Cardiac troponin I specific antibody, kit and application

Abstract

The invention provides an antibody specifically targeting cardiac troponin I. The invention further provides a preparation method of the antibody. The invention further provides an antibody pair comprising the antibody and a kit. The invention further provides the use of these antibodies to detect the level of cardiac troponin I and to diagnose myocardial damage.

Description

Technical Field

[0001] The present invention belongs to the field of molecular immunology and specifically relates to an antibody that specifically targets cardiac troponin I, an antibody pair comprising the antibody, a kit, and uses thereof. Background Art

[0002] Troponin (Tn) is a regulatory protein for muscle contraction. Located on the thin filaments of the contractile proteins, it plays a crucial role in regulating muscle contraction and relaxation. Troponin consists of three isoforms: fast-acting, slow-acting, and cardiac troponin (cTn). The first two are associated with skeletal muscle, while cardiac troponin is present only in cardiomyocytes. It is a complex composed of three subunits: cardiac troponin T (cTnT), cardiac troponin I (cTnI), and troponin C (TnC). Cardiac troponin is degraded from myocardial fibers and released into the circulation upon cardiomyocyte injury. Elevated cTn levels in the blood reflect myocardial cell damage. Cardiac troponin I is commonly used to detect myocardial injury, such as in myocardial infarction, coronary artery disease, myocarditis, and pericarditis.

[0003] Cummins et al. developed a method for measuring cardiac troponin I in serum (Cummins et al., Am Heart Journal 113:1333-1344 (1987)). However, the polyclonal antibody used in this method significantly cross-reacts with skeletal muscle troponin I (sTnI). Bodar et al. developed a sandwich ELISA method for measuring cardiac troponin I (Bodar et al., Clinical Chemistry 38:2203-2214 (1992); US 7285418), but its accuracy is low.

[0004] Therefore, there is a need for an immunoassay that can detect cardiac troponin I with high sensitivity and accuracy, and that does not cross-react with skeletal muscle troponin I. Such an immunoassay would allow treating physicians to use appropriate treatments to provide the best possible prognosis for affected patients. The present invention satisfies these needs. Summary of the Invention

[0005] In view of this, the present invention discloses an antibody or an antigen-binding fragment thereof that specifically targets cardiac troponin I, and related compositions, reagents, and methods.

[0006] In a first aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to cardiac troponin I, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region and the light chain comprises a light chain variable region;

[0007] in,

[0008] the heavy chain variable region comprises:

[0009] a. CDR-H1 comprising a sequence as set forth in any one of SEQ ID NOs. 1-11;

[0010] b. CDR-H2 comprising a sequence as set forth in any one of SEQ ID NOs. 12-22; and

[0011] c. CDR-H3 comprising a sequence as set forth in any one of SEQ ID NOs. 23-33;

[0012] the light chain variable region comprises:

[0013] a. CDR-L1 comprising a sequence as set forth in any one of SEQ ID NOs. 34-44;

[0014] b. CDR-L2 comprising a sequence as set forth in any one of SEQ ID NOs. 45-55; and

[0015] c. CDR-L3 comprising a sequence as set forth in any one of SEQ ID NOs. 56-66.

[0016] In some specific embodiments, the antibody or antigen-binding fragment thereof, wherein,

[0017] the heavy chain variable region comprises:

[0018] CDR-H1 as set forth in SEQ ID NO. 1, CDR-H2 as set forth in SEQ ID NO. 12, and CDR-H3 as set forth in SEQ ID NO. 23; or

[0019] CDR-H1 as set forth in SEQ ID NO. 2, CDR-H2 as set forth in SEQ ID NO. 13, and CDR-H3 as set forth in SEQ ID NO. 24; or

[0020] CDR-H1 as set forth in SEQ ID NO. 3, CDR-H2 as set forth in SEQ ID NO. 14, and CDR-H3 as set forth in SEQ ID NO. 25; or

[0021] CDR-H1 as set forth in SEQ ID NO. 4, CDR-H2 as set forth in SEQ ID NO. 15, and CDR-H3 as set forth in SEQ ID NO. 26; or

[0022] CDR-H1 as shown in SEQ ID NO.5, CDR-H2 as shown in SEQ ID NO.16, and CDR-H3 as shown in SEQ ID NO.27; or

[0023] CDR-H1 as shown in SEQ ID NO.6, CDR-H2 as shown in SEQ ID NO.17, and CDR-H3 as shown in SEQ ID NO.28; or

[0024] CDR-H1 as shown in SEQ ID NO.7, CDR-H2 as shown in SEQ ID NO.18, and CDR-H3 as shown in SEQ ID NO.29; or

[0025] CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; or

[0026] CDR-H1 as shown in SEQ ID NO.9, CDR-H2 as shown in SEQ ID NO.20, and CDR-H3 as shown in SEQ ID NO.31; or

[0027] CDR-H1 as shown in SEQ ID NO.10, CDR-H2 as shown in SEQ ID NO.21, and CDR-H3 as shown in SEQ ID NO.32; or

[0028] CDR-H1 shown as SEQ ID NO.11, CDR-H2 shown as SEQ ID NO.22, and CDR-H3 shown as SEQ ID NO.33.

[0029] In some specific embodiments, the antibody or antigen-binding fragment thereof, wherein

[0030] The light chain variable region comprises:

[0031] CDR-L1 as shown in SEQ ID NO.34, CDR-L2 as shown in SEQ ID NO.45, and CDR-L3 as shown in SEQ ID NO.56; or

[0032] CDR-L1 as shown in SEQ ID NO.35, CDR-L2 as shown in SEQ ID NO.46, and CDR-L3 as shown in SEQ ID NO.57; or

[0033] CDR-L1 as shown in SEQ ID NO.36, CDR-L2 as shown in SEQ ID NO.47, and CDR-L3 as shown in SEQ ID NO.58; or

[0034] CDR-L1 as shown in SEQ ID NO.37, CDR-L2 as shown in SEQ ID NO.48, and CDR-L3 as shown in SEQ ID NO.59; or

[0035] CDR-L1 as shown in SEQ ID NO.38, CDR-L2 as shown in SEQ ID NO.49, and CDR-L3 as shown in SEQ ID NO.60; or

[0036] CDR-L1 as shown in SEQ ID NO.39, CDR-L2 as shown in SEQ ID NO.50, and CDR-L3 as shown in SEQ ID NO.61; or

[0037] CDR-L1 as shown in SEQ ID NO.40, CDR-L2 as shown in SEQ ID NO.51, and CDR-L3 as shown in SEQ ID NO.62; or

[0038] CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63; or

[0039] CDR-L1 as shown in SEQ ID NO.42, CDR-L2 as shown in SEQ ID NO.53, and CDR-L3 as shown in SEQ ID NO.64; or

[0040] CDR-L1 as shown in SEQ ID NO.43, CDR-L2 as shown in SEQ ID NO.54, and CDR-L3 as shown in SEQ ID NO.65; or

[0041] CDR-L1 as shown in SEQ ID NO.44, CDR-L2 as shown in SEQ ID NO.55, and CDR-L3 as shown in SEQ ID NO.66.

[0042] In some specific embodiments, the antibody or antigen-binding fragment thereof, wherein

[0043] The heavy chain of the antibody or antigen-binding fragment thereof includes the three heavy chain CDRs described above, namely CDR-H1, CDR-H2, and CDR-H3.

[0044] In some specific embodiments, the antibody or antigen-binding fragment thereof, wherein,

[0045] The light chain of the antibody or antigen-binding fragment thereof includes the three light chain CDRs described above, namely CDR-L1, CDR-L2, and CDR-L3.

[0046] In some specific embodiments, the antibody or antigen-binding fragment thereof comprises:

[0047] CDR-H1 as shown in SEQ ID NO. 1, CDR-H2 as shown in SEQ ID NO. 12, and CDR-H3 as shown in SEQ ID NO. 23; and

[0048] CDR-L1 as shown in SEQ ID NO.34, CDR-L2 as shown in SEQ ID NO.45, and CDR-L3 as shown in SEQ ID NO.56; or

[0049] CDR-H1 as shown in SEQ ID NO. 2, CDR-H2 as shown in SEQ ID NO. 13, and CDR-H3 as shown in SEQ ID NO. 24; and

[0050] CDR-L1 as shown in SEQ ID NO.35, CDR-L2 as shown in SEQ ID NO.46, and CDR-L3 as shown in SEQ ID NO.57; or

[0051] CDR-H1 as shown in SEQ ID NO. 3, CDR-H2 as shown in SEQ ID NO. 14, and CDR-H3 as shown in SEQ ID NO. 25; and

[0052] CDR-L1 as shown in SEQ ID NO.36, CDR-L2 as shown in SEQ ID NO.47, and CDR-L3 as shown in SEQ ID NO.58; or

[0053] CDR-H1 as shown in SEQ ID NO.4, CDR-H2 as shown in SEQ ID NO.15, and CDR-H3 as shown in SEQ ID NO.26; and

[0054] CDR-L1 as shown in SEQ ID NO.37, CDR-L2 as shown in SEQ ID NO.48, and CDR-L3 as shown in SEQ ID NO.59; or

[0055] CDR-H1 as shown in SEQ ID NO.5, CDR-H2 as shown in SEQ ID NO.16, and CDR-H3 as shown in SEQ ID NO.27; and

[0056] CDR-L1 as shown in SEQ ID NO.38, CDR-L2 as shown in SEQ ID NO.49, and CDR-L3 as shown in SEQ ID NO.60; or

[0057] CDR-H1 as shown in SEQ ID NO.6, CDR-H2 as shown in SEQ ID NO.17, and CDR-H3 as shown in SEQ ID NO.28; and

[0058] CDR-L1 as shown in SEQ ID NO.39, CDR-L2 as shown in SEQ ID NO.50, and CDR-L3 as shown in SEQ ID NO.61; or

[0059] CDR-H1 as shown in SEQ ID NO.7, CDR-H2 as shown in SEQ ID NO.18, and CDR-H3 as shown in SEQ ID NO.29; and

[0060] CDR-L1 as shown in SEQ ID NO.40, CDR-L2 as shown in SEQ ID NO.51, and CDR-L3 as shown in SEQ ID NO.62; or

[0061] CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and

[0062] CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63; or

[0063] CDR-H1 as shown in SEQ ID NO.9, CDR-H2 as shown in SEQ ID NO.20, and CDR-H3 as shown in SEQ ID NO.31; and

[0064] CDR-L1 as shown in SEQ ID NO.42, CDR-L2 as shown in SEQ ID NO.53, and CDR-L3 as shown in SEQ ID NO.64; or

[0065] CDR-H1 as shown in SEQ ID NO.10, CDR-H2 as shown in SEQ ID NO.21, and CDR-H3 as shown in SEQ ID NO.32; and

[0066] CDR-L1 as shown in SEQ ID NO.43, CDR-L2 as shown in SEQ ID NO.54, and CDR-L3 as shown in SEQ ID NO.65; or

[0067] CDR-H1 as shown in SEQ ID NO.11, CDR-H2 as shown in SEQ ID NO.22, and CDR-H3 as shown in SEQ ID NO.33; and

[0068] CDR-L1 as shown in SEQ ID NO.44, CDR-L2 as shown in SEQ ID NO.55,

[0069] and CDR-L3 as shown in SEQ ID NO.66.

[0070] In some specific embodiments, the antibody or antigen-binding fragment thereof has a KD value of less than 20 nM. For example, a KD value of less than 15 nM, 12 nM, 10 nM, 8 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or 0.5 nM. Preferably, the KD value is measured by biofilm interferometry.

[0071] In some specific embodiments, the antibody is a monoclonal antibody.

[0072] In some specific embodiments, the antibody or antigen-binding fragment thereof comprises a non-CDR region.

[0073] In some specific embodiments, the non-CDR region may be homologous or non-homologous to the CDR region.

[0074] In some specific embodiments, the antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody (e.g., scFv), humanized antibody, chimeric antibody or diabody.

[0075] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof as described above.

[0076] In some specific embodiments, the nucleic acid molecule comprises nucleic acid molecule A:

[0077] It encodes the heavy chain of the antibody or antigen-binding fragment thereof as described above, wherein the heavy chain includes CDR-H1, CDR-H2, and CDR-H3.

[0078] In some specific embodiments, the nucleic acid molecule comprises nucleic acid molecule B:

[0079] It encodes the light chain of the antibody or antigen-binding fragment thereof as described above, wherein the light chain includes CDR-L1, CDR-L2, and CDR-L3.

[0080] In some specific embodiments, the nucleic acid molecule comprises nucleic acid molecule C:

[0081] The aforementioned nucleic acid molecule A, and the aforementioned nucleic acid molecule B; optionally, the nucleic acid molecule C further comprises a connecting sequence, which is used to connect the nucleic acid molecule A and the nucleic acid molecule B.

[0082] In another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

[0083] In some specific embodiments, the vector includes the above-mentioned nucleic acid molecule A, nucleic acid molecule B, or nucleic acid molecule C.

[0084] In another aspect, the present invention provides a host cell comprising the above-mentioned nucleic acid molecule or vector.

[0085] In some specific embodiments, the host cell includes the aforementioned nucleic acid molecule A, nucleic acid molecule B, nucleic acid molecule C, or the aforementioned vector.

[0086] In another aspect, the present invention provides a conjugate comprising a monoclonal antibody or an antigen-binding fragment thereof and a conjugated moiety, wherein the monoclonal antibody is the antibody or antigen-binding fragment thereof described in any one of the present invention, and the conjugated moiety is a detectable label; preferably, the conjugated moiety is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

[0087] On the other hand, the present invention provides an antibody pair comprising a capture antibody and a detection antibody, wherein the capture antibody and the detection antibody target different epitopes of cardiac troponin I, wherein the capture antibody and the detection antibody are selected from the antibodies or antigen-binding fragments thereof as described above, known antibody 1, known antibody 2, or known antibody 3.

[0088] In some specific embodiments, the known antibody 1 is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 67 to 69 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 70 to 72.

[0089] In some specific embodiments, the known antibody 2 is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 73 to 75 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 76 to 78.

[0090] In some specific embodiments, the known antibody 3 is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 79 to 81 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 82 to 84.

[0091] The capture antibody and the detection antibody can be interchanged, that is, a certain antibody can be used as both a detection antibody and a capture antibody.

[0092] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody being an antibody comprising CDR-H1 to 3 as shown in SEQ ID NOs. 73 to 75 and CDR-L1 to 3 as shown in SEQ ID NOs. 76 to 78; the detection antibody being an antibody comprising CDR-H1 as shown in SEQ ID NO. 9, CDR-H2 as shown in SEQ ID NO. 20, and CDR-H3 as shown in SEQ ID NO. 31; and CDR-L1 as shown in SEQ ID NO. 42, CDR-L2 as shown in SEQ ID NO. 53, and CDR-L3 as shown in SEQ ID NO. 64.

[0093] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody being an antibody comprising CDR-H1 to 3 as shown in SEQ ID NOs. 73 to 75 and CDR-L1 to 3 as shown in SEQ ID NOs. 76 to 78; the detection antibody being an antibody comprising CDR-H1 as shown in SEQ ID NO. 6, CDR-H2 as shown in SEQ ID NO. 17, and CDR-H3 as shown in SEQ ID NO. 28; and CDR-L1 as shown in SEQ ID NO. 39, CDR-L2 as shown in SEQ ID NO. 50, and CDR-L3 as shown in SEQ ID NO. 61.

[0094] Preferably, the antibody pair comprises a capture antibody which is an antibody comprising CDR-H1 to 3 as shown in SEQ ID NO. 67 to 69 and CDR-L1 to 3 as shown in SEQ ID NO. 70 to 72; and a detection antibody which is an antibody comprising CDR-H1 as shown in SEQ ID NO. 5, CDR-H2 as shown in SEQ ID NO. 16, and CDR-H3 as shown in SEQ ID NO. 27; and CDR-L1 as shown in SEQ ID NO. 38, CDR-L2 as shown in SEQ ID NO. 49, and CDR-L3 as shown in SEQ ID NO. 60.

[0095] Preferably, the antibody pair comprises a capture antibody which is an antibody comprising CDR-H1 to 3 as shown in SEQ ID NO. 79 to 81 and CDR-L1 to 3 as shown in SEQ ID NO. 82 to 84; and a detection antibody which is an antibody comprising CDR-H1 as shown in SEQ ID NO. 8, CDR-H2 as shown in SEQ ID NO. 19, and CDR-H3 as shown in SEQ ID NO. 30; and CDR-L1 as shown in SEQ ID NO. 41, CDR-L2 as shown in SEQ ID NO. 52, and CDR-L3 as shown in SEQ ID NO. 63.

[0096] Preferably, the antibody pair comprises a capture antibody which is an antibody comprising CDR-H1 to 3 as shown in SEQ ID NO. 79 to 81 and CDR-L1 to 3 as shown in SEQ ID NO. 82 to 84; and a detection antibody which is an antibody comprising CDR-H1 as shown in SEQ ID NO. 11, CDR-H2 as shown in SEQ ID NO. 22, and CDR-H3 as shown in SEQ ID NO. 33; and CDR-L1 as shown in SEQ ID NO. 44, CDR-L2 as shown in SEQ ID NO. 55, and CDR-L3 as shown in SEQ ID NO. 66.

[0097] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody being an antibody comprising CDR-H1 as set forth in SEQ ID NO. 3, CDR-H2 as set forth in SEQ ID NO. 14, and CDR-H3 as set forth in SEQ ID NO. 25; and CDR-L1 as set forth in SEQ ID NO. 36, CDR-L2 as set forth in SEQ ID NO. 47, and CDR-L3 as set forth in SEQ ID NO. 58; and the detection antibody being an antibody comprising CDR-H1 as set forth in SEQ ID NO. 11, CDR-H2 as set forth in SEQ ID NO. 22, and CDR-H3 as set forth in SEQ ID NO. 33; and CDR-L1 as set forth in SEQ ID NO. 44, CDR-L2 as set forth in SEQ ID NO. 55, and CDR-L3 as set forth in SEQ ID NO. 66.

[0098] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody being an antibody comprising CDR-H1 as set forth in SEQ ID NO. 4, CDR-H2 as set forth in SEQ ID NO. 15, and CDR-H3 as set forth in SEQ ID NO. 26; and CDR-L1 as set forth in SEQ ID NO. 37, CDR-L2 as set forth in SEQ ID NO. 48, and CDR-L3 as set forth in SEQ ID NO. 59; and the detection antibody being an antibody comprising CDR-H1 as set forth in SEQ ID NO. 5, CDR-H2 as set forth in SEQ ID NO. 16, and CDR-H3 as set forth in SEQ ID NO. 27; and CDR-L1 as set forth in SEQ ID NO. 38, CDR-L2 as set forth in SEQ ID NO. 49, and CDR-L3 as set forth in SEQ ID NO. 60.

[0099] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody being an antibody comprising CDR-H1 as set forth in SEQ ID NO. 1, CDR-H2 as set forth in SEQ ID NO. 12, and CDR-H3 as set forth in SEQ ID NO. 23; and CDR-L1 as set forth in SEQ ID NO. 34, CDR-L2 as set forth in SEQ ID NO. 45, and CDR-L3 as set forth in SEQ ID NO. 56; and the detection antibody being an antibody comprising CDR-H1 as set forth in SEQ ID NO. 9, CDR-H2 as set forth in SEQ ID NO. 20, and CDR-H3 as set forth in SEQ ID NO. 31; and CDR-L1 as set forth in SEQ ID NO. 42, CDR-L2 as set forth in SEQ ID NO. 53, and CDR-L3 as set forth in SEQ ID NO. 64.

[0100] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody being an antibody comprising CDR-H1 as set forth in SEQ ID NO. 3, CDR-H2 as set forth in SEQ ID NO. 14, and CDR-H3 as set forth in SEQ ID NO. 25; and CDR-L1 as set forth in SEQ ID NO. 36, CDR-L2 as set forth in SEQ ID NO. 47, and CDR-L3 as set forth in SEQ ID NO. 58; and the detection antibody being an antibody comprising CDR-H1 as set forth in SEQ ID NO. 5, CDR-H2 as set forth in SEQ ID NO. 16, and CDR-H3 as set forth in SEQ ID NO. 27; and CDR-L1 as set forth in SEQ ID NO. 38, CDR-L2 as set forth in SEQ ID NO. 49, and CDR-L3 as set forth in SEQ ID NO. 60.

[0101] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.2, CDR-H2 as shown in SEQ ID NO.13, and CDR-H3 as shown in SEQ ID NO.24; and CDR-L1 as shown in SEQ ID NO.35, CDR-L2 as shown in SEQ ID NO.46, and CDR-L3 as shown in SEQ ID NO.57; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63.

[0102] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.10, CDR-H2 as shown in SEQ ID NO.21, and CDR-H3 as shown in SEQ ID NO.32; and CDR-L1 as shown in SEQ ID NO.43, CDR-L2 as shown in SEQ ID NO.54, and CDR-L3 as shown in SEQ ID NO.65; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63.

[0103] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.11, CDR-H2 as shown in SEQ ID NO.22, and CDR-H3 as shown in SEQ ID NO.33; and CDR-L1 as shown in SEQ ID NO.44, CDR-L2 as shown in SEQ ID NO.55, and CDR-L3 as shown in SEQ ID NO.66; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63.

[0104] Preferably, the antibody pair comprises a capture antibody and a detection antibody, the capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.12, and CDR-H3 as shown in SEQ ID NO.23; and CDR-L1 as shown in SEQ ID NO.34, CDR-L2 as shown in SEQ ID NO.45, and CDR-L3 as shown in SEQ ID NO.56; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63.

[0105] In another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof according to any one of the present invention, or the conjugate of the present invention.

[0106] Preferably, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

[0107] The present invention provides a kit comprising the antibody pair of the present invention; optionally, the detection antibody comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

[0108] In another aspect, the present application provides use of an antibody or an antigen binding fragment thereof according to any one of the present application or an antibody pair according to the present application or a conjugate according to the present application in the manufacture of a kit for detecting the presence or level of cardiac troponin I in a sample from a subject.

[0109] In one embodiment, the present application provides use of an antibody or an antigen binding fragment thereof according to any one of the present application or an antibody pair according to the present application or a conjugate according to the present application in the manufacture of a kit for aiding in the diagnosis of myocardial injury.

[0110] In some embodiments, the myocardial injury is caused by myocardial infarction, coronary syndrome, myocarditis, pericarditis, etc.

[0111] In one embodiment, the present application provides use of an antibody or an antigen binding fragment thereof according to any one of the present application or a conjugate according to the present application in the manufacture of a kit for aiding in the diagnosis of myocardial infarction.

[0112] In another aspect, the present application provides a method of producing an antibody or an antigen binding fragment thereof according to any one of the present application, comprising the steps of culturing the host cell according to the present application under suitable conditions and recovering the antibody or the antigen binding fragment thereof from the cell culture.

[0113] In another aspect, the present application provides an antibody or an antigen binding fragment thereof, comprising an antibody or an antigen binding fragment thereof obtained by the method of producing an antibody or an antigen binding fragment thereof as described above.

[0114] In some embodiments, the present application provides an antibody or an antigen binding fragment thereof, comprising culturing E. coli according to the present application, obtaining the molecular genetic construct therefrom and transferring the molecular genetic construct into a host cell, culturing the host cell and recovering the antibody or the antigen binding fragment thereof from the cell culture.

[0115] The molecular genetic construct can be a single molecular genetic construct encoding the entire sequence of the antibody, such as the molecular genetic construct obtained by culturing E. coli having accession number VKPM B-14631, etc.

[0116] The molecular genetic construct can also be two molecular genetic constructs encoding the sequence of the light chain and the sequence of the heavy chain of the antibody, respectively, such as the molecular genetic constructs obtained by culturing E. coli having accession numbers VKPM B-14668 and VKPM B-14669.

[0117] In another aspect, the present application provides a method of aiding in the diagnosis of myocardial injury, comprising the step of using an antibody or an antigen binding fragment thereof according to any one of the present application on a sample from a subject.

[0118] The deposit status of the antibodies of the present invention is as follows:

[0119] The Escherichia coli plasmid encoding the complete sequence of the novel antibody 2 was deposited with the Russian National Collection of Industrial Microorganisms under the accession number VKPM B-14631 on December 14, 2023. The taxonomic designation is: Escherichia coli RosettaTM (DE3) pLysS plasmid encoding the novel antibody 2 (NA2).

[0120] The Escherichia coli strain harboring the plasmid encoding the light chain sequence of the novel antibody 3 was deposited with the Russian National Collection of Industrial Microorganisms under the accession number VKPM B-14669 on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA3) VL.

[0121] Escherichia coli containing a plasmid encoding the heavy chain sequence of the novel antibody 3, with the accession number VKPM B-14668, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA3) VH.

[0122] The Escherichia coli bacteria harboring the plasmid encoding the light chain sequence of the novel antibody 4 were deposited with the Russian National Collection of Industrial Microorganisms under the accession number VKPM B-14671 on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA4) VL.

[0123] The Escherichia coli bacteria harboring the plasmid encoding the heavy chain sequence of the novel antibody 4 were deposited with the Russian National Collection of Industrial Microorganisms under the accession number VKPM B-14670 on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA4) VH.

[0124] Escherichia coli containing a plasmid encoding the light chain sequence of the novel antibody 5, with the accession number VKPM B-14673, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA5) VL.

[0125] Escherichia coli containing a plasmid encoding the heavy chain sequence of the novel antibody 5, with the accession number VKPM B-14672, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA5) VH.

[0126] Escherichia coli harboring a plasmid encoding the light chain sequence of the novel antibody 6, with accession number VKPM B-14675, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA6) VL.

[0127] Escherichia coli containing a plasmid encoding the heavy chain sequence of the novel antibody 6, with the accession number VKPM B-14674, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA6) VH.

[0128] Escherichia coli containing a plasmid encoding the light chain sequence of the novel antibody 7, with the accession number VKPM B-14677, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA7) VL.

[0129] Escherichia coli containing a plasmid encoding the heavy chain sequence of the novel antibody 7, with the accession number VKPM B-14676, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA7) VH.

[0130] Escherichia coli containing a plasmid encoding the light chain sequence of the novel antibody 8, with the accession number VKPM B-14679, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA8) VL.

[0131] Escherichia coli containing a plasmid encoding the heavy chain sequence of the novel antibody 8, with the accession number VKPM B-14678, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA8) VH.

[0132] The Escherichia coli plasmid encoding the complete sequence of the novel antibody 9 was deposited with the Russian National Collection of Industrial Microorganisms under the accession number VKPM B-14632 on December 14, 2023. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS plasmid encoding the novel antibody 9 (NA9).

[0133] The Escherichia coli plasmid encoding the complete sequence of the novel antibody 10 was deposited with the Russian National Collection of Industrial Microorganisms under the accession number VKPM B-14633 on December 14, 2023. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS plasmid encoding the novel antibody 10 (NA10).

[0134] Escherichia coli harboring a plasmid encoding the light chain sequence of the novel antibody 11, with the accession number VKPM B-14681, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA11) VL.

[0135] Escherichia coli harboring a plasmid encoding the heavy chain sequence of novel antibody 11, with accession number VKPM B-14680, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA11) VH.

[0136] Escherichia coli harboring a plasmid encoding the light chain sequence of the novel antibody 12, with accession number VKPM B-14683, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA12) VL.

[0137] Escherichia coli harboring a plasmid encoding the heavy chain sequence of the novel antibody 12, with accession number VKPM B-14682, was deposited at the Russian National Collection of Industrial Microorganisms on February 7, 2024. The taxonomic designation is: Escherichia coli Rosetta™ (DE3) pLysS (NA12) VH. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 The results of the detection performance of different types of samples using the antibody pairs of the present invention are as follows;

[0139] Figure 2The results of the prognostic monitoring performance of the antibody of the present invention on patients with acute myocardial injury are as follows;

[0140] Figure 3 The results of the prognosis monitoring performance of the antibody of the present invention on patients with acute myocardial injury are as follows;

[0141] Figure 4 The present invention shows the prognostic monitoring performance test results of the antibody of the present invention on patients with acute myocardial injury. DETAILED DESCRIPTION

[0142] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0143] Unless otherwise indicated, the term "isolated molecule" (wherein the molecule is, for example, a polypeptide, polynucleotide, or antibody, or fragment thereof) is a molecule that, by virtue of the origin or source from which it is derived, is (1) not associated with naturally associated components with which it is naturally associated, (2) is substantially free of other molecules from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates would be "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by separation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a variety of methods well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For some purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0144] An "antibody" is an immunoglobulin molecule capable of specific binding to a target analyte, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, situated in the variable region of the immunoglobulin molecule. As used herein, unless otherwise specified, the term includes not only intact polyclonal or monoclonal antibodies, but also any antigen binding portion, fusion protein comprising an antigen binding portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. Antigen binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, a domain antibody (dAb, e.g., shark and camelid antibodies), a fragment comprising a complementarity determining region (CDR), a single chain variable fragment antibody (scFv), a maxibody, a minibody, an intrabody, a diabody, a triabody, a tetrabody, a v-NAR, and a bis-scFv, as well as polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Antibodies include any species or antigen binding fragment thereof, such as IgG, IgA, or IgM (or subtypes thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant regions of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0145] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply, "antibody portion"), used interchangeably, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., cardiac troponin). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which consists of the VH and CH1 domains; (iv) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) isolated complementarity determining regions (CDRs), disulfide-linked Fvs (dsFv) and anti-idiotypic (anti-Id) antibodies and intrabodies. In addition, although the two domains of the Fv fragment (VL and VH) are encoded by separate genes, they can be connected using recombinant methods through a synthetic linker that enables them to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv)). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites.

[0146] Antibodies can be derived from any mammal, including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g., chickens), fish (e.g., sharks), and camelids (e.g., llamas).

[0147] The "variable region" of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. As is known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FR) connected by three complementarity determining regions (CDRs) (also referred to as hypervariable regions), and contribute to the formation of the antigen binding site of the antibody. If a variant of the subject variable region is desired, particularly one with substitutions of amino acid residues outside the CDR region (i.e., in the framework region), appropriate amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the subject variable region with the variable regions of other antibodies containing CDR1 and CDR2 sequences in the same typical species as the subject variable region.

[0148] Variant antibodies may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions and / or insertions from the specific sequences and fragments discussed above. "Deletion" variants may include deletions of individual amino acids; deletions of small groups of amino acids, such as 2, 3, 4 or 5 amino acids; or deletions of larger amino acid regions, such as deletions of specific amino acid domains or other features. "Insertion" variants may include insertions of individual amino acids; insertions of small groups of amino acids, such as 2, 3, 4 or 5 amino acids; or insertions of larger amino acid regions, such as insertions of specific amino acid domains or other features. "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, an amino acid can be substituted with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid.

[0149] As outlined elsewhere herein, certain positions of the antibody molecule may vary. As used herein, "position" refers to the location in the sequence of a protein. Positions may be numbered sequentially or according to established formats, for example, the EU index and the Kabat index may be used to number the amino acid residues of an antibody. The corresponding position is generally determined by comparison with other parental sequences.

[0150] In general, the term "epitope" refers to an area or site of an antigen to which an antibody specifically binds, such as an area or site containing contact residues that interact with the antibody. Thus, the term "epitope" refers to a portion of a molecule that can be recognized and bound by an antibody on one or more antibody antigen-binding regions. Typically, epitopes are defined in the context of molecular interactions between an antibody or its antigen-binding fragment and its corresponding antigen. Epitopes typically consist of surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0151] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid chains of any length. The chain may be linear or branched, may comprise modified amino acids, and / or may be interrupted by non-amino acids. The term also encompasses amino acid chains that have been modified naturally or by insertion; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It is understood that a polypeptide may exist as a single chain or in an associated chain.

[0152] The terms "dissociation constant" and "equilibrium dissociation constant" (KD) are sometimes used interchangeably and refer to the value obtained by titrating at equilibrium or by dividing the dissociation rate constant (koff) by the association rate constant (Kon). The association rate constant, dissociation rate constant, and equilibrium dissociation constant are used to express the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art.

[0153] The binding affinity between two molecules, such as an antibody or its fragment and an antigen, through a monovalent interaction can be quantified by measuring the dissociation constant (KD). Furthermore, KD can be measured by measuring the kinetics of complex formation and dissociation using, for example, a surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and dissociation of a monovalent complex refer to the association rate constant Ka (or Kon) and the dissociation rate constant Kd (or Koff), respectively. KD is associated with Ka and Kd by the equation KD=Kd / Ka. The value of the dissociation constant can be directly determined by known methods, and even the dissociation constant value of a complex mixture can be calculated by methods such as those shown in Caceci et al. (1984, Byte9:340-362).

[0154] As used herein, "vector" means a construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0155] "Individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, livestock (e.g., cows, pigs, horses, chickens, etc.), sports animals, pets, primates, horses, dogs, cats, mice, and rats.

[0156] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0157] Example 1. Screening of Anti-Cardial Troponin I Antibodies

[0158] Recombinant soluble cardiac troponin I was expressed in E. coli and further purified. The resulting purified protein solution was concentrated, stored below -80°C, and its purity was confirmed by SDS-PAGE and sieving chromatography (SEC).

[0159] Recombinant soluble cardiac troponin I was used to immunize rabbits, rats, and pigs. The immunized hybridoma cells were screened, and 11 hybridoma cell lines producing anti-cardiac troponin I were obtained (the antibodies produced were named: Neoantibody 2, NeoAntibody 3, NeoAntibody 4, NeoAntibody 5, NeoAntibody 6, NeoAntibody 7, NeoAntibody 8, NeoAntibody 9, NeoAntibody 10, NeoAntibody 11, and NeoAntibody 12, respectively; NeoAntibody 2, NeoAntibody 3, NeoAntibody 4, NeoAntibody 5 were rabbit-derived, NeoAntibody 6, NeoAntibody 7, NeoAntibody 8, NeoAntibody 9, NeoAntibody 10 were rat-derived, and NeoAntibody 11 and NeoAntibody 12 were pig-derived). RNA was extracted from the screened hybridoma cells, and the sequences of the antibodies produced were sequenced. Partial sequencing results are shown in Table 1 below (amino acid sequences).

[0160] Table 1

[0161]

[0162]

[0163] Example 2. Production of Anti-Cardial Troponin I Antibodies

[0164] The novel antibody 2, novel antibody 3, novel antibody 4, novel antibody 5, novel antibody 6, novel antibody 7, novel antibody 8, novel antibody 9, novel antibody 10, novel antibody 11, and novel antibody 12 antibodies were obtained in a recombinant expression form. Appropriate molecular genetic constructs were designed for all recombinantly expressed antibodies. To develop molecular genetic constructs for rat and porcine antibodies, the variable domains of novel antibody 6, novel antibody 7, novel antibody 8, novel antibody 9, novel antibody 10, novel antibody 11, and novel antibody 12 antibodies and the constant domain sequences of the heavy chain IgG1 isotype and light chain λ isotype of human immunoglobulins were used. To develop molecular genetic constructs for rabbit antibodies, the variable domains of novel antibody 2, novel antibody 3, novel antibody 4, and novel antibody 5 antibodies and the constant domain sequences of the heavy chain IgG isotype and light chain λ isotype of rabbit immunoglobulins were used.

[0165] The molecular genetic construct of the recombinant antibody is obtained in preparative quantities in bacterial cells and purified.

[0166] The molecular genetic construct of the recombinant antibody prepared above is used to transfect the mammalian cell line Expi293F. For example, the light chain and heavy chain gene sequences are transferred to the open reading frame (ORF) of the expression vector by homologous recombination or restriction enzyme digestion, and ultimately a "promoter-antibody light chain gene-terminator" and a "promoter-antibody heavy chain gene-terminator" structure are formed on the two expression vectors, respectively. Then, the two expression vectors (antibody light chain expression vector and heavy chain expression vector) are simultaneously transferred into the mammalian cell line Expi293F for expression by biological, physical, and chemical methods. Ultimately, the two light chains and two heavy chains are reassembled into a complete antibody in the cell and secreted into the culture supernatant. Alternatively, the expression vector is modified so that one expression vector contains two open reading frames, and then the light chain and heavy chain gene sequences are respectively inserted into the two open reading frames (ORF, Open Reading Frame) of the expression vector through homologous recombination or restriction enzyme cutting, forming a "promoter-antibody light chain gene-terminator-vector sequence-promoter-antibody heavy chain gene-terminator" or "promoter-antibody heavy chain gene-terminator-vector sequence-promoter-antibody light chain gene-terminator" structure. Then, this expression vector containing both the antibody light chain and heavy chain is transferred into the mammalian cell line Expi293F for expression through biological, physical, and chemical methods. Ultimately, the two light chains and two heavy chains are reassembled into a complete antibody in the cells and secreted into the culture supernatant.

[0167] Antibodies were purified from conditioned medium (Neo Antibody 2, Neo Antibody 3, Neo Antibody 4, Neo Antibody 5, Neo Antibody 6, Neo Antibody 7, Neo Antibody 8, Neo Antibody 9, Neo Antibody 10, Neo Antibody 11, Neo Antibody 12) by protein A affinity chromatography. Affinity chromatography resins were from GE Healthcare Life Sciences (Piscataway, NJ) and purified according to the manufacturer's instructions. Purified monoclonal antibodies were stored as a suspension in 50% ammonium sulfate at 4°C.

[0168] Example 3: Characterization of Antibody Binding Kinetics

[0169] The antibodies prepared in Example 2 were dissolved in PBS (5 μg / mL) and applied to the sensor, which was then blocked. The antigen (cardiac troponin complex) was then administered at concentrations of 10, 30, and 90 nM, respectively. Finally, the affinity of the antibodies was measured using biomembrane interferometry, and the results are shown in Table 2. As can be seen from Table 2, all 11 antibodies of the present invention had good affinity for the target cardiac troponin.

[0170] Table 2

[0171] Antibody Ka(1 / Ms) Kd(1 / s) KD(M) New Antibody 2 1.11E+05 1.33E-03 1.38E-08 New Antibody 3 1.63E+05 5.64E-04 3.70E-09 New Antibody 4 1.77E+05 2.16E-04 1.23E-09 New Antibody 5 1.10E+05 3.26E-04 2.95E-09 New Antibody 6 1.18E+05 4.50E-04 3.85E-09 New Antibody 7 8.82E+04 4.75E-04 5.78E-09 New Antibody 8 1.24E+05 1.50E-03 1.20E-08 New Antibody 9 1.35E+05 2.37E-04 1.97E-09 New Antibody 10 9.11E+04 4.10E-04 4.76E-09 New Antibody 11 1.56E+05 1.71E-03 1.09E-08 New Antibody 12 6.99E+04 7.23E-04 1.08E-08

[0172] Example 4, Epitope specificity testing of antibodies

[0173] Epitope specificity of the monoclonal antibodies was determined by ELISA using short peptides (15-20 amino acids, from amino acids 18-200 of the protein sequence). Peptides were conjugated to BSA using sulfo-SMCC obtained from Pierce (Rockford, IL) according to the manufacturer's instructions. BSA was linked to the peptide through an additional cysteine residue from the N-terminus. For conjugation, 2.5 mg of bovine serum albumin (BSA) obtained from Sigma Chemicals, St. Louise, MO was dissolved in PBS to a concentration of 10 mg / mL to obtain a protein solution. 2 mg of sulfo-SMCC dissolved in 0.1 mL of dimethyl sulfoxide was added to the protein solution. The BSA activation reaction was carried out at room temperature for 2 hours. Excess sulfo-SMCC was removed by gel filtration using a NAP-5 column obtained from GE Healthcare Life Sciences, Piscataway, NJ. The NAP-5 column was pre-equilibrated with 10 mM KH2PO4, 150 mM NaCl, pH 7.2. 2 mg of a synthetic peptide designed according to the sequence of cardiac troponin I was then added to the modified protein solution to start the conjugation. The reaction was carried out on ice for 2 hours with constant shaking. Unreacted synthetic peptide was removed from the BSA-synthetic peptide conjugate by gel filtration using a NAP-5 column pre-equilibrated with PBS. Binding of the short peptide to the appropriate BSA was confirmed by an increase in protein molecular weight of 3-5 kDa shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conjugate was aliquoted and stored at -20°C until use.

[0174] Dilute the BSA-synthetic peptide to a 5 μg / mL solution in 0.1 M NaHCO₃ (pH 9.6) and add 100 μL per well of a high-absorption 96-well plate. Incubate overnight at 4°C. Drain the solution and add 400 μL of 1% BSA in PBS to each well. Block the plate at room temperature for 3 hours. Drain the solution and wash three times with PBS containing 0.05% Tween-20. Dilute 11 antibodies to a 100 μg / mL solution in PBS containing 1% BSA, add 100 μL to each well, and incubate at 37°C for 1.5 hours. Drain the solution and wash three times with PBS containing 0.05% Tween-20. Prepare a 1 / 2000 dilution of goat anti-mouse IgG-HRP solution in PBS containing 1% BSA, add 100 μL to each well, and incubate at 37°C for 1.5 hours. Drain the solution and wash three times with PBS containing 0.05% Tween-20. Add 100 μL of TMB substrate solution to each well and incubate at room temperature for 30 minutes. Terminate with 100 μL of 0.16 M sulfuric acid solution. Read the absorbance at OD450 on a microplate reader.

[0175] According to the above method, 11 antibodies were tested and the results are as follows:

[0176] Neoantibody 2 recognizes a polypeptide of amino acid residues 78 to 93 and a polypeptide of amino acid residues 83 to 100. Therefore, the epitope of Neoantibody 2 is within amino acid residues 83 to 93.

[0177] Neoantibody 3 and Neoantibody 5 recognize the polypeptide of amino acid residues 39-54, therefore, the epitopes of Neoantibody 3 and Neoantibody 5 are within amino acid residues 39-54.

[0178] Neoantibody 4 and Neoantibody 8 recognize the polypeptide of amino acid residues 22-40, therefore, the epitopes of Neoantibody 4 and Neoantibody 8 are within amino acid residues 22-40.

[0179] Neoantibody 6 and Neoantibody 9 recognize a polypeptide of amino acid residues 161-178, and therefore, the epitopes of Neoantibody 6 and Neoantibody 9 are within amino acid residues 161-178.

[0180] Neoantibody 7 and Neoantibody 10 recognize the polypeptide of amino acid residues 174-192, and therefore, the epitopes of Neoantibody 6 and Neoantibody 9 are within amino acid residues 174-192.

[0181] Neoantibody 11 and Neoantibody 12 recognize a polypeptide of amino acid residues 174 to 192 and a polypeptide of amino acid residues 182 to 197. Therefore, the epitopes of Neoantibody 1 and Neoantibody 2 are within amino acid residues 182 to 192.

[0182] Example 5. Sandwich immunoassay using antibody pairs

[0183] To identify the optimal antibody pair, a sandwich immunoassay was used. Antibodies recognizing different epitopes were combined to test their ability to detect human cardiac troponin. In addition to the antibodies used in this study, known antibodies 1, 2, and 3 (cat# RC4T21) from HyTest were also used. Their antibody sequences are shown in Table 3.

[0184] Table 3

[0185]

[0186]

[0187] The detection capabilities of the antibody pairs were determined using dissociation-enhanced lanthanide fluorescence immunoassay and chemiluminescence immunoassay.

[0188] For the sake of et al., 2010, using a dissociation-enhanced lanthanide fluorescence immunoassay using a stable Eu 3+ Chelate-labeled detection antibody. The capture antibody in this assay is unlabeled. 100 μL (2 μg / mL) of capture antibody per well in PBS was incubated in a 96-well immunoassay plate at room temperature for 30 minutes, then continuously shaken. The plate was washed with 10 mM Tris-HCl (pH 7.8) buffer supplemented with 0.15 M NaCl, 0.025% Tween 20, and 0.5 g / L NaN3 (Washing Solution, WS). After washing with 0.05 mL of buffer B (50 mM Tris-HCl buffer, pH 7.8, 0.9% NaCl, 0.01% Tween 40, 0.5% BSA, and 0.05% NaN3), the detection antibody solution (2 mg / L) was added to the assay plate. Immediately after detection, add 25 μL of antigen solution (native human cardiac troponin complex) standard solution in 50 mM Tris-HCl buffer (pH 7.8) supplemented with 150 mM KCl, 5 mM CaCl2, 7.5% BSA, and 0.15% NaN3. Incubate the plate at room temperature for 30 minutes while shaking continuously. After washing, add 0.1 ml of Enhancement solution (Perkin Elmer, Finland) and incubate with gentle shaking at room temperature for 10 minutes. 3+ The fluorescence was measured on a Victor 1420 multilabel counter (Wallac-Perkin Elmer, Finland). Fluorescence is expressed in counts per second (CPS).

[0189] To perform the sandwich chemiluminescent immunoassay (CLIA), the detection antibody is labeled with a biotin derivative. The capture antibody (unlabeled) (100 μL per well (2 μg / mL) in phosphate-buffered saline (PBS)) is incubated in a 96-well immunoassay plate at room temperature for 30 minutes, followed by continuous shaking. The plate is washed with PBS supplemented with 0.025% Tween 20 and 0.05% ProClin (PBST). After washing with 0.05 ml of buffer B, a solution of the detection antibody (2 mg / L) in CLIA assay buffer (PBST, containing 75 g / L BSA) is added to the plate. Immediately after detection, 25 μL of a standard solution of the antigen solution (native human cardiac troponin complex) in 50 mM Tris-HCl buffer, pH 7.8, supplemented with 150 mM KCl, 5 mM CaCl2, 7.5% BSA, and 0.15% NaN3 is added. The plate is incubated at room temperature for 30 minutes while continuously shaking. After washing with PBST, 0.1 ml of Pierce TM Streptavidin Poly-HRP (5 ng / mL in CLIA assay buffer) was added and incubated with gentle shaking at room temperature for 5 minutes. After washing with PBST (6 times), SuperSignal TM ELISA Femto Substrate. Incubation time was 1 minute at room temperature with gentle agitation. Chemiluminescence was measured on an EnVision 2105 multimode plate reader (Perkin Elmer, Finland). Chemiluminescence was expressed as counts per second (CPS).

[0190] The above immunoassay can only detect cTnI and has no cross-reactivity (or less than 0.03%) with the skeletal muscle isoform of TnI.

[0191] Limit of blank (LOB) and LOD were determined according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). The results are shown in Tables 4 and 5.

[0192] This indicates that the sensitivity of these antibody pairs is below 10 pg / mL, which can meet the performance requirements for developing a high-sensitivity troponin test kit.

[0193] Table 4. LOD of antibody pairs in DELFIA

[0194] Capture antibody Detection of antibodies LOD, pg / mL Known Antibody 2 New Antibody 10 2.0 Known Antibody 2 New Antibody 7 0.9 New Antibody 4 New Antibody 12 0.7 New Antibody 5 New Antibody 6 0.8 Known Antibody 1 New Antibody 6 2.5 Known Antibody 3 New Antibody 9 4.3 Known Antibody 3 New Antibody 12 2.4 New Antibody 2 New Antibody 10 3.3 New Antibody 4 New Antibody 6 2.4

[0195] Table 5. LOD values ​​of antibody pairs in CLIA

[0196]

[0197]

[0198] Example 6. Gender-Specific 99th Percentile of hs-cTnI Specific Immunoassay

[0199] To determine the clinical performance of the hs-cTnI-specific sandwich chemiluminescent immunoassay, the sex-specific 99th percentile was determined according to the Clinical and Laboratory Standards Institute (CLSI) recommendations (Defining, Establishing, and Validating Reference Intervals in Clinical Laboratories; Approved Guidelines—Third Edition; EPC28-A3 Vol. 28 No. 30).

[0200] Briefly, serum or plasma samples from 400 healthy men and 400 healthy women (excluding those with NT-proBNP ≥ 125 ng / L, HbA1c > 6.5%, and eGFR < 60 mL / min) were tested using a sandwich immunoassay using the Novel Antibody 2 / Novel Antibody 9 antibody pair on a Mindray chemiluminescent immunoassay analyzer. The results are shown in Table 6 below, demonstrating high sensitivity in both men and women.

[0201] Table 6. 99th percentile of sex-specificity of the CLIA novel antibody 2 / novel antibody 9 antibody pair

[0202] 99th percentile cTnI value (ng / L) Detection rate (%) overall 26.5 75％ female 17.6 65％ male 33.7 80％

[0203] Example 7: Clinical diagnosis of different myocardial injuries using hs-cTnI specific immunoassay

[0204] To determine the diagnostic performance of the hs-cTnI sandwich immunoassay, serum or plasma samples from patients with various myocardial injuries (including acute myocardial infarction (AMI), myocardial surgery, coronary artery disease, heart failure, cardiomyopathy, angina pectoris, arrhythmia, aortic dissection, etc.) and other non-myocardial injury patients (including kidney disease and diabetes) were evaluated. Immunoassays of the above samples were performed using the new antibody 2 / new antibody 9 antibody pair. The results showed that cardiac troponin could not be detected in samples from patients with kidney disease and diabetes, while cardiac troponin could be detected in samples from patients with other relevant myocardial injuries, such as Figure 1 Therefore, it is shown that hs-cTnI detection can significantly distinguish myocardial injury diseases from other non-myocardial injury diseases.

[0205] Example 8: Prognostic performance of hs-cTnI-specific immunoassay for acute myocardial injury

[0206] To determine the prognosis of hs-cTnI sandwich immunoassay, serum or plasma samples from different patients with acute myocardial infarction were dynamically monitored at different time points. The results showed that hs-cTnI values ​​were highly consistent with disease stage progression. The specific results are as follows:

[0207] Patient 1: An 11-year-old female was admitted to the ICU due to ventricular fibrillation, arrhythmia, and respiratory arrest. At this time, the first test was performed using antibodies to known antibodies 2 / new antibodies 10; cardiopulmonary resuscitation and electrocardiogram tests were subsequently performed. At this time, a second test was performed, and it was found that the cardiac troponin level increased, indicating myocardial damage. After entering the ICU, treatment began, including defibrillation and assisted endotracheal intubation, ventilator assistance, arteriovenous catheterization, and ECMO. A third test was performed before treatment, at which time the myocardial damage was more severe. After a period of treatment, subsequent tests 4-11 were performed, and the cardiac troponin value continued to decrease. Figure 2 shown.

[0208] Patient 2: A 57-year-old female with a history of hypertension and icteric hepatitis was diagnosed with aortic dissection after admission. At this time, the first test using antibodies to new antibody 4 / new antibody 6 showed a weakly positive cTn. A series of surgeries that caused myocardial damage were performed. At this time, a second test was performed, and the cTnI value increased significantly. Subsequently, venous thromboembolism prevention was performed, and a third test was performed, and the cTnI was relatively stable. Another thoracotomy was performed, and the cTnI value increased again in the 4th and 5th tests. At the 6th measurement, there was a large area of ​​embolism in the left brain, but the cTnI was relatively stable. Subsequent brain surgery was performed, the blood circulation was stable, and the cTnI gradually decreased in the 7th and 8th tests. After the brain surgery, the heart function and renal function were stable, and the cTnI continued to decrease in the 9th-12th tests, such as Figure 3 shown.

[0209] Patient 3: History of hypertension, oral cancer. He was diagnosed with acute non-ST-segment elevation myocardial infarction and was admitted to the hospital. At this time, the first test using antibodies for new antibody 11 / new antibody 9 showed very high cTn. He subsequently received a series of treatments and used medication to control high blood pressure, heart rate, and blood coagulation. A second test was performed, which showed a decrease compared to the first. He subsequently underwent heart surgery that caused myocardial damage. Therefore, cTnI increased in the 3rd and 4th tests. He continued to use medication to control high blood pressure, heart rate, and blood coagulation, and local ischemia was relieved. Therefore, cTnI decreased in the 5th to 7th tests, as shown in Figure 2. Figure 4 shown.

[0210] Example 9: Determination of Antibody Specificity

[0211] To assess the potential cross-reactivity of the antibodies, each of the following substances was spiked at a concentration (1000 ng / ml) into a sample with a known cTnI concentration (0.7 ng / L). The results of the spiked samples were compared with those of an unspiked control sample. All antibodies demonstrated high specificity for cTnI, as shown in Table 7.

[0212] The percent cross-reactivity was calculated as follows:

[0213]

[0214] Table 7

[0215]

[0216] ND a =Not detected.

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to cardiac troponin I, comprising a heavy chain and a light chain, wherein: The heavy chain includes a heavy chain variable region, and the light chain includes a light chain variable region; in, The heavy chain variable region comprises: a. CDR-H1, comprising a sequence as shown in any one of SEQ ID NOs. 1 to 11; b. CDR-H2, comprising a sequence as shown in any one of SEQ ID NOs. 12 to 22; and c. CDR-H3, comprising a sequence as shown in any one of SEQ ID NOs. 23 to 33; The light chain variable region comprises: a. CDR-L1 comprising a sequence as shown in any one of SEQ ID NOs. 34 to 44; b. CDR-L2, comprising a sequence as shown in any one of SEQ ID NOs. 45 to 55; and c. CDR-L3 comprising the sequence shown in any one of SEQ ID NOs. 56 to 66.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The isolated antibody or antigen-binding fragment thereof comprises: CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.12, and CDR-H3 as shown in SEQ ID NO.23; and CDR-L1 as shown in SEQ ID NO.34, CDR-L2 as shown in SEQ ID NO.45, and CDR-L3 as shown in SEQ ID NO.56; or CDR-H1 as shown in SEQ ID NO.2, CDR-H2 as shown in SEQ ID NO.13, and CDR-H3 as shown in SEQ ID NO.24; and CDR-L1 as shown in SEQ ID NO.35, CDR-L2 as shown in SEQ ID NO.46, and CDR-L3 as shown in SEQ ID NO.57; or CDR-H1 as shown in SEQ ID NO.3, CDR-H2 as shown in SEQ ID NO.14, and CDR-H3 as shown in SEQ ID NO.25; and CDR-L1 as shown in SEQ ID NO.36, CDR-L2 as shown in SEQ ID NO.47, and CDR-L3 as shown in SEQ ID NO.58; or CDR-H1 as shown in SEQ ID NO.4, CDR-H2 as shown in SEQ ID NO.15, and CDR-H3 as shown in SEQ ID NO.26; and CDR-L1 as shown in SEQ ID NO.37, CDR-L2 as shown in SEQ ID NO.48, and CDR-L3 as shown in SEQ ID NO.59; or CDR-H1 as shown in SEQ ID NO.5, CDR-H2 as shown in SEQ ID NO.16, and CDR-H3 as shown in SEQ ID NO.27; and CDR-L1 as shown in SEQ ID NO.38, CDR-L2 as shown in SEQ ID NO.49, and CDR-L3 as shown in SEQ ID NO.60; or CDR-H1 as shown in SEQ ID NO.6, CDR-H2 as shown in SEQ ID NO.17, and CDR-H3 as shown in SEQ ID NO.28; and CDR-L1 as shown in SEQ ID NO.39, CDR-L2 as shown in SEQ ID NO.50, and CDR-L3 as shown in SEQ ID NO.61; or CDR-H1 as shown in SEQ ID NO.7, CDR-H2 as shown in SEQ ID NO.18, and CDR-H3 as shown in SEQ ID NO.29; and CDR-L1 as shown in SEQ ID NO.40, CDR-L2 as shown in SEQ ID NO.51, and CDR-L3 as shown in SEQ ID NO.62; or CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63; or CDR-H1 as shown in SEQ ID NO.9, CDR-H2 as shown in SEQ ID NO.20, and CDR-H3 as shown in SEQ ID NO.31; and CDR-L1 as shown in SEQ ID NO.42, CDR-L2 as shown in SEQ ID NO.53, and CDR-L3 as shown in SEQ ID NO.64; or CDR-H1 as shown in SEQ ID NO.10, CDR-H2 as shown in SEQ ID NO.21, and CDR-H3 as shown in SEQ ID NO.32; and CDR-L1 as shown in SEQ ID NO.43, CDR-L2 as shown in SEQ ID NO.54, and CDR-L3 as shown in SEQ ID NO.65; or CDR-H1 shown in SEQ ID NO.11, CDR-H2 shown in SEQ ID NO.22, and CDR-H3 shown in SEQ ID NO.33; and CDR-L1 shown in SEQ ID NO.44, CDR-L2 shown in SEQ ID NO.55, and CDR-L3 shown in SEQ ID NO.

66.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The isolated antibody or antigen-binding fragment thereof has a KD value of less than 20 nM.

4. The isolated antibody or antigen-binding fragment thereof according to claim 3, wherein The isolated antibody or antigen-binding fragment thereof has a KD value of less than 5 nM.

5. The isolated antibody or antigen-binding fragment thereof according to claim 4, wherein The isolated antibody or antigen-binding fragment thereof has a KD value of less than 2 nM.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. A vector comprising the nucleic acid molecule according to claim 6 .

8. A host cell comprising the nucleic acid molecule according to claim 6 or the vector according to claim 7.

9. A conjugate comprising a monoclonal antibody or an antigen-binding fragment thereof and a conjugated moiety, wherein: The monoclonal antibody is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and the coupling portion is a detectable label.

10. An antibody pair comprising a capture antibody and a detection antibody, wherein: The capture antibody and the detection antibody target different epitopes of cardiac troponin I, wherein the capture antibody and the detection antibody are selected from the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, known antibody 1, known antibody 2, or known antibody 3, wherein The known antibody 1 is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 67 to 69 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 70 to 72; The known antibody 2 is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 73 to 75 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 76 to 78; The known antibody 3 is an antibody comprising CDR-H1 to CDR-3 shown in SEQ ID NOs. 79 to 81 and CDR-L1 to CDR-3 shown in SEQ ID NOs. 82 to 84.

11. The antibody pair according to claim 10, wherein The antibody pair comprises a capture antibody and a detection antibody, wherein The capture antibody is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 73 to 75 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 76 to 78; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO. 9, CDR-H2 as shown in SEQ ID NO. 20, and CDR-H3 as shown in SEQ ID NO. 31; and CDR-L1 as shown in SEQ ID NO. 42, CDR-L2 as shown in SEQ ID NO. 53, and CDR-L3 as shown in SEQ ID NO. 64; or The capture antibody is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 73 to 75 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 76 to 78; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO. 6, CDR-H2 as shown in SEQ ID NO. 17, and CDR-H3 as shown in SEQ ID NO. 28; and CDR-L1 as shown in SEQ ID NO. 39, CDR-L2 as shown in SEQ ID NO. 50, and CDR-L3 as shown in SEQ ID NO. 61; or The capture antibody is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 67 to 69 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 70 to 72; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO. 5, CDR-H2 as shown in SEQ ID NO. 16, and CDR-H3 as shown in SEQ ID NO. 27; and CDR-L1 as shown in SEQ ID NO. 38, CDR-L2 as shown in SEQ ID NO. 49, and CDR-L3 as shown in SEQ ID NO. 60; or The capture antibody is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 79 to 81 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 82 to 84; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO. 8, CDR-H2 as shown in SEQ ID NO. 19, and CDR-H3 as shown in SEQ ID NO. 30; and CDR-L1 as shown in SEQ ID NO. 41, CDR-L2 as shown in SEQ ID NO. 52, and CDR-L3 as shown in SEQ ID NO. 63; or The capture antibody is an antibody comprising CDR-H1 to CDR-3 as shown in SEQ ID NOs. 79 to 81 and CDR-L1 to CDR-3 as shown in SEQ ID NOs. 82 to 84; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO. 11, CDR-H2 as shown in SEQ ID NO. 22, and CDR-H3 as shown in SEQ ID NO. 33; and CDR-L1 as shown in SEQ ID NO. 44, CDR-L2 as shown in SEQ ID NO. 55, and CDR-L3 as shown in SEQ ID NO. 66; or The capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.3, CDR-H2 as shown in SEQ ID NO.14, and CDR-H3 as shown in SEQ ID NO.25; and CDR-L1 as shown in SEQ ID NO.36, CDR-L2 as shown in SEQ ID NO.47, and CDR-L3 as shown in SEQ ID NO.58; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.11, CDR-H2 as shown in SEQ ID NO.22, and CDR-H3 as shown in SEQ ID NO.33; and CDR-L1 as shown in SEQ ID NO.44, CDR-L2 as shown in SEQ ID NO.55, and CDR-L3 as shown in SEQ ID NO.66; or The capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.4, CDR-H2 as shown in SEQ ID NO.15, and CDR-H3 as shown in SEQ ID NO.26; and CDR-L1 as shown in SEQ ID NO.37, CDR-L2 as shown in SEQ ID NO.48, and CDR-L3 as shown in SEQ ID NO.59; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.5, CDR-H2 as shown in SEQ ID NO.16, and CDR-H3 as shown in SEQ ID NO.27; and CDR-L1 as shown in SEQ ID NO.38, CDR-L2 as shown in SEQ ID NO.49, and CDR-L3 as shown in SEQ ID NO.60; or The capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.12, and CDR-H3 as shown in SEQ ID NO.23; and CDR-L1 as shown in SEQ ID NO.34, CDR-L2 as shown in SEQ ID NO.45, and CDR-L3 as shown in SEQ ID NO.56; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.9, CDR-H2 as shown in SEQ ID NO.20, and CDR-H3 as shown in SEQ ID NO.31; and CDR-L1 as shown in SEQ ID NO.42, CDR-L2 as shown in SEQ ID NO.53, and CDR-L3 as shown in SEQ ID NO.64; or The capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.3, CDR-H2 as shown in SEQ ID NO.14, and CDR-H3 as shown in SEQ ID NO.25; and CDR-L1 as shown in SEQ ID NO.36, CDR-L2 as shown in SEQ ID NO.47, and CDR-L3 as shown in SEQ ID NO.58; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.5, CDR-H2 as shown in SEQ ID NO.16, and CDR-H3 as shown in SEQ ID NO.27; and CDR-L1 as shown in SEQ ID NO.38, CDR-L2 as shown in SEQ ID NO.49, and CDR-L3 as shown in SEQ ID NO.60; or The capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.2, CDR-H2 as shown in SEQ ID NO.13, and CDR-H3 as shown in SEQ ID NO.24; and CDR-L1 as shown in SEQ ID NO.35, CDR-L2 as shown in SEQ ID NO.46, and CDR-L3 as shown in SEQ ID NO.57; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63; or The capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.10, CDR-H2 as shown in SEQ ID NO.21, and CDR-H3 as shown in SEQ ID NO.32; and CDR-L1 as shown in SEQ ID NO.43, CDR-L2 as shown in SEQ ID NO.54, and CDR-L3 as shown in SEQ ID NO.65; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63; or The capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.11, CDR-H2 as shown in SEQ ID NO.22, and CDR-H3 as shown in SEQ ID NO.33; and CDR-L1 as shown in SEQ ID NO.44, CDR-L2 as shown in SEQ ID NO.55, and CDR-L3 as shown in SEQ ID NO.66; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.63; or The capture antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.12, and CDR-H3 as shown in SEQ ID NO.23; and CDR-L1 as shown in SEQ ID NO.34, CDR-L2 as shown in SEQ ID NO.45, and CDR-L3 as shown in SEQ ID NO.56; the detection antibody is an antibody comprising CDR-H1 as shown in SEQ ID NO.8, CDR-H2 as shown in SEQ ID NO.19, and CDR-H3 as shown in SEQ ID NO.30; and CDR-L1 as shown in SEQ ID NO.41, CDR-L2 as shown in SEQ ID NO.52, and CDR-L3 as shown in SEQ ID NO.

63.

12. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the conjugate according to claim 9.

13. The kit according to claim 12, wherein The kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. A kit comprising the antibody pair according to claim 10 or 11.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody pair according to claim 10 or 11, or the conjugate according to claim 9 in the preparation of a kit for detecting the presence or level of cardiac troponin I in a sample from a subject.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody pair according to claim 10 or 11, or the conjugate according to claim 9 in the preparation of a kit for assisting in the diagnosis of myocardial injury.

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