Anti-cardiac troponin I antibody and application thereof

Abstract

The invention discloses an anti-cardiac troponin I antibody and application thereof, and relates to the field of antibodies. The anti-cardiac troponin I antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detection of cardiac troponin I, and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-cardiac troponin I antibody and an application thereof. Background Art

[0002] Before the 1980s, the World Health Organization (WHO) had always used myocardial enzyme spectrum activity as one of the diagnostic criteria for acute myocardial infarction (AMI). In the late 1980s, researchers found that the sensitivity and specificity of troponin (Tn) were higher than biomarkers such as creatine phosphokinase (CK), creatine phosphokinase isoenzyme (CK-MB), lactate dehydrogenase and aspartate aminotransferase. Cardiac troponin (cTnI) exists only in the myocardium and is a marker of myocardial cells. Its abnormal changes can affect the systolic and diastolic function of the heart and can be used to diagnose myocardial necrosis and judge myocardial damage. It has become one of the most sensitive and specific markers of myocardial cell damage and is recognized as a major biochemical marker for rapid diagnosis of AMI and acute coronary syndrome (ACS), as well as for assisting ACS risk stratification and reflecting its prognosis.

[0003] The level of cTnI in normal human blood is generally lower than 0.3 μg / L. When the integrity of myocardial cell membrane is damaged due to ischemia or hypoxia, free cTnI can quickly penetrate the cell membrane and enter the bloodstream. Therefore, rapid, sensitive and accurate determination of cTnI in human blood and its changing trend in the early stage of the disease are of great significance for the diagnosis of acute myocardial infarction, risk stratification of acute coronary syndrome, and monitoring of myocardial damage caused by various factors.

[0004] Immunological detection methods are a means of detection based on the specific reaction of antibodies and antigens. Currently, immunological detection methods for detecting cTnI include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold, etc. Any of the above immunological detection methods requires antibodies against cTnI when used for cTnI detection. Therefore, those skilled in the art have a strong demand for anti-cTnI antibodies with good performance. Summary of the invention

[0005] The present application provides an anti-cardiac troponin I antibody or an antigen-binding fragment thereof, which provides an important source of raw materials for the detection of cardiac troponin I and has good activity or affinity.

[0006] In order to achieve the above-mentioned object, according to one aspect of the present invention, an anti-cardiac troponin I antibody or an antigen-binding fragment thereof is provided, wherein the anti-cardiac troponin I antibody or an antigen-binding fragment thereof comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence of any one of SEQ ID NOs: 19 and 20 and three complementarity determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NOs: 23 and 24.

[0007] In order to achieve the above object, according to a second aspect of the present invention, an anti-cardiac troponin I antibody or an antigen-binding fragment thereof is provided, wherein the anti-cardiac troponin I antibody or an antigen-binding fragment thereof comprises the following complementarity determining regions:

[0008] HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1;

[0009] HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2;

[0010] HCDR3, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 3 and 17;

[0011] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0012] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and

[0013] LCDR3 comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 6 and 18.

[0014] In order to achieve the above-mentioned object, according to the third aspect of the present invention, an anti-cardiac troponin I antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 19 and 20; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 23 and 24.

[0015] In order to achieve the above-mentioned object, according to the fourth aspect of the present invention, there is provided an anti-cardiac troponin I antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 21 and 22; and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 25 and 26.

[0016] In order to achieve the above object, according to the fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above anti-cardiac troponin I antibody or an antigen-binding fragment thereof.

[0017] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or a kit is provided, wherein the reagent or the kit comprises the above-mentioned anti-cardiac troponin I antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0018] To achieve the above-mentioned object, according to the seventh aspect of the present invention, there is provided a method for detecting cardiac troponin I, comprising: a) contacting the above-mentioned anti-cardiac troponin I antibody or its antigen-binding fragment, antibody conjugate, or reagent or kit with cardiac troponin I in a sample to be detected to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0019] In order to achieve the above object, according to the eighth aspect of the present invention, a nucleic acid is provided, which encodes the above-mentioned anti-cardiac troponin I antibody or its antigen-binding fragment.

[0020] In order to achieve the above object, according to the ninth aspect of the present invention, a vector is provided, wherein the vector comprises the above nucleic acid.

[0021] To achieve the above object, according to the tenth aspect of the present invention, a cell is provided, wherein the cell comprises the above nucleic acid, vector or expresses the above anti-cardiac troponin I antibody or antigen-binding fragment thereof.

[0022] In order to achieve the above object, according to the eleventh aspect of the present invention, a method for preparing the above anti-cardiac troponin I antibody or its antigen-binding fragment is provided, the method comprising culturing the above cells.

[0023] In order to achieve the above-mentioned object, according to the twelfth aspect of the present invention, there is provided a use of the above-mentioned anti-cardiac troponin I antibody or its antigen-binding fragment, antibody conjugate, reagent or kit in the preparation of a product for detecting cardiac troponin I. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 1 The results of reducing SDS-PAGE of Anti-CTNI 8D10 Rmb1 to Anti-CTNI 8D10 Rmb3 are shown. DETAILED DESCRIPTION

[0026] In the first aspect, an embodiment of the present invention provides an anti-cardiac troponin I antibody or an antigen-binding fragment thereof, wherein the anti-cardiac troponin I antibody or an antigen-binding fragment thereof comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence of any one of SEQ ID NOs: 19 and 20 and three complementarity determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NOs: 23 and 24.

[0027] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the anti-cardiac troponin I antibody or its antigen-binding fragment described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the anti-cardiac troponin I antibody or its antigen-binding fragment described in the first aspect.

[0028] For example, the HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:19; the LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:23.

[0029] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0030] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0031] In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0032] The definition methods of CDR are well known in the art, and the CDR definition methods include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., US Pept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above schemes, but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or extended based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also belong to the scope of protection of the present disclosure.

[0033] Table 1: CDR Definition 1

[0034]

[0035]

[0036] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with the amino acid numbering on the heavy chain represented by "H+number" and the amino acid numbering on the light chain represented by "L+number". One of ordinary skill in the art can unambiguously map the Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Patent. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0037] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0038] 3If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0039] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0040] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0041] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

[0042] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0043] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0044] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0045] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0046] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0047] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0048] According to an embodiment of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, Chothia, AbM or IMGT systems are as follows:

[0049]

[0050]

[0051] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions:

[0052] HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1;

[0053] HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2;

[0054] HCDR3, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 3 and 17;

[0055] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0056] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and

[0057] LCDR3 comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 6 and 18.

[0058] In a second aspect, an embodiment of the present invention provides an anti-cardiac troponin I antibody or an antigen-binding fragment thereof, wherein the anti-cardiac troponin I antibody or an antigen-binding fragment thereof comprises the following complementary determining regions:

[0059] HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1;

[0060] HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2;

[0061] HCDR3, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 3 and 17;

[0062] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0063] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and

[0064] LCDR3 comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 6 and 18.

[0065] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0066] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0067] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0068] In an optional embodiment, the anti-cardiac troponin I antibody or antigen-binding fragment thereof of the first aspect or the second aspect further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

[0069] The HFR1 comprises / is SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

[0070] The HFR2 comprises / is SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;

[0071] The HFR3 comprises / is SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

[0072] The HFR4 comprises / is SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0073] The LFR1 comprises / is SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;

[0074] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0075] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto;

[0076] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0077] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the anti-cardiac troponin I antibodies or antigen-binding fragments thereof provided by the present invention may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0078] In an optional embodiment, the anti-cardiac troponin I antibody or antigen-binding fragment thereof has a KD of less than 8.19×10 -8 M binds cardiac troponin I with high affinity.

[0079] In an optional embodiment, the anti-cardiac troponin I antibody or antigen-binding fragment thereof has a KD≤10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M, KD≤10 -12 M, KD≤10 -13 M binds cardiac troponin I with high affinity.

[0080] In an optional embodiment, the anti-cardiac troponin I antibody or antigen-binding fragment thereof has a KD of ≤1.89×10 -9 M binds cardiac troponin I with high affinity.

[0081] There are many methods for determining antibody affinity (KD), which can be divided into thermodynamic detection methods, kinetic detection methods and dynamic equilibrium detection methods according to the detection principle. Among them, thermodynamic detection methods are commonly used, such as isothermal titration calorimetry (ITC); kinetic detection methods are commonly used, such as surface plasmon resonance (SPR) and biomembrane interferometry (BLI); dynamic equilibrium detection methods are commonly used, such as enzyme-linked immunosorbent assay (ELISA).

[0082] In an alternative embodiment, KD is determined using a kinetic assay; optionally, surface plasmon resonance, for example by using a kinetic assay such as Biosensor system of the system.

[0083] In a third aspect, an embodiment of the present invention provides an anti-cardiac troponin I antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 19 and 20, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 23 and 24.

[0084] In an optional embodiment, the heavy chain variable region and the light chain variable region described in the first aspect or the third aspect are selected from any combination of the following:

[0085] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:19 SEQ ID NO:23 2 SEQ ID NO:20 SEQ ID NO:23 3 SEQ ID NO:19 SEQ ID NO:24 .

[0086] In an optional embodiment, the antibody or antigen-binding fragment thereof described in the first aspect, the second aspect or the third aspect comprises any combination of the following heavy chain variable regions and light chain variable regions:

[0087] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:19 SEQ ID NO:23 2 SEQ ID NO:20 SEQ ID NO:23 3 SEQ ID NO:19 SEQ ID NO:24 .

[0088] In an optional embodiment, the anti-cardiac troponin I antibody or the antigen-binding fragment thereof of the first aspect, the second aspect or the third aspect further comprises a constant region.

[0089] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0090] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0091] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0092] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0093] In an alternative embodiment, the light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.

[0094] In an alternative embodiment, the constant region is derived from a species of cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0095] In an alternative embodiment, the species origin of the constant region is rabbit.

[0096] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:16.

[0097] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0098] In an optional embodiment, the antigen-binding fragment is selected from any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0099] The antigen-binding fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for those skilled in the art to understand based on the contents described in the present invention that the antigen-binding fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, those skilled in the art can easily obtain the above antigen-binding fragments.

[0100] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0101] In a fourth aspect, the present invention provides an anti-cardiac troponin I antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 21 and 22, and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 25 and 26.

[0102] In an optional embodiment, the antibody or antigen-binding fragment thereof of the first, second, third or fourth aspect above comprises any combination of the following heavy chains and light chains:

[0103] combination Heavy chain Light chain 1 SEQ ID NO:21 SEQ ID NO:25 2 SEQ ID NO:22 SEQ ID NO:25 3 SEQ ID NO:21 SEQ ID NO:26

[0104] In a fifth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned anti-cardiac troponin I antibody or an antigen-binding fragment thereof.

[0105] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the anti-cardiac troponin I antibody or its antigen-binding fragment.

[0106] In an optional embodiment, the antibody conjugate further comprises a label coupled to the anti-cardiac troponin I antibody or its antigen-binding fragment.

[0107] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0108] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0109] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0110] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0111] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0112] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0113] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0114] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0115] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0116] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0117] In an optional embodiment, the colloidal metal is colloidal gold.

[0118] In an optional embodiment, the antibody conjugate further comprises a solid phase carrier coupled to the anti-cardiac troponin I antibody or its antigen-binding fragment.

[0119] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0120] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.

[0121] In a sixth aspect, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned anti-cardiac troponin I antibody or its antigen-binding fragment or the above-mentioned antibody conjugate.

[0122] As mentioned above, the anti-cardiac troponin I antibody or its antigen-binding fragment in some embodiments or examples of the present invention can effectively bind to cardiac troponin I, therefore, the reagent or kit comprising the anti-cardiac troponin I antibody or its antigen-binding fragment can effectively detect cardiac troponin I qualitatively or quantitatively. The reagent or kit provided by the present invention can be used, for example, for detection involving the specific binding properties of cardiac troponin I and its antibody, such as immunoblotting and immunoprecipitation. As mentioned above, the anti-cardiac troponin I antibody or its antigen-binding fragment in some embodiments or examples of the present invention has a higher binding activity or affinity with cardiac troponin I, therefore, the reagent or kit comprising the anti-cardiac troponin I antibody or its antigen-binding fragment has a higher detection sensitivity or specificity.

[0123] In a seventh aspect, the present invention provides a method for detecting cardiac troponin I, comprising: a) contacting the above-mentioned anti-cardiac troponin I antibody or its antigen-binding fragment, antibody conjugate, reagent or kit with cardiac troponin I in a sample to be tested to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample;

[0124] In an optional embodiment, the immune complex further comprises a second antibody, which binds to the anti-cardiac troponin I antibody or an antigen-binding fragment thereof.

[0125] In an alternative embodiment, the immune complex further comprises a second antibody, which binds to cardiac troponin I.

[0126] In an eighth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned anti-cardiac troponin I antibody or its antigen-binding fragment.

[0127] In a ninth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0128] In a tenth aspect, the present invention provides a cell containing the above-mentioned vector.

[0129] In an eleventh aspect, the present invention provides a method for preparing an anti-cardiac troponin I antibody or an antigen-binding fragment thereof, comprising: culturing the cells as described above.

[0130] In a twelfth aspect, the present invention provides use of the above-mentioned anti-cardiac troponin I antibody or its antigen-binding fragment, antibody conjugate or the above-mentioned reagent or kit in the preparation of a product for detecting cardiac troponin I.

[0131] Based on the amino acid sequence of the anti-cardiac troponin I antibody or its antigen-binding fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-cardiac troponin I antibody or its antigen-binding fragment, for example, separating and purifying the anti-cardiac troponin I antibody or its antigen-binding fragment from the culture product of a recombinant cell capable of recombinantly expressing the anti-cardiac troponin I antibody or its antigen-binding fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the anti-cardiac troponin I antibody or its antigen-binding fragment of the present invention, it belongs to the protection scope of the present invention.

[0132] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0133] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0134] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0135] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0136] Example 1 Preparation of Anti-CTNI 8D10 Monoclonal Antibody

[0137] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. Rabbit monoclonal antibodies against cardiac troponin can be obtained by hybridoma technology, phage display technology or single B cell cloning technology to obtain antibody sequences. The specific implementation method of phage display technology is as follows:

[0138] 1. Gene acquisition

[0139] (1) Rabbit immunization

[0140] An emulsified preparation of cardiac troponin prepared with incomplete Freund's adjuvant was injected subcutaneously to stimulate the immune response of New Zealand white rabbits aged 4 to 6 weeks, and pre- and post-immunization sera were collected on days 0, 14, 28, 42, and 69, respectively. The spleens of the rabbits were then surgically removed and spleen cell suspensions were prepared.

[0141] (2) Construction of phage library

[0142] RNA was extracted from rabbit spleen cells and reverse transcribed into cDNA. Specifically designed rabbit antibody gene amplification primers were used to amplify VH and VL gene fragments using cDNA as templates. Then, the VH and VL gene fragments were inserted into the phage vector V02 in sequence by restriction ligation. Finally, the ligated phage plasmid was electrotransformed into TG1 competent cells. The next day, monoclonal colonies were selected for PCR identification and antibody gene sequencing to evaluate the quality of the phage library. After passing the test, the phage library was screened.

[0143] (3) Screening of phage libraries

[0144] The phage library TG1 bacterial solution was inoculated into a shake flask, and when the bacterial solution reached an appropriate concentration (OD600 of 0.8-1.0), helper phage was added for infection for 1 hour, and then the culture was continued overnight. The bacterial solution was collected the next day for centrifugation, and the supernatant was taken and purified by salting-out precipitation to obtain the displayed phage library.

[0145] The phage library was panned for 3-4 rounds using the magnetic bead panning method, and then monoclonal phage-infected colonies were selected for antibody supernatant expression. The monoclonal phage antibody expression supernatant was then screened and identified using the ELISA screening method to obtain monoclonal phage against cardiac troponin.

[0146] Phage antibody gene sequencing: Anti-cardiac troponin monoclonal phage was sequenced for antibody gene. Through sequence analysis, duplicate and invalid sequences were removed to obtain a unique rabbit monoclonal antibody sequence.

[0147] 2. Expression Plasmid Construction

[0148] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has been modified to introduce multiple cloning restriction sites and is hereinafter referred to as 3.4A expression vector; based on the variable region genes obtained in the above experiment, VL and VH gene-specific primers were designed, with restriction endonuclease sites and protective bases at both ends, respectively, and 0.71Kb Light Chain gene fragment and 1.35Kb Heavy Chain gene fragment were amplified by PCR amplification method.

[0149] The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was double-digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of the Heavy Chain and Light Chain, respectively.

[0150] 3. Recombinant Antibody Sample Preparation

[0151] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, the cell density reaches the selected antibody concentration and cells, and the cell viability is >95%; the cells are washed by centrifugation and re-dissolved with culture medium, and the cell density is adjusted to 2.9×10 6 cells / ml, wash the cells, and re-dissolve them with culture medium, and use it as a cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to the cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of the cells after transfection, and place them in a 35℃ constant temperature incubator for culture, with a speed of 120rmp and a CO2 content of 8%. Centrifuge and collect samples after 13 days. The centrifuged supernatant was affinity purified using a proteinA affinity chromatography column. Take 6ug layer of purified antibodies for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands are shown, one Mr is 50KD (heavy chain) and the other Mr is 28KD (light chain).

[0152] The obtained antibody was named Anti-CTNI 8D10Rmb1. Anti-CTNI 8D10Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table:

[0153] Table 2 Antibody sequences

[0154] Antibody Name Heavy chain Light chain Anti-CTNI 8D10Rmb1 SEQ ID NO:21 SEQ ID NO:25 Anti-CTNI 8D10Rmb2 SEQ ID NO:22 SEQ ID NO:25 Anti-CTNI 8D10Rmb3 SEQ ID NO:21 SEQ ID NO:26

[0155] Example 2 Performance testing of antibodies

[0156] 1. Affinity Analysis

[0157] The purified antibody was diluted in advance, and the CTNI recombinant antigen was gradiently diluted at the same time; the binding and dissociation curves of the antigen and antibody were tested on the Biacore 8K+ device using a CM5 chip that had been pre-coupled with goat anti-rabbit IgG, and the instrument automatically fitted the affinity constant, binding rate, and dissociation rate (KD represents the equilibrium dissociation constant, i.e., affinity constant; ka represents the binding rate; and kd represents the dissociation rate).

[0158] Table 3 Affinity data

[0159] Sample name KD(M) ka kd Comparison 8.19E-08 1.14E+04 9.34E-04 Anti-CTNI 8D10Mb1 1.27E-09 3.81E+05 4.83E-04 Anti-CTNI 8D10Mb2 1.89E-09 2.79E+05 5.27E-04 Anti-CTNI 8D10Mb3 1.46E-09 3.02E+05 4.40E-04

[0160] 2. Activity identification

[0161] The coating solution (main component NaHCO3) was used to dilute the CTNI recombinant antigen to 0.5ug / ml, 100uL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with the washing solution (main component Na2HPO4+Nacl), and the wells were patted dry; the blocking solution (20% BSA+80% PBS) was added, 120uL per well, incubated at 37°C for 1h, and the wells were patted dry; the diluted purified antibody and control antibody were added, 100uL / well, incubated at 37°C for 30min; the cells were washed 5 times with the washing solution, and the wells were patted dry; the goat anti-rabbit IgG-HRP was added, 100uL per well, incubated at 37°C for 30min; the cells were washed 5 times with the washing solution, and the wells were patted dry; the colorimetric solution A (50uL / well) and the colorimetric solution B (50uL / well) were added, and the wells were 10min; the stop solution was added, 50uL / well; the OD value was read at 450nm (reference 630nm) on the microplate reader.

[0162] Note: Solution A (main ingredients: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main ingredients: citric acid + EDTA·2Na + TMB + concentrated HCL); Stop solution (EDTA·2Na + concentrated H2SO4).

[0163] Table 4 Activity data

[0164] Concentration (ng / ml) 6.25 3.13 1.56 0.78 0.39 0.00 Comparison 1.313 0.785 0.443 0.232 0.122 0.009 Anti-CTNI 8D10Rmb1 1.844 1.381 0.795 0.414 0.235 0.008 Anti-CTNI 8D10Rmb2 1.822 1.312 0.725 0.425 0.257 0.010 Anti-CTNI 8D10Rmb3 1.835 1.377 0.776 0.478 0.236 0.009

[0165] 3. Stability assessment

[0166] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant incubator) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies are stable. Table 5 below shows the OD results of the enzyme immunoassay activity test of the antibody Anti-CTNI 8D10Rmb1 for 21 days.

[0167] Table 5 Stability data

[0168] Sample concentration (ng / ml) 3.13 1.56 0.00 4℃, 21-day samples 1.355 0.766 0.012 -80℃, 21 days sample 1.364 0.752 0.014 37℃, 21 days sample 1.378 0.774 0.013

[0169] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0170] Some of the amino acid sequences involved in this application are shown in Table 6:

[0171]

[0172]

Claims

1. An anti-cardiac troponin I antibody or an antigen-binding fragment thereof, wherein the anti-cardiac troponin I antibody or an antigen-binding fragment thereof comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence of any one of SEQ ID NOs: 19 and 20 and three complementary determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NOs: 23 and 24.

2. The anti-cardiac troponin I antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An anti-cardiac troponin I antibody or an antigen-binding fragment thereof, characterized in that: The anti-cardiac troponin I antibody or its antigen-binding fragment comprises the following complementarity determining region: HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; HCDR3, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 3 and 17; LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and LCDR3, which comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 6 and 18; Optionally, the anti-cardiac troponin I antibody or antigen-binding fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; The HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto; In an optional embodiment, the anti-cardiac troponin I antibody or antigen-binding fragment thereof has a KD of less than 8.19×10 - 8 M binds cardiac troponin I with high affinity.

4. An anti-cardiac troponin I antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 19 and 20; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 23 and 24; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations: ； Optionally, the anti-cardiac troponin I antibody or antigen-binding fragment thereof further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is rabbit; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto; Optionally, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

5. An anti-cardiac troponin I antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 21 and 22; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 25 and 26.

6. An antibody conjugate, characterized in that: The antibody conjugate comprises the anti-cardiac troponin I antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative coupled to the anti-cardiac troponin I antibody or antigen-binding fragment thereof; Optionally, the antibody conjugate further comprises a label coupled to the anti-cardiac troponin I antibody or antigen-binding fragment thereof; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the anti-cardiac troponin I antibody or its antigen-binding fragment.

7. A reagent or a kit, characterized in that: The reagent or kit comprises the anti-cardiac troponin I antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. A method for detecting cardiac troponin I, characterized in that: include: a) contacting the anti-cardiac troponin I antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with cardiac troponin I in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the anti-cardiac troponin I antibody or an antigen-binding fragment thereof; Optionally, the immune complex further comprises a second antibody, which binds to cardiac troponin I.

9. A nucleic acid, a vector, a cell or a method for preparing an anti-cardiac troponin I antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The nucleic acid encodes the anti-cardiac troponin I antibody or its antigen-binding fragment according to any one of claims 1 to 5; the vector contains the above-mentioned nucleic acid; the cell contains the above-mentioned nucleic acid or vector; and the method includes culturing the above-mentioned cell.

10. Use of the anti-cardiac troponin I antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 in the preparation of a product for detecting cardiac troponin I.

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