL) gene fragments were amplified. The VH and VL gene fragments were then sequentially inserted into the phage vector V02 (developed in our laboratory) using enzyme digestion and ligation. Finally, the ligated phage plasmids were electroporated into TG1 competent cells. The following day, single clones were selected for PCR identification and antibody gene sequencing to assess the quality of the phage library. Only qualified clones were screened for phage library screening.

[0134] 3. Screening of phage libraries

[0135] Inoculate the TG1 phage library into a shake flask and, when the culture reaches an appropriate concentration (OD600 of 0.8-1.0), add helper phage for infection for one hour. Then, continue incubating overnight. The next day, collect the culture and centrifuge. The supernatant is then purified using salting-out precipitation to obtain the displayed phage library.

[0136] The phage library was panned for 3-4 rounds using the magnetic bead panning method. Then, monoclonal phage-infected colonies were selected for antibody supernatant expression. The monoclonal phage antibody expression supernatant was then screened and identified using the ELISA screening method to obtain anti-CTNI monoclonal phage.

[0137] 4. Phage antibody gene sequencing:

[0138] The anti-CTNI monoclonal phage was sequenced for antibody genes. Through sequence analysis, duplicate and invalid sequences were removed to obtain a unique sheep monoclonal antibody sequence. The obtained anti-CTNI sheep monoclonal antibody sequence was then verified by eukaryotic recombinant expression.

[0139] Example 2 Preparation of monoclonal antibodies

[0140] 1. Construction of recombinant antibody expression plasmid

[0141] pcDNA TM 3.4 Vector is a constructed recombinant antibody eukaryotic expression vector, which has been modified to introduce multiple cloning restriction enzyme sites and is subsequently referred to as 3.4A expression vector. Based on the variable region genes obtained in the above experiment, VL and VH gene-specific primers were designed, with restriction endonuclease sites and protective bases at both ends, respectively. PCR amplification was used to amplify a 0.72Kb light chain gene fragment and a 1.40Kb heavy chain gene fragment.

[0142] The heavy chain and light chain gene fragments and the 3.4A vector were double-digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the heavy chain gene and the light chain gene were respectively connected to the 3.4A expression vector to obtain recombinant expression plasmids of the heavy chain and light chain, respectively.

[0143] 2. Recombinant Antibody Preparation

[0144] Resuscitate HEK293 cells in advance and subculture them into 200 mL system to make the cell density reach 3-5×10 6 cells / ml cell density reaches the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 Cells were washed with 100 cells / ml and re-dissolved in culture medium, which was also used as a cell diluent. Plasmid DNA and transfection reagent diluents were prepared separately using culture medium. The transfection reagent diluent was added to the plasmid DNA diluent, mixed thoroughly, and allowed to stand at room temperature for 15 minutes. This mixture was slowly added to the cell diluent over 1 minute, mixed thoroughly, and samples were taken and counted. The viability of the cells after transfection was recorded and observed, and the cells were placed in a 35°C constant temperature incubator at 120 rpm and 8% CO2. After 13 days, the samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity chromatography column to obtain purified antibodies.

[0145] The resulting antibody was named Anti-CTNI 8H5Rmb1. Anti-CTNI 8H5Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table:

[0146] Table 2: Antibody sequences

[0147] Example 2 Performance testing of antibodies

[0148] 1. Affinity Analysis

[0149] Purified antibody was diluted in advance, and recombinant cTnI antigen (from Feipeng Bio) was serially diluted. Binding and dissociation curves were measured on a Biacore 8K+ instrument using a CM5 chip pre-coupled with mouse anti-goat IgG. The instrument automatically fitted the affinity constant, association rate, and dissociation rate. (KD represents the equilibrium dissociation constant, also known as the affinity constant; ka represents the association rate; and kd represents the dissociation rate.)

[0150] Table 3: Affinity data

[0151] 2. Activity Identification

[0152] cTnI recombinant antigen (from Feipeng Bio) was diluted to 3 μg / ml in coating solution (main component NaHCO3), 100 L per well, and incubated at 4°C overnight; the next day, the cells were washed twice with washing solution (main component Na2HPO4+NaCl) and patted dry; blocking solution (20% BSA+80% PBS) was added at 120 μL per well and incubated at 37°C for 1 hour, and patted dry; diluted purified antibody and control antibody were added at 100 μL / well and incubated at 37°C for 30 minutes; the cells were washed five times with washing solution and patted dry; mouse anti-sheep IgG-HRP was added at 100 μL per well and incubated at 37°C for 30 minutes; the cells were washed five times with washing solution and patted dry; colorimetric solution A (50 μL / well) and colorimetric solution B (50 μL / well) were added for 10 minutes; stop solution was added at 50 μL / well; and the OD value was read at 450 nm (reference 630 nm) on a microplate reader.

[0153] Note: Solution A (main ingredients: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main ingredients: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0154] Table 4: Activity data

[0155] 3. Stability assessment

[0156] The above-mentioned antibodies were placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected on days 7, 14, and 21 for status observation, and the 21-day sample was tested for activity. Table 5 below shows the OD results of the enzyme immunoassay for Anti-CTNI 8H5Rmb5 over 21 days.

[0157] Table 5: Stability data

[0158] Conclusion: The results showed that three Under the test conditions, no obvious changes in protein status were observed after the antibodies were placed for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies are stable.

[0159] 4. Antibody Performance in Chemiluminescence Assays (Exemplary Performance Test Results of Anti-CTNI 8H5RMb6)

[0160] 4.1 Antibody Coating Process

[0161] Wash 10mg / mL carboxyl magnetic beads three times with MES buffer. Resuspend the beads in MES buffer and add EDC to a final concentration of 1mg / mL. Mix on a shaker at 25°C and incubate for 30 minutes. Resuspend the beads in MES buffer and add Anti-CTNI-A (from Feipeng Bio) to a final concentration of 0.2mg / mL. This solution serves as the magnetic particle working solution. Mix on a shaker at 37°C and incubate for 120 minutes. Store in Tris buffer at 2-8°C.

[0162] 4.2 Acridinium ester labeling antibody process

[0163] Anti-CTNI 8H5RMb6 antibody was desalted using a Zeba desalting column (10K MWCO) and replaced with PBS (100 mM PB, 50 mM NaCl, pH 8.0). Acridinium ester was prepared in DMSO to a 4 mM solution. The antibody solution and acridinium ester solution were mixed at a 1:10 molar ratio and reacted at 25°C for 2 hours. Excess reagents were removed by desalting, and glycerol was added to a final concentration of 50% before storage at -20°C.

[0164] 4.3 Detection process

[0165] Use diluent to dilute the magnetic bead working solution from step 4.1 to 0.2 mg / mL. Dilute the acridinium ester working solution from step 4.2 to 0.5 μg / mL. Detection was performed on a Yingkai Shine i2910 fully automatic chemiluminescence immunoassay using a double antibody sandwich assay. To perform the assay, 100 μL of sample and 50 μL of magnetic bead working solution were added sequentially. After incubation for 10 minutes, the sample was washed and then 50 μL of acridinium ester working solution was added. Mix thoroughly and incubate for 10 minutes. After incubation, the reaction mixture was rinsed, and the pre-excitation solution and the excitation solution were added. Relative luminescence units (RLU) were measured, and the corresponding concentration values ​​were calculated using a four-parameter fitting method.

[0166] Among them, the samples include:

[0167] ① Samples without cTnI;

[0168] ② Beckman cTnI fixed value sample;

[0169] ③ Samples containing 1000 ng / mL of skeletal muscle troponin I (sTnI), troponin C (TnC), and troponin T (TnT) in the diluent matrix and plasma matrix were all from Feipeng Bio.

[0170] 4.4 Test results

[0171] (1) Blank limit: The sample without cTnI was tested 60 times, and the average value (AV) and standard deviation (SD) were calculated. The corresponding concentration and blank limit were calculated. The test results are shown in Table 6. The results show that the blank limit of the reagent composed of the antibody Anti-CTNI 8H5RMb6 is 0.15 pg / mL, which has high sensitivity.

[0172] Table 6: Test results of samples without cTnI

[0173] (2) Clinical relevance:

[0174] The paired antibody reagent consisting of Anti-CTNI 8H5RMb6 as the labeled antibody and Anti-CTNI-A as the coating antibody was used on the luminescence platform to detect samples with Beckman cTnI values. The test results are shown in Table 7, and the clinical relevance standard curve is shown in Figure 1. The results showed that the use of Anti-CTNI 8H5RMb6 on the luminescence platform had high sensitivity and clinical relevance, with a correlation coefficient R 2 =0.9928.

[0175] Table 7: Test results of Beckman fixed value samples on the luminescence platform

[0176] (2) Cross-reactivity:

[0177] The cross-reactivity detected using the reagent composed of the antibody Anti-CTNI 8H5RMb6 is shown in Table 8. The results show that in the diluent matrix and plasma matrix, the cross-reactivity of the reagent composed of the antibody Anti-CTNI 8H5RMb6 with skeletal muscle troponin I (sTnI) was less than 0.5%, and the cross-reactivity with both troponin C (TnC) and troponin T (TnT) was less than 0.1%, indicating low cross-reactivity and good specificity.

[0178] Table 8: Cross-reactivity results

[0179] Some of the amino acid sequences involved in this application are shown in Table 9:

[0180] Table 9: Amino acid sequence listing Industrial Applicability

[0181] The anti-cTnI antibodies provided in the present disclosure can specifically recognize and bind to cTnI, have high detection sensitivity and specificity, and can be used for the detection of cTnI. Therefore, the anti-cTnI antibodies provided in the present disclosure have excellent practical performance and broad market application prospects.

[0182] The foregoing description is merely a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure.

Claims

1. An anti-cTnI antibody, comprising three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 22, 23, 24, and 25, and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 30, 31, 32, and 33.

2. The antibody according to claim 1, characterized in that The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems, or a combination of multiple systems.

3. An anti-cTnI antibody, characterized in that: The antibody comprises the following complementarity determining regions: HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1; HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2; HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3; LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 6; Optionally, the antibody further comprises HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; optionally, the HFR1 comprises SEQ ID NO: 7 or an amino acid sequence at least 80% identical thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence at least 80% identical thereto; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto; Optionally, the antibody has a KD of <4.04×10 -8 M binds cTnI with high affinity.

4. An anti-cTnI antibody comprising a heavy chain variable region and / or a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 22, 23, 24, and 25; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 30, 31, 32, and 33; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations: Optionally, the antibody further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is sheep; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto.

5. An anti-cTnI antibody comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 26, 27, 28, and 29; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 34, 35, 36, and 37.

6. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a marker or purification tag coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

7. A reagent or kit, characterized in that: The reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 in detecting cTnI, diagnosing cTnI-related diseases, or preparing a product for detecting cTnI.

9. The use according to claim 8, characterized in that The cTnI-related diseases include myocardial injury, myocardial failure, myocardial infarction, acute coronary syndrome, left ventricular hypertrophy due to essential hypertension, myocardial lysis due to non-ischemic heart failure, skeletal muscle injury accompanied by myocardial injury, or chronic renal failure accompanied by myocardial injury.

10. A method for diagnosing whether a subject has a cTnI-related disease, characterized in that: The method comprises: a) contacting the antibody of any one of claims 1 to 5, the antibody conjugate of claim 6, or the reagent or kit of claim 7 with cTnI in a test sample from a subject under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample; and c) if the antigen is present in the test sample, or the antigen is present in the test sample and the level of the antigen is higher than that in the control sample, determining that the patient suffers from the cTnI-related disease; if the antigen is not present in the test sample, or the antigen is present in the test sample and the level of the antigen is lower than that in the control sample, determining that the patient does not suffer from the cTnI-related disease; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to cTnI.

11. A method for detecting cTnI, characterized in that: The method comprises: a) contacting the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with cTnI in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to cTnI.

12. A nucleic acid, characterized in that The nucleic acid encodes the antibody according to any one of claims 1 to 5.

13. A carrier, characterized in that The vector comprises the nucleic acid of claim 12.

14. A cell, characterized in that The cell comprises the nucleic acid of claim 12 or the vector of claim 13.

15. A method for preparing the antibody according to any one of claims 1 to 5, characterized in that: The method comprises culturing the cell of claim 14.

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