Anti-cTnl antibodies and test kits

Abstract

The application discloses an anti-cTnI antibody and a detection kit, and relates to the technical field of antibodies.The anti-cTnI antibody disclosed by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the antibody provides an important raw material source for detection of cTnI, and can be used for qualitative or quantitative detection of cTnI.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202211678406.6, filed on December 26, 2022, entitled "Anti-cTnI antibody or a functional fragment thereof and a detection kit", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and more specifically, to anti-cTnI antibodies and detection kits. Background Technology

[0004] Cardiac troponin (CTN) is a regulatory protein for cardiac muscle contraction. cTn is composed of subunits of three different genes: cardiac troponin T (CTNT), cardiac troponin I (cTnI), and troponin C (TNC).

[0005] Cardiac troponin I (cTnI) is found only in the myocardium and is a marker of cardiomyocytes. Abnormal changes in cTnI can affect the systolic and diastolic functions of the heart and can be used to diagnose myocardial necrosis and assess myocardial injury. It has become one of the most sensitive and specific markers of myocardial cell injury and is a recognized major biochemical marker for the rapid diagnosis of acute myocardial infarction (AMI) and acute coronary syndrome (ACS), as well as for assisting in ACS risk stratification and reflecting its prognosis.

[0006] When the integrity of myocardial cell membranes is disrupted due to ischemia or hypoxia, free cTnI can rapidly permeate 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 are of significant clinical importance for the diagnosis of acute myocardial infarction, risk stratification of acute coronary syndrome, and monitoring of myocardial damage caused by various factors.

[0007] Clinically, methods for detecting cTnI levels include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, and colloidal gold assays, each requiring antibodies targeting cTnI. Therefore, there is a strong demand in this field for high-performance cTnI antibodies. Summary of the Invention

[0008] The purpose of this invention is to provide anti-cTnI antibodies and detection kits.

[0009] In a first aspect, embodiments of the present invention provide an 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 the amino acid sequence shown in SEQ ID NO:1 or is based thereon with any one or more of the following mutations: K in K30 is mutated to S, D in D55 is mutated to N, E in E57 is mutated to S, E in E63 is mutated to A, and Q in Q65 is mutated to R or V; the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:2 or is based thereon with the following mutation: S in S96 is mutated to T; the heavy chain variable region and the light chain variable region have at least one of the above mutations.

[0010] Secondly, embodiments of the present invention provide an antibody comprising CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3, wherein: CDR-VH1, CDR-VH2, and CDR-VH3 comprise or have amino acid sequences consistent with CDR1, CDR2, and CDR3 in the heavy chain variable region described in the foregoing embodiments; and CDR-VL1, CDR-VL2, and CDR-VL3 comprise or have amino acid sequences consistent with CDR1, CDR2, and CDR3 in the light chain variable region described in the foregoing embodiments.

[0011] Thirdly, embodiments of the present invention provide an antibody conjugate comprising the antibody described in the foregoing embodiments.

[0012] Fourthly, embodiments of the present invention provide a reagent or kit comprising the antibody or antibody conjugate described in the foregoing embodiments.

[0013] Fifthly, embodiments of the present invention provide a method for detecting cTnI, comprising: mixing the antibody described in the foregoing embodiments with a sample to be tested, so that the antibody comes into contact with cTnI in the sample to be tested to form an immune complex.

[0014] Sixthly, embodiments of the present invention provide an isolated nucleic acid that encodes the antibody described in the foregoing embodiments.

[0015] In a seventh aspect, embodiments of the present invention provide a carrier containing the isolated nucleic acid described in the foregoing embodiments.

[0016] Eighthly, embodiments of the present invention provide a cell containing the isolated nucleic acid described in the foregoing embodiments or the vector described in the foregoing embodiments.

[0017] Ninthly, embodiments of the present invention provide a method for preparing the antibodies described in the foregoing embodiments, comprising: culturing the cells described in the foregoing embodiments.

[0018] In a tenth aspect, embodiments of the present invention provide the use of antibodies, antibody conjugates, or reagents or kits as described in the foregoing embodiments in detecting cTnI or preparing products for detecting cTnI.

[0019] In one aspect, embodiments of the present invention provide a method for screening antibodies against cTnI, the method comprising:

[0020] a) Design primers to replace at least one amino acid site of CDR-VH1, CDR-VH2 and CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 as described in the foregoing embodiments;

[0021] b) Using the nucleic acid, vector, or cell described in the foregoing embodiments as templates, construct a mutant library using the primers described in a);

[0022] c) Screen for anti-cTnI antibodies from the mutant library.

[0023] In a twelfth aspect, embodiments of the present invention provide a mutant library, the mutant library comprising the antibodies described in the foregoing embodiments.

[0024] The anti-cTnI antibody disclosed in this invention includes the above-mentioned heavy chain complementarity-determining region and light chain complementarity-determining region. This antibody provides an important source of raw materials for the detection of cTnI and has improved activity or affinity. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0026] This invention provides an 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 the amino acid sequence shown in SEQ ID NO:1 or is based thereon with any one or more of the following mutations: K in K30 is mutated to S, D in D55 is mutated to N, E in E57 is mutated to S, E in E63 is mutated to A, and Q in Q65 is mutated to R or V.

[0027] The amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:2 or based on it with the following mutation: S in S96 is mutated to T;

[0028] The heavy chain variable region and the light chain variable region have at least one of the above-mentioned mutations.

[0029] It should be noted that the mutation sites in this article are numbered by numbering the amino acid sequences shown in SEQ ID NO:1 or SEQ ID NO:2 from the N-terminus to the C-terminus. For example, position 30 refers to the 30th position of the amino acid sequence shown in SEQ ID NO:1 starting from the N-terminus; "K30S" means that the lysine at position 30 of the amino acid sequence shown in SEQ ID NO:1 is replaced by serine.

[0030] In this invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or functional fragments, as long as they exhibit the desired antigen-binding activity.

[0031] In this article, the terms "full-length antibody," "full-length monoclonal antibody," or "full-length monoclonal antibody" refer to antibody molecules composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). They typically consist of a lighter light chain and a heavier heavy chain, with the heavy chain (H chain) and light chain (L chain) linked by disulfide bonds. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies considerably and is called the variable region (V region); the carboxyl terminus (C-terminus) is relatively stable and changes very little, and is called the constant region (C region). The V regions of the L chain and H chain are referred to as VL and VH, respectively.

[0032] In this document, the terms "polyclonal antibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes. For example, antibodies that recognize two antigenic epitopes (bispecific antibodies, or simply biantibodies), three antigenic epitopes, or four antigenic epitopes are used in a broad sense, and their specific structures are not limited, as long as they can recognize multiple antigenic epitopes. In this invention, at least one of the multiple antigenic epitopes is derived from cTnI.

[0033] In this document, the term "functional fragment" refers to a fragment containing part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but still possesses the performance activity of specifically binding to an antigen. For example, the fragment may contain part or all of an antibody CDR. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include Fv fragments, disulfide-stabilized Fv fragments (dsFv), F(ab')2 fragments, Fab' fragments, Fab fragments, F(ab)2 fragments, scFv fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, Fv-Fc fusion proteins, multispecific antibodies formed from functional fragments, single-domain antibodies, VHH nanobodies, domain antibodies, bivalent domain antibodies, or at least one of the smallest recognition units. Such fragments can be generated by recombinant nucleic acid technology or by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0034] In this paper, the term "at least 80% homology" refers to at least 80% homology with each reference sequence, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. The term "at least 90% homology" refers to at least 90% homology with each reference sequence, which can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0035] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of the following mutation combinations 1 to 31:

[0036]

[0037]

[0038] The amino acid sequence of the wild-type WT in the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the wild-type WT in the light chain variable region is shown in SEQ ID NO: 2.

[0039] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 1 to 17 and 19 to 31.

[0040] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 1-4, 5-17, 20, 24-31.

[0041] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 1-2, 4, 5-8, 10-13, 15, 20, 24-31.

[0042] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 1 to 2, 4, 5, 10, 15, 24 to 31.

[0043] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of mutation combinations 1, 4, 5, 15, 25 to 31.

[0044] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 25 to 31.

[0045] On the other hand, embodiments of the present invention also provide an antibody comprising CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3, wherein:

[0046] CDR-VH1, CDR-VH2, and CDR-VH3 include or are amino acid sequences consistent with CDR1, CDR2, and CDR3 in the heavy chain variable region described in any of the foregoing embodiments;

[0047] CDR-VL1, CDR-VL2, and CDR-VL3 comprise or are amino acid sequences consistent with CDR1, CDR2, and CDR3 in the light chain variable region described in any of the foregoing embodiments.

[0048] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0049] In this invention, the heavy chain complementarity determination region is represented by CDR-VH, and the three CDRs contained in the heavy chain variable region include CDR-VH1 (HCDR1), CDR-VH2 (HCDR2), and CDR-VH3 (HCDR3); the light chain complementarity determination region is represented by CDR-, and the three CDRs contained in the light chain variable region include CDR-VL1 (LCDR1), CDR-VL2 (LCDR2), and CDR-VL3 (LCDR3).

[0050] Commonly used CDR numbering schemes in this field include: Kabat numbering, Chothia numbering, IMGT numbering, Chothia and Martin numbering, and AHo and Lesk numbering. CDR definition schemes include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, “Kabat numbering” and “Kabat definition” refer to the numbering and definition system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). For “Chothia definition,” see Chothia et al., J Mol Biol 196: 901-917 (1987). Exemplary defined CDRs are listed in Table 1 below. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR.

[0051] Table 1: CDR Definition 1

[0052] CDR Kabat <![CDATA[AbM 2 ]]> IMGT HCDR1 31-35 26-35 26-35 HCDR2 50-65 50-58 51-56 HCDR3 95-102 95-102 93-102 LCDR1 24-34 24-34 27-32 LCDR2 50-56 50-56 50-51 LCDR3 89-97 89-97 89-97

[0053] 1 The numbers for all CDR definitions in Table 1 are based on the Kabat numbering system (see below).

[0054] As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0055] The polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system. However, those skilled in the art are fully capable of converting the sequence listing sequence numbers to Kabat numbers.

[0056] In some embodiments, the CDR1 to CDR3 in the heavy chain variable region and / or the light chain variable region are defined by any one of the schemes Kabat, Chothia, AbM, Contact, and IMGT, or by a combination of multiple schemes.

[0057] In some embodiments, CDR-VH1, CDR-VH2, and CDR-VH3 sequentially comprise or are amino acid sequences at positions 26-35, 50-65, and 95-102 of the heavy chain variable region under Kabat numbering; CDR-VL1, CDR-VL2, and CDR-VL3 respectively comprise or are amino acid sequences at positions 24-34, 50-56, and 89-97 of the light chain variable region under Kabat numbering. It should be noted that CDRs defined by other methods not limited to those in Table 1 are also within the scope of this disclosure.

[0058] In some embodiments, CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 comprise or are as shown in the following amino acid sequences:

[0059] CDR-VH1: GFNI-X1-DYYMH;

[0060] CDR-VH2: RIDPE-X2-G-X3-TKYAP-X4-F-X5-G;

[0061] CDR-VH3: YYSSYVPFVY;

[0062] CDR-VL1: RSSQSLIYSNRHTYLH;

[0063] CDR-VL2: QVSNRFS;

[0064] CDR-VL3: SQ-X6-THIPFT;

[0065] Where X1 is K or S, X2 is D or N, X3 is E or S, X4 is E or A, X5 is Q, R or V, and X6 is S or T;

[0066] X1~X6 excludes combinations of X1 / X2 / X3 / X4 / X5 / X6 that are K / D / E / E / Q / S; that is, X1, X2, X3, X4, X5, X6 are not simultaneously K, D, E, E, Q, S.

[0067] In some embodiments, X1 / X2 / X3 / X4 / X5 / X6 is selected from any one of the following combinations 1-31:

[0068]

[0069]

[0070] In some embodiments, X1 / X2 / X3 / X4 / X5 / X6 are selected from any one of combinations 1 to 17 and 19 to 31.

[0071] In some embodiments, X1 / X2 / X3 / X4 / X5 / X6 are selected from any one of combinations 1 to 4, 5 to 17, 20, and 24 to 31.

[0072] In some embodiments, X1 / X2 / X3 / X4 / X5 / X6 are selected from any combination of 1 to 2, 4, 5 to 8, 10 to 13, 15, 20, 24 to 31.

[0073] In some embodiments, X1 / X2 / X3 / X4 / X5 / X6 are selected from any combination of 1 to 2, 4, 5, 10, 15, 24 to 31.

[0074] In some embodiments, X1 / X2 / X3 / X4 / X5 / X6 are selected from any one of combinations 1, 4, 5, 15, 25 to 31.

[0075] In some embodiments, X1 / X2 / X3 / X4 / X5 / X6 are selected from any one of combinations 25 to 31.

[0076] In some embodiments, the heavy chain variable region and light chain variable region of the antibody further include a framework region (FR region).

[0077] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0078] In some embodiments, the antibody further includes at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4. At least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is derived from any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, and humans.

[0079] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0080] In some embodiments, the HFR1 comprises an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having at least 80% homology with it;

[0081] The HFR2 comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least 80% homology with it;

[0082] The HFR3 comprises an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 80% homology with it;

[0083] The HFR4 comprises an amino acid sequence as shown in SEQ ID NO:6 or an amino acid sequence having at least 80% homology with it;

[0084] The LFR1 comprises an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology with it;

[0085] The LFR2 comprises an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% homology with it;

[0086] The LFR3 comprises an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology with it;

[0087] The LFR4 comprises an amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology with it;

[0088] In some embodiments, the antibody includes the heavy chain variable region and / or light chain variable region described in any of the foregoing embodiments.

[0089] In some embodiments, the antibody further comprises a constant region. The constant region includes at least one of a heavy chain constant region and a light chain constant region.

[0090] In some embodiments, the species source of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, and humans.

[0091] In some embodiments, the species source of the constant region is mice.

[0092] In some embodiments, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0093] In some embodiments, the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

[0094] In some embodiments, the heavy chain constant region sequence is as shown in SEQ ID NO:11 or has at least 80% identity with it.

[0095] In some embodiments, the light chain constant region sequence is as shown in SEQ ID NO:12 or has at least 80% identity with it.

[0096] On the other hand, embodiments of the present invention also provide an antibody conjugate comprising the antibody described in any of the foregoing embodiments.

[0097] In some embodiments, the antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody.

[0098] In some embodiments, the antibody conjugate further includes a solid-phase support conjugated to the antibody. In the antibody conjugate, the antibody is conjugated to the solid-phase support.

[0099] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0100] In optional embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0101] In an optional embodiment, the solid support is a nitrocellulose membrane.

[0102] In some embodiments, the antibody conjugate further includes a marker conjugated to the antibody.

[0103] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0104] In some embodiments, the marker is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0105] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0106] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0107] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0108] In optional embodiments, 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.

[0109] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0110] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0111] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, dispersed dyes, dye-labeled microspheres, and latexes.

[0112] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold or colloidal silver.

[0113] In an optional embodiment, the colloidal metal is colloidal gold.

[0114] On the other hand, embodiments of the present invention also provide a reagent or kit comprising the antibody or antibody conjugate described in any of the foregoing embodiments.

[0115] As previously mentioned, the antibodies in some embodiments or examples of this invention can effectively bind to cTnI. Therefore, reagents or kits containing these antibodies can effectively perform qualitative or quantitative detection of cTnI. The reagents or kits provided by this invention can be used, for example, for detections involving the specific binding properties of cTnI and its antibodies, such as immunoblotting and immunoprecipitation. As previously mentioned, the antibodies in some embodiments or examples of this invention have higher binding activity or affinity for cTnI; therefore, reagents or kits containing these antibodies have higher detection sensitivity or specificity.

[0116] On the other hand, embodiments of the present invention also provide a method for detecting cTnI, which includes:

[0117] The antibody described in any of the foregoing embodiments is mixed with the sample to be tested, so that the antibody comes into contact with cTnI in the sample to be tested to form an immune complex.

[0118] In some embodiments, the presence of cTnI or the content of cTnI in the sample to be tested is determined based on the signal of the immune complex.

[0119] In some embodiments, the immune complex further includes a second antibody that binds to the antibody.

[0120] In some embodiments, the immune complex further includes a second antibody that binds to cTnI.

[0121] On the other hand, embodiments of the present invention also provide an isolated nucleic acid that encodes the antibody described in any of the foregoing embodiments.

[0122] On the other hand, embodiments of the present invention also provide a carrier containing the isolated nucleic acid described in any of the foregoing embodiments.

[0123] On the other hand, embodiments of the present invention also provide a cell containing the isolated nucleic acid or the vector described in any of the foregoing embodiments.

[0124] On the other hand, embodiments of the present invention also provide a method for preparing the antibodies described in any of the foregoing embodiments, which includes: culturing the cells described in any of the foregoing embodiments.

[0125] On the other hand, embodiments of the present invention also provide the application of antibodies, antibody conjugates, or reagents or kits as described in any of the foregoing embodiments in detecting cTnI or preparing products for detecting cTnI.

[0126] On the other hand, embodiments of the present invention also provide the use of the antibodies or antibody conjugates or reagents or kits described in any of the foregoing embodiments in the preparation of products having at least one of the following uses: diagnosing or assisting in the diagnosis of cTnI metabolism-related diseases, predicting or assisting in the prediction of the prognostic efficacy of cTnI metabolism-related diseases.

[0127] In an optional implementation, the cTnI metabolism-related diseases include at least one of myocardial injury, acute coronary syndrome, myocardial infarction (acute myocardial infarction), primary hypertension with left ventricular hypertrophy, non-ischemic heart failure with myocardial lysis, skeletal muscle injury with myocardial injury, or chronic renal failure with myocardial injury.

[0128] In an optional implementation, the product includes a reagent or a kit.

[0129] Based on the disclosure of the amino acid sequence of the antibody in this invention, those skilled in the art will readily conceive of preparing the antibody using genetic engineering or other techniques (chemical synthesis, recombinant expression), such as isolating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody of this invention, it falls within the protection scope of this invention.

[0130] On the other hand, embodiments of the present invention also provide a method for screening antibodies against cTnI, the method comprising:

[0131] a) Design primers for replacing at least one amino acid site of CDR-VH1, CDR-VH2 and CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 as described in any of the foregoing embodiments;

[0132] b) Using the nucleic acid, vector, or cell described in any of the foregoing embodiments as a template, construct a mutant library using the primers described in a);

[0133] c) Screen for anti-cTnI antibodies from the mutant library.

[0134] In some embodiments, the primers are used to replace amino acids at sites 1, 2, 3, 4, 5, or 6 of K30, D55, E57, E63, Q65, and S96 in any of the foregoing embodiments.

[0135] In some embodiments, the mutant library is a single-point saturation mutant library.

[0136] Furthermore, embodiments of the present invention also provide a mutant library, which includes the antibodies described in any of the foregoing embodiments.

[0137] In some embodiments, the mutant library is constructed by the steps of constructing a mutant library described in any of the foregoing embodiments.

[0138] The amino acid or nucleotide sequences involved in this article are shown in the table below. Mutation sites in the heavy chain variable region or HCDRs are referenced to the amino acid sequence shown in SEQ ID NO:1 or the HCDRs in SEQ ID NO:1, while mutation sites in the light chain variable region or LCDRs are referenced to the amino acid sequence shown in SEQ ID NO:2 or the LCDRs in SEQ ID NO:2. WT indicates no mutation.

[0139] amino acid sequence listing

[0140]

[0141]

[0142] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0144] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0145] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0146] In this embodiment, restriction endonucleases, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs, Taq DNA polymerase was purchased from TaKaRa, the V01 expression vector was constructed in our laboratory, gel extraction kits and plasmid extraction kits were commercially available, and primer synthesis and gene sequencing were outsourced. The cTnI monoclonal antibody (hereinafter referred to as WT antibody) sequence was obtained from mouse hybridoma cell sequencing.

[0147] Example 1: Construction and screening of affinity maturation mutant libraries

[0148] 1. Construction of WT template plasmid

[0149] (1) Antibody gene synthesis

[0150] The VH and VL sequences of the WT antibody were optimized using E. coli codons, and the antibody gene sequence was then outsourced to a company for gene synthesis. The VH and VL amino acid sequences of the WT antibody are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0151] (2) Amplification of WT antibody gene fragment

[0152] The synthesized antibody sequence was amplified by PCR using DNA polymerase, and then the antibody bands were separated by agarose gel electrophoresis. The antibody gene fragment was then purified using a gel recovery kit.

[0153] (3) Enzyme digestion and ligation of WT antibody gene fragment

[0154] The antibody gene fragment and the VO1 vector plasmid were simultaneously digested with restriction endonucleases, and then purified using a gel extraction kit to obtain the antibody gene fragment and VO1 vector with sticky ends. Next, the antibody gene fragment and VO1 vector were ligated with T4 DNA ligase at 22°C for 4 hours. The ligation product was recovered and purified, and the DNA concentration was measured. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 E. coli competent cells, and the entire bacterial culture was plated on ampicillin-resistant plates and incubated overnight at 37°C.

[0155] (4) Extraction and sequencing verification of WT template plasmid

[0156] The following day, 10 single-clone colonies were selected, and colony PCR and gel electrophoresis were performed using Taq DNA polymerase. The bacteria with the correct inserted antibody gene sequence were selected for culture and amplification. The plasmid template plasmid was obtained using a plasmid extraction kit and sent to a sequencing company for gene sequencing verification.

[0157] 2. Construction of single-point mutation libraries

[0158] (1) Primer design and synthesis

[0159] Using degenerate base codons, 69 pairs of upstream and downstream primers for single-point saturation mutations in the full CDR regions (69 amino acid sites) of VH and VL were designed and outsourced to a company for primer synthesis.

[0160] (2) PCR amplification of single-point saturation mutant plasmid

[0161] The PCR method was used, and the reaction system was prepared according to Table 2. Then, the PCR reaction conditions in Table 3 were used to amplify and prepare single-point saturated mutant library plasmids. Finally, the WT template plasmid was digested with restriction endonuclease at 37°C for 1 hour to obtain plasmids of 69 mutant libraries.

[0162] Table 2: PCR System

[0163] WT template plasmid 50ng DNA polymerase 1μl DNA polymerase buffer 10μl dNTP (2.5mM) 4μl Upstream primer (10 μM) 1μl Downstream primer (10uM) 1μl <![CDATA[ddH2O]]> Adjust the volume to 50 μl

[0164] Table 3: PCR reaction conditions

[0165] Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ time 5min 30s 30s 2min 5min ∞

[0166] Step 2 to Step 4: 22 cycles.

[0167] (3) Single-point saturation mutant plasmid transformation

[0168] Take 10 μl of the reaction product and transform it into 100 μl of TG1 Escherichia coli competent cells. Spread the entire bacterial culture onto an ampicillin-resistant plate and incubate overnight at 37°C.

[0169] 3. Screening of single-point mutation libraries

[0170] (1) Antibody expression in mutant library

[0171] The following day, 500 μl of culture medium was added to each 96-well plate beforehand. For each single-point mutation library, 92 single-clone colonies were selected, and WT, negative, and blank control colonies were set up. After incubation at 37°C for 5-6 hours, the bacterial culture was transferred to a new 96-well plate and incubated at 37°C for 1-2 hours. Finally, induction medium was added, and the plates were incubated overnight at 37°C to express antibodies, resulting in antibody expression supernatants from 69 mutant libraries.

[0172] (2) Mutant library screening and sequencing

[0173] Using a double-antibody sandwich method, CTNI antibody was added to 69 ELISA plates at a concentration of 0.0625 μg / ml, 100 μl / well, and incubated overnight at 4°C. The next day, the plates were blocked with 1%–2% skim milk powder. The antibody expression supernatant from the 69 mutant libraries was added to the wells of the ELISA plates at a concentration of 100 μl / well, and WT, negative, and blank controls were set up. After incubation at room temperature for 1 hour, ascites fluid containing CTNI was added to the wells of the ELISA plates at a concentration of 1:800, 100 μl / well, and incubated at room temperature for 1 hour. Then, HRP-labeled paired antibody was added, and the plates were washed, developed, and read using the standard ELISA detection method. The data results were then processed and analyzed, and clones with improved mutations were sent for sequencing. Finally, the sequencing results were analyzed, and the mutation sites of 7 unique mutant candidate clones were selected (see Table 4) for the construction of combined mutant libraries. (Ratio value explanation: The ratio value represents the degree of affinity enhancement. When the ratio value is equal to 1, it means that the affinity of the mutant clone is the same as that of WT; when the ratio value is greater than 1, it means that the affinity has been enhanced.)

[0174] Table 4: Screening results and mutation sites of candidate clones

[0175]

[0176] 4. Construction of combined mutant libraries

[0177] (1) Library primer design and synthesis

[0178] Based on the mutation sites on VH and VL, amplification primers for the combined mutant library were designed and outsourced to a company for primer synthesis.

[0179] (2) Fragment amplification and ligation

[0180] According to the PCR system in Table 2 and the PCR reaction conditions in Table 3, the antibody mutant fragments were amplified, and then the antibody mutant fragments were recovered by gel electrophoresis. The antibody mutant fragments were then spliced ​​into complete antibody fragments using the overlap PCR method.

[0181] Finally, the antibody fragment was inserted into the V01 vector using an enzyme digestion and ligation method to form a complete antibody expression plasmid: the method is the same as that for "WT antibody gene fragment digestion and ligation". 100 ng of the plasmid was transformed into 100 μl of TG1 E. coli competent cells, and the entire bacterial culture was plated on ampicillin-resistant plates and incubated overnight at 37°C.

[0182] 5. Screening of combined mutant libraries

[0183] Twenty-four randomly selected combined mutant antibodies were subjected to expression in the supernatant, ELISA screening, and sequencing analysis of positive clones. The Ratio values ​​are shown in Table 5.

[0184] Table 5: Information on candidate clones with combined mutations

[0185]

[0186] Example 2: Validation of antibody recombinant expression in eukaryotes

[0187] 1. Construction of eukaryotic recombinant expression plasmids

[0188] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the variable region gene sequences of the 24 candidate clones (see Table 5) obtained by screening the above combined mutant library, VL and VH gene-specific amplification primers and constant region (amino acid sequences of the heavy chain constant region and the light chain constant region are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively, wherein the N-terminus of the heavy chain constant region is connected to the C-terminus of VH to form the heavy chain, and the N-terminus of the light chain constant region is connected to the C-terminus of VL to form the light chain) overlap primers are designed. The primers at both ends are equipped with HindIII and EcoRI restriction sites and protective bases, respectively. The 0.73kb Light Chain gene fragment and the 1.40kb Heavy Chain gene fragment are amplified by PCR amplification.

[0189] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery, the Heavy Chain and Light Chain genes were ligated into the 3.4A expression vector and transformed into DH5α competent cells. After bacterial growth, single colonies were picked for PCR identification of positive clones. Positive clones were then sequenced to confirm sequence accuracy. Plasmids were extracted from correctly sequenced clones for later use.

[0190] 2. Sample preparation of recombinant antibodies

[0191] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6Cells / ml, cell viability >95%; centrifuge to wash cells, rehydrate with culture medium, and adjust cell density to 2.9 × 10⁶ cells / ml. 6 Cells / ml were used as cell dilution buffers. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. Antibodies were obtained by affinity purification using a protein A affinity chromatography column.

[0192] 3. Affinity Analysis

[0193] Wild-type antibodies (heavy chain amino acid sequence as shown in SEQ ID NO:13, light chain amino acid sequence as shown in SEQ ID NO:14), candidate clone antibodies with 7 single-site mutations obtained in step 3 of Example 1, and 24 mutant antibodies obtained in step 2 of Example 2 were subjected to affinity detection analysis. The specific steps were as follows: the binding and dissociation curves of the antigen and antibody were tested on a Biacore 8K+ instrument, and the instrument automatically fitted and obtained the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; the smaller the KD value, the higher the affinity; ka represents the binding rate; kd represents the dissociation rate). The results showed that the mutant antibodies had improved affinity for cTnI compared to the wild-type antibodies. Table 6 below shows the affinity detection results of some mutant antibodies as an example.

[0194] Table 6. Results of Antibody Affinity Detection

[0195]

[0196]

[0197] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody against cTnI, characterized in that, It includes: Heavy chain variable region and light chain variable region, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:1 or is based thereon with any one or more of the following mutations; The amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:2 or a mutation thereof; The heavy chain variable region and the light chain variable region are selected from any one of the following mutation combinations 1 to 31: The amino acid sequence of the wild-type WT in the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the wild-type WT in the light chain variable region is shown in SEQ ID NO:

2.

2. The antibody according to claim 1, characterized in that, The heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 1 to 17 and 19 to 31.

3. The antibody according to claim 2, characterized in that, The heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 1-4, 5-17, 20, 24-31.

4. The antibody according to claim 3, characterized in that, The heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 1-2, 4, 5-8, 10-13, 15, 20, 24-31.

5. The antibody according to claim 4, characterized in that, The heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 1-2, 4, 5, 10, 15, 24-31.

6. The antibody according to claim 5, characterized in that, The heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 1, 4, 5, 15, 25 to 31.

7. The antibody according to claim 6, characterized in that, The heavy chain variable region and the light chain variable region are selected from any one of the mutation combinations 25 to 31.

8. An antibody against cTnI, characterized in that, It includes CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3, among which: CDR-VH1, CDR-VH2, and CDR-VH3 are amino acid sequences identical to CDR1, CDR2, and CDR3 in the heavy chain variable region of any one of claims 1 to 7; CDR-VL1, CDR-VL2, and CDR-VL3 are amino acid sequences identical to CDR1, CDR2, and CDR3 in the light chain variable region of any one of claims 1 to 7; The CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 are defined by any one of the following schemes: Kabat, Chothia, AbM, Contact, and IMGT.

9. An antibody against cTnI, characterized in that, It includes CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3; wherein CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 are as shown in the following amino acid sequences: CDR-VH1: GFNI-X1-DYYMH; CDR-VH2: RIDPE-X2-G-X3-TKYAP-X4-F-X5-G; CDR-VH3: YYSSYVPFVY; CDR-VL1: RSSQSLIYSNRHTYLH; CDR-VL2: QVSNRFS; CDR-VL3: SQ-X6-THIPFT; X1 / X2 / X3 / X4 / X5 / X6 is selected from any one of the following combinations 1-31: 。 10. The antibody according to claim 9, characterized in that, X1 / X2 / X3 / X4 / X5 / X6 are selected from any one of combinations 1 to 17 and 19 to 31.

11. The antibody according to claim 10, characterized in that, X1 / X2 / X3 / X4 / X5 / X6 are selected from any combination of 1~4, 5~17, 20, 24~31.

12. The antibody according to claim 11, characterized in that, X1 / X2 / X3 / X4 / X5 / X6 are selected from any combination of 1~2, 4, 5~8, 10~13, 15, 20, 24~31.

13. The antibody according to claim 12, characterized in that, X1 / X2 / X3 / X4 / X5 / X6 are selected from any combination of 1~2, 4, 5, 10, 15, 24~31.

14. The antibody according to claim 13, characterized in that, X1 / X2 / X3 / X4 / X5 / X6 are selected from any one of combinations 1, 4, 5, 15, 25 to 31.

15. The antibody according to claim 14, characterized in that, X1 / X2 / X3 / X4 / X5 / X6 are selected from any one of combinations 25 to 31.

16. The antibody according to any one of claims 9 to 15, characterized in that, The antibody also includes at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

17. The antibody according to claim 16, characterized in that, At least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is derived from any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, and humans.

18. The antibody according to claim 16, characterized in that, The HFR1 includes an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having at least 80% homology with it; The HFR2 comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least 80% homology with it; The HFR3 comprises an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 80% homology with it; The HFR4 comprises an amino acid sequence as shown in SEQ ID NO:6 or an amino acid sequence having at least 80% homology with it; The LFR1 includes an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology with it; The LFR2 comprises an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% homology with it; The LFR3 comprises an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology with it; The LFR4 comprises an amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology with it.

19. The antibody according to any one of claims 1-15 and 17-18, characterized in that, The antibody also includes a constant region; The constant region includes at least one of the heavy chain constant region and the light chain constant region.

20. The antibody according to claim 19, characterized in that, The species source of the constant region is any one of the following: cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, and humans.

21. The antibody according to claim 20, characterized in that, The species source of the constant region is mice.

22. The antibody according to claim 19, characterized in that, The heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

23. The antibody according to claim 19, characterized in that, The light chain constant region includes light chain constant regions selected from κ-type or λ-type.

24. The antibody according to claim 19, characterized in that, The heavy chain constant region sequence is as shown in SEQ ID NO:11 or has at least 80% identity with it.

25. The antibody according to claim 19, characterized in that, The light chain constant region sequence is as shown in SEQ ID NO:12 or has at least 80% identity with it.

26. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises the antibody as described in any one of claims 1 to 25 and at least one of biotin, a solid-phase carrier, and a labeling agent conjugated thereto.

27. The antibody conjugate according to claim 26, characterized in that, The marker is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

28. The antibody conjugate according to claim 26, characterized in that, The marker is colloidal gold.

29. A reagent or kit, characterized in that, It includes the antibody as described in any one of claims 1 to 25 or the antibody conjugate as described in any one of claims 26 to 28.

30. The use of the antibody according to any one of claims 1 to 25 in the preparation of a product for detecting cTnI, characterized in that, The detection includes: mixing the antibody according to any one of claims 1 to 25 with the sample to be tested, so that the antibody comes into contact with cTnI in the sample to be tested to form an immune complex.

31. The application according to claim 30, characterized in that, Based on the signal from the immune complex, it is determined whether the sample to be tested contains cTnI or the amount of cTnI.

32. The application according to claim 30, characterized in that, The immune complex further includes a second antibody, which binds to the antibody.

33. The application according to claim 30, characterized in that, The immune complex also includes a second antibody that binds to cTnI.

34. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antibody according to any one of claims 1 to 25.

35. A carrier, characterized in that, The vector contains the isolated nucleic acid as described in claim 34.

36. A cell characterized by, The cell contains the isolated nucleic acid as described in claim 34 or the vector as described in claim 35.

37. A method for preparing the antibody according to any one of claims 1 to 25, characterized in that, The method includes: culturing the cells of claim 36.

38. The use of the antibody as described in any one of claims 1 to 25, the antibody conjugate as described in any one of claims 26 to 28, or the reagent or kit as described in claim 29 in the preparation of products for detecting cTnI or in non-diagnostic destinations for detecting cTnI.

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