Anti-cardiac troponin I antibody and application thereof

Abstract

The invention discloses an antibody for resisting cardiac troponin I, 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 specificity and activity.

Description

[0001] Cross-referencing:

[0002] This application claims priority to Chinese Patent Application No. 202411081257.4, filed on August 7, 2024, entitled "An Antibody Against Cardiac Troponin I and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and more specifically, to an antibody against cardiac troponin I and its application. Background Technology

[0004] In the late 1980s, cTnI and cTnT were successively recommended as biomarkers for cardiomyocyte death. Today, both biomarkers are recommended by guidelines for the auxiliary diagnosis of clinicopathological myocardial injury such as acute myocardial infarction (AMI), postoperative myocardial trauma, and chemotherapy-induced cardiotoxicity, as well as myocardial injury associated with other diseases. In the late 1990s, the existing cTnI and cTnT reagents could detect troponin in patients at levels in ng / mL (μg / L). This level of sensitivity meant that troponin could only be reliably detected 3-6 hours after the onset of ischemic symptoms such as chest pain. This limited troponin to a late-stage biomarker for AMI.

[0005] Troponin complex is an important protein involved in the contraction of striated muscle and myocardium. In the heart, cardiac troponin complex is composed of myocardium-specific cTnI, cTnT, and cTnC (cardiac troponin C). After a myocardial infarction, necrotic myocardial tissue releases troponin complex into the bloodstream. The detection of cTnI and cTnT is now widely used. Troponin-I (TnI) is the regulatory subunit of the troponin complex and is associated with actin filaments in muscle cells. Three different tissue-specific TnI isoforms have been identified in skeletal and cardiac muscle. The cardiac isoform shares only 60% similarity with the skeletal muscle isoform; the molecular weight of cardiac troponin-I (cTnI) is approximately 24,000 Daltons. Clinical trials have demonstrated that cTnI is released into the bloodstream within hours of myocardial infarction (MI) or ischemic injury.2,3 Elevated cTnI levels can be detected in serum within 4-6 hours of chest pain onset (higher than those in samples from non-myocardial infarction patients), peaking within approximately 8-28 hours and remaining at a high level for 3-10 days after myocardial infarction. Compared to other existing markers of myocardial necrosis such as CK-MB, myoglobin, and lactate dehydrogenase, cardiac troponin has higher sensitivity and superior tissue specificity, making it the preferred diagnostic tool for myocardial injury. In diagnostic testing, the troponin complex ITC (a complex of cTnI, cTnT, and cTnC) and IC (a complex of cTnI and cTnC) are also targets.

[0006] Currently, all clinical methods for detecting cTnI, whether enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, or colloidal gold assay, require cTnI antibodies with high specificity, activity, and sensitivity. Therefore, developing antibodies with high specificity, activity, sensitivity, and clinical relevance is of great significance. Among these, antibodies that can simultaneously bind to ITC and IC antibodies can achieve more accurate detection and improve detection sensitivity. Summary of the Invention

[0007] This application provides an antibody against cardiac troponin I, which provides an important source of raw materials for the detection of cardiac troponin I. Moreover, the antibody has high detection sensitivity, strong specificity, and high clinical relevance.

[0008] To achieve the above objectives, according to one aspect of the present invention, an antibody against cardiac troponin I is provided, the antibody comprising three complementary determining regions having a heavy chain variable region having an amino acid sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:3 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:4 to SEQ ID NO:6.

[0009] To achieve the above objective, according to a second aspect of the present invention, an antibody against cardiac troponin I is provided, the antibody comprising the following complementarity-determining region:

[0010] HCDR1, which contains or consists of any of the following amino acid sequences: amino acids 30 to 34 of SEQ ID NO:1, amino acids 30 to 34 of SEQ ID NO:2 and amino acids 30 to 34 of SEQ ID NO:3;

[0011] HCDR2, which contains or consists of any of the following amino acid sequences: amino acids 49 to 63 of SEQ ID NO:1, amino acids 49 to 65 of SEQ ID NO:2, and amino acids 49 to 64 of SEQ ID NO:3;

[0012] HCDR3, which contains or consists of any of the following amino acid sequences: amino acids 94 to 103 of SEQ ID NO:1, amino acids 97 to 106 of SEQ ID NO:2, and amino acids 97 to 108 of SEQ ID NO:3;

[0013] LCDR1, which contains or consists of any of the following amino acid sequences: amino acids 24 to 36 of SEQ ID NO:4, amino acids 24 to 34 of SEQ ID NO:5 and amino acids 24 to 34 of SEQ ID NO:6;

[0014] LCDR2, which contains or consists of any of the following amino acid sequences: amino acids 52 to 58 of SEQ ID NO:4, amino acids 50 to 56 of SEQ ID NO:5 and amino acids 50 to 56 of SEQ ID NO:6;

[0015] LCDR3, which contains or consists of any of the following amino acid sequences: amino acids 91 to 100 of SEQ ID NO:4, amino acids 89 to 100 of SEQ ID NO:5, and amino acids 89 to 102 of SEQ ID NO:6.

[0016] To achieve the above objectives, according to a third aspect of the present invention, an antibody against cardiac troponin I is provided, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is shown as any one of SEQ ID NO:1 to SEQ ID NO:3; and the amino acid sequence of the light chain variable region is shown as any one of SEQ ID NO:4 to SEQ ID NO:6.

[0017] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody against cardiac troponin I is provided, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain of the aforementioned antibody is shown as any one of SEQ ID NO:33 to SEQ ID NO:35; and the amino acid sequence of the light chain is shown as any one of SEQ ID NO:36 to SEQ ID NO:38.

[0018] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the antibodies described above.

[0019] To achieve the above objectives, according to a sixth aspect of the present invention, a reagent or kit is provided, the reagent or kit comprising the antibody or antibody conjugate described above.

[0020] To achieve the above objectives, according to a seventh aspect of the present invention, the use of the above-described antibody, antibody-drug conjugate, reagent, or kit in the preparation of a product for detecting cardiac troponin I is provided.

[0021] To achieve the above objectives, the present invention also provides a nucleic acid molecule, a vector, a cell, and a method for preparing the above-mentioned antibody. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Clinical sample standard curve of chemiluminescence platform

[0024] Figure 2 Clinical sample standard curve of fluorescence platform Detailed Implementation

[0025] In a first aspect, embodiments of the present invention provide an antibody against cardiac troponin I, the aforementioned antibody comprising three complementary determining regions having a heavy chain variable region having an amino acid sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:3 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:4 to SEQ ID NO:6.

[0026] In an optional implementation, the complementary decision region described in the first aspect above is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0027] In this invention, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. An antigen-binding fragment is a substance containing an antibody CDR that lacks some amino acids present in the full-length chain but can still specifically bind to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), and the smallest unit of antibody recognition. The above-mentioned antigen-binding fragments can bind to the same antigen as the parent antibody.

[0028] Antigen-binding fragments typically possess the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the aforementioned antigen-binding fragments can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0029] Antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0030] In this invention, the term "cardiac troponin I" includes at least one form of CTNI. Besides CTNI itself, CTNI can also exist as ITC and IC, which are complexes of cTnI, cTnT, and cTnC, and IC is a complex of cTnI and cTnC. Antibodies capable of simultaneously binding to various forms of CTNI can achieve more accurate detection and improve detection sensitivity.

[0031] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of antibodies.

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

[0033] Methods for defining CDRs are well-known in the art and 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., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow any 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 lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below; definitions vary slightly in different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0034] Table 1: CDR Definition 1

[0035] CDR Kabat AbM2 IMGT Chothia <![CDATA[Contact 6 ]]> HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> H30--H35 HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 H47--H58 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 H93--H101 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 L30--L36 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 L46--L55 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97 L89--L96

[0036] 1In Table 1, except for the Contact definition system where the CDR is based on the Chothia numbering system, the CDRs defined in other definition systems are based on the Kabat numbering system (see below). Amino acid numbers on the heavy chain are represented by "H + number," and amino acid numbers on the light chain are represented by "L + number." Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia numbering" as used herein refers to the numbering system described by Al-Lazikani et al., (1997) JMB 273, 927-948.

[0037] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0038] 3 If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0039] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0040] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0041] 6 The CDR numbering shown in Table 1 under the Contact definition system is based on the Chothia numbering system, and the Contact definition system is only applicable to the Chothia or Martin numbering system.

[0042] According to an embodiment of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 are numbered by the Kabat system.

[0043] According to embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

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

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

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

[0047] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0048] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0049] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0050] Secondly, embodiments of the present invention provide an antibody against cardiac troponin I, wherein the antibody comprises the following complementarity-determining region:

[0051] HCDR1, which contains or consists of any of the following amino acid sequences: amino acids 30 to 34 of SEQ ID NO:1, amino acids 30 to 34 of SEQ ID NO:2 and amino acids 30 to 34 of SEQ ID NO:3;

[0052] HCDR2, which contains or consists of any of the following amino acid sequences: amino acids 49 to 63 of SEQ ID NO:1, amino acids 49 to 65 of SEQ ID NO:2, and amino acids 49 to 64 of SEQ ID NO:3;

[0053] HCDR3, which contains or consists of any of the following amino acid sequences: amino acids 94 to 103 of SEQ ID NO:1, amino acids 97 to 106 of SEQ ID NO:2, and amino acids 97 to 108 of SEQ ID NO:3;

[0054] LCDR1, which contains or consists of any of the following amino acid sequences: amino acids 24 to 36 of SEQ ID NO:4, amino acids 24 to 34 of SEQ ID NO:5 and amino acids 24 to 34 of SEQ ID NO:6;

[0055] LCDR2, which contains or consists of any of the following amino acid sequences: amino acids 52 to 58 of SEQ ID NO:4, amino acids 50 to 56 of SEQ ID NO:5 and amino acids 50 to 56 of SEQ ID NO:6;

[0056] LCDR3, which contains or consists of any of the following amino acid sequences: amino acids 91 to 100 of SEQ ID NO:4, amino acids 89 to 100 of SEQ ID NO:5, and amino acids 89 to 102 of SEQ ID NO:6.

[0057] According to an embodiment of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0058] It should be noted that the above position numbering was obtained by sequentially numbering the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:6 from the N-terminus to the C-terminus. For example, amino acids 30 to 34 of SEQ ID NO:1 refer to the amino acid residues corresponding to positions 30 to 34 from the N-terminus of the amino acid sequence shown in SEQ ID NO:1.

[0059] In an optional embodiment, the complementarity-determining region of the antibody includes any one of (a) to (c):

[0060] (a) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are as follows: amino acids 30-34, 49-63, and 94-103 of SEQ ID NO:1; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as follows: amino acids 24-36, 52-58, and 91-100 of SEQ ID NO:4.

[0061] (b) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are as follows: amino acids 30–34, 49–65, and 97–106 of SEQ ID NO:2; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as follows: amino acids 24–34, 50–56, and 89–100 of SEQ ID NO:5; and

[0062] (c) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are as follows: amino acids 30-34, 49-64, and 97-108 of SEQ ID NO:3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as follows: amino acids 24-34, 50-56, and 89-102 of SEQ ID NO:6.

[0063] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0064] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0065] In optional embodiments, the antibodies described in the first and second aspects further comprise HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0066] In an optional embodiment, the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 of the above-mentioned antibody include any one of (a') to (c'):

[0067] (a') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:7 to SEQ ID NO:10, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:11 to SEQ ID NO:14; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions.

[0068] (b') The amino acid sequences are as shown in SEQ ID NO:15 to SEQ ID NO:18, HFR1, HFR2, HFR3, HFR4, and the amino acid sequences are as shown in SEQ ID NO:19 to SEQ ID NO:22, LFR1, LFR2, LFR3, LFR4; or amino acid sequences having at least 80% identity with the sequences of each of the frame regions; and

[0069] (c') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:23 to SEQ ID NO:26, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:27 to SEQ ID NO:30; or the amino acid sequences have at least 80% identity with the sequences of each of the frame regions.

[0070] In this invention, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0071] In other embodiments, the amino acid sequences of each frame region of the anti-cardiac troponin I antibody provided by the present invention 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 corresponding frame regions described above.

[0072] Thirdly, embodiments of the present invention provide an antibody against cardiac troponin I, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is shown as any one of SEQ ID NO:1 to SEQ ID NO:3; and the amino acid sequence of the light chain variable region is shown as any one of SEQ ID NO:4 to SEQ ID NO:6.

[0073] In an optional implementation, the heavy chain variable region and the light chain variable region described in the third aspect above are selected from any combination of (A) to (C):

[0074] (A) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:4;

[0075] (B) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:5; and

[0076] (C) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:3 and the light chain variable region with the amino acid sequence shown in SEQ ID NO:6.

[0077] In an optional implementation, the aforementioned antibody comprises any one of F(ab')2, Fab', Fab, Fv, and scFv.

[0078] In optional embodiments, the antibodies described in the first, second, or third aspects above further include a constant region.

[0079] In an optional implementation, the aforementioned constant region includes a heavy chain constant region and a light chain constant region.

[0080] In an optional embodiment, the aforementioned 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.

[0081] In an optional embodiment, the aforementioned heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM, and / or the tail peptide of IgM.

[0082] In this paper, the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) are all components of the heavy chain constant region, which is located at the C-terminus of the heavy chain of the antibody molecule. Each heavy chain constant region, from the N-terminus to the C-terminus, includes the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional). Different types of antibodies (such as IgG, IgA, IgM, etc.) have different amino acid sequences and structures in their heavy chain constant regions, but they all have relatively conserved structural features. These conserved structures enable the heavy chain constant region to perform its biological function. The heavy chain constant region, CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) of different species and subclasses are well known in the art, and their amino acid sequences can be determined based on bioinformatics databases, such as the IMGT database (https: / / www.imgt.org / IMGTrepertoire / Proteins / ). It should be understood that different bioinformatics databases or software may not have completely consistent results in the division and sequence identification of constant regions. However, those skilled in the art have a general and unified understanding of the concept, division and sequence identification of constant regions and their segments. Therefore, the constant regions that those skilled in the art can identify and divide using common knowledge and ordinary methods are all within the scope of protection of this invention.

[0083] For example, the amino acid sequence of the corresponding segment (such as the IgM CH2 region) divided by the IMGT database can be used as the reference sequence. The start or end position of the reference sequence can be moved forward by several amino acid residues (i.e., moved towards the IgM CH1 region) or backward by several amino acid residues (i.e., moved towards the IgM CH3 region) to obtain a sequence of the corresponding segment that is longer or shorter than the reference sequence.

[0084] In this document, the term "hinge region" refers to a polypeptide that links the CH1 and CH2 domains within the constant region of the heavy chain of an antibody. This region is rich in proline, thus allowing for stretching and bending, and typically contains at least one proline (P). Hinge regions are usually dimers, consisting of two polypeptides with the same amino acid sequence. Specific amino acid sequences are not limited and are all within the scope of protection of this application. Hinge regions of different species and subclasses are well known.

[0085] In this paper, the term "tail peptide" refers to a short peptide sequence of about a dozen amino acid residues at the end of the CH3 or CH4 region of an antibody. Tail peptides from different species are well known.

[0086] In this document, the term "IgM tail peptide" refers to a short peptide sequence at the end of the CH4 region of an IgM antibody, located at the C-terminus of the CH4 region. The specific amino acid sequence is not limited and is within the scope of protection of this application. The IgM tail peptide contains cysteine ​​residues that participate in polymer formation and can also bind to the J chain to further stabilize the multimeric structure.

[0087] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0088] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0089] In an optional implementation, the species source of the aforementioned constant region is cattle, horses, pigs, sheep, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0090] In an optional implementation, the species source of the aforementioned constant region is rabbit.

[0091] In this paper, the partitioning of the variable and constant regions is based on the IMGT partitioning method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:housemouse(Mus musculus)IGHC,IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org .Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. theinternational ImMunoGenetics information http: / / www.imgt.org Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. The variable regions delineated by different methods may differ in some amino acids from the C-terminus of the variable region delineated by IMGT or the N-terminus of the constant region. Variable regions or constant regions delineated by other methods known in the art are also within the scope of protection of this invention.

[0092] In an optional implementation, the antibody includes the following constant region:

[0093] The amino acid sequence CH as shown in SEQ ID NO:31; and the amino acid sequence CL as shown in SEQ ID NO:32; or an amino acid sequence having at least 80% identity with each of the constant regions.

[0094] In other embodiments, the above-described 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 constant region (SEQ ID NO: 179, 180, 181).

[0095] Fourthly, embodiments of the present invention provide an antibody against cardiac troponin I, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain of the aforementioned antibody is shown as any one of SEQ ID NO:33 to SEQ ID NO:35; and the amino acid sequence of the light chain is shown as any one of SEQ ID NO:36 to SEQ ID NO:38.

[0096] In an optional embodiment, the heavy and light chains of the antibodies described in the first, second, third, or fourth aspects above are selected from any combination of (A') to (C'):

[0097] (A') The heavy chain with an amino acid sequence as shown in SEQ ID NO:33, and the light chain with an amino acid sequence as shown in SEQ ID NO:36;

[0098] (B') The amino acid sequence of the heavy chain as shown in SEQ ID NO:34, and the amino acid sequence of the light chain as shown in SEQ ID NO:37; and

[0099] (C') The heavy chain has an amino acid sequence as shown in SEQ ID NO:35, and the light chain has an amino acid sequence as shown in SEQ ID NO:38.

[0100] Fifthly, the present invention provides an antibody conjugate comprising the antibodies described above.

[0101] In an optional embodiment, the antibody conjugate includes biotin or a biotin derivative.

[0102] In an optional embodiment, the antibody conjugate may further include a marker or purification tag.

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

[0104] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

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

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

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

[0108] In optional embodiments, the aforementioned radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

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

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

[0111] In optional embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0112] In optional embodiments, the colloidal metals mentioned above include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0113] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated with the antibody.

[0114] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0115] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon and nitrocellulose membranes.

[0116] In this study, the antibody performance was not affected after conjugation with the conjugate.

[0117] In a sixth aspect, embodiments of the present invention provide a reagent or kit, wherein the aforementioned reagent or kit includes the above-described antibody or antibody conjugate.

[0118] As previously stated, the antibodies in some specific embodiments or examples of the present invention can bind to cardiac troponin I. Therefore, reagents or kits containing said antibodies can effectively perform qualitative or quantitative detection of cardiac troponin I. The reagents or kits provided by the present invention can be used, for example, for detections involving the specific binding properties of cardiac troponin I and its antibodies, such as immunoblotting and immunoprecipitation. As previously stated, the antibodies of the present invention have improved cardiac troponin I binding activity, affinity, stability, or specificity; therefore, reagents or kits containing said antibodies have improved detection sensitivity or specificity.

[0119] The above kit may contain any one or more of the following: processing solution, anti-cardiac troponin I antibody, cardiac troponin I quality control, anti-IgG antibody, instructions for use, or literature. Anti-cardiac troponin I antibody can be used for various types of diagnostic tests, such as detecting the presence of various diseases, drugs, or other proteins in vitro or in vivo. For example, it can be used to test for related diseases by detecting the serum or blood of the subject.

[0120] In a seventh aspect, embodiments of the present invention provide a method for detecting cardiac troponin I, comprising: a) under conditions sufficient to induce an antibody / antigen binding reaction, first binding a test sample with a cardiac troponin I antibody to form a complex; and b) detecting the presence of the complex, wherein the presence of the complex 2 indicates the presence of cardiac troponin I in the test sample.

[0121] In an optional embodiment, the immune complex further includes a second antibody that binds to the antibody.

[0122] In an optional embodiment, the immune complex further includes a second antibody that binds to cardiac troponin I.

[0123] Eighthly, embodiments of the present invention provide the use of the above-mentioned anti-cardiac troponin I antibody and antibody-drug conjugate in the preparation of products for detecting cardiac troponin I.

[0124] It should be noted that the products of this invention include, but are not limited to, reagents, kits, test strips, or reagent plates.

[0125] Ninthly, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0126] In a tenth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0127] In the eleventh aspect, the present invention provides cells containing the above-described carrier.

[0128] In a twelfth aspect, the present invention provides a method for preparing an antibody against cardiac troponin I, comprising: culturing cells as described above.

[0129] In this invention, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, it may optionally encode the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0130] In this invention, the term "vector" refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. For example, vectors include plasmids, phage particles, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors.

[0131] In this invention, the term "recombinant cell" refers to a cell into which exogenous polynucleotides and / or vectors can be introduced, or have already been introduced. The exogenous polynucleotides may or may not be integrated into the genome of the "recombinant cell." When the recombinant cell contains a vector, the vector can be introduced into mammalian cells to construct recombinant cells, which are then used to express the antibodies or antigen-binding fragments provided by this invention. The corresponding antibodies can be obtained by culturing the recombinant cells. Suitable mammalian cells include CHO cells, etc.

[0132] Based on the amino acid sequence of the anti-cardiac troponin I antibody disclosed in this invention, those skilled in the art will readily conceive of preparing the anti-cardiac troponin I antibody using genetic engineering or other techniques (chemical synthesis, recombinant expression), for example, by isolating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing any of the above-mentioned antibodies. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the anti-cardiac troponin I antibody of this invention, it falls within the protection scope of this invention.

[0133] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0134] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0135] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0136] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0137] Example 1: Discovery of Monoclonal Antibodies

[0138] Rabbit monoclonal antibodies against cTnI can be obtained through single B cell cloning technology, and the specific implementation method is as follows:

[0139] 1. Rabbit Immunity

[0140] An emulsion of cTnI protein prepared with incomplete Freund's adjuvant was injected subcutaneously into 4-6 week old New Zealand white rabbits to stimulate an immune response. Serum was collected before and after immunization at days 0, 14, 28, 42, and 69 for serum titer testing. Rabbits with acceptable titers were selected, and their spleens were surgically removed to prepare spleen cell suspensions. Fresh individual spleen cells were then isolated and cultured overnight in B cell culture medium.

[0141] 2. Specific single B cell acquisition

[0142] 1) Prepare a fresh cell suspension by diluting spleen cells with dissolved oxygen in PBS containing 2-3% fetal bovine serum and 1 mM EDTA.

[0143] 2) Single B cells were specifically sorted for cTnI antigen using a Sony MA900 flow cytometer (Sonybiotechnology, Japan) and placed in each well of a 96-well plate.

[0144] 3) Add primary B cells with cTnI specificity to B cell culture medium and then culture them at 37°C and 5.5% CO2 for 7-10 days.

[0145] 4) At the end of the primary B cell culture, the B cell culture supernatant was screened and identified by ELISA. Finally, three B cell positive clones with superior detection performance against cTnI were selected and named Anti-cTnI-11F13, Anti-cTnI-6G17 and Anti-cTnI-5H13, respectively.

[0146] 3. B-cell antibody gene sequencing:

[0147] RNA was extracted from Anti-cTnI-11F13, Anti-cTnI-6G17, and Anti-cTnI-5H13 positive clones and reverse transcribed into cDNA. Then, the antibody gene fragment was amplified by PCR. Next, the antibody gene fragment was inserted into a sequencing T vector (purchased from Takara). Finally, the antibody gene was sequenced to obtain the gene sequence of the antibody variable region.

[0148] The anti-cTnI rabbit monoclonal antibody sequence obtained by the above method was expressed via eukaryotic recombinant expression.

[0149] Example 2: Preparation of Recombinant Antibody

[0150] 1. Expression plasmid construction

[0151] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector that has been modified to introduce polyclonal restriction enzyme sites, and will be referred to as the 3.4A expression vector. Based on the variable region gene obtained in the above experiments, gene-specific primers for the light chain variable region and the heavy chain variable region were designed, with restriction endonuclease sites and protective bases at both ends, respectively. The light chain gene fragment and the heavy chain gene fragment were amplified by PCR.

[0152] The heavy chain and light chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was also double-digested with restriction endonucleases. After purification and recovery of the fragments and vector, the heavy chain gene and light chain gene were respectively ligated into the 3.4A expression vector to obtain recombinant expression plasmids of the heavy chain and light chain, respectively.

[0153] 2. Recombinant cell preparation

[0154] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cells / ml, cell viability >95%; centrifuged to wash cells, reconstituted with culture medium, cell counted, density 3.1 × 10⁶ cells / ml. 6 Cells / ml. Prepare plasmid DNA and transfection reagent dilutions separately using culture medium. Add the transfection reagent dilution to the plasmid DNA dilution, mix well, and let stand at room temperature for 15 min. Slowly add this mixture to the cell dilution over 1 min, mix well, and then sample and count the cells. Record and observe the cell viability after transfection. Incubate at 35℃ with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, centrifuge and collect the samples. Perform ELISA on the cell supernatant.

[0155] 2.1 Indirect ELISA method for detecting supernatant

[0156] 1) Coating: CTNI antigen, ITC antigen, IC antigen, CTNC antigen, and CTNT antigen (all antigens are from Feipeng Biotechnology) are coated at a concentration of 1ug / ml and incubated overnight at 4℃.

[0157] 2) Wash twice with PBST, blot dry, block with 120ul / well of 1% casein, incubate at 37°C for 1 hour, and blot dry.

[0158] 3) Dilute the cell supernatant with 1% casein 10 times and 50 times respectively, add 100ul / well to the ELISA plate in step 2), and incubate at 37°C for 30min.

[0159] 4) Wash 5 times with PBST, pat dry, add 100ul / well of goat anti-rabbit IgG-HRP (1% casein diluted 5000 times), and incubate at 37℃ for 30min.

[0160] 5) Wash 5 times with PBST, pat dry, add 50 μL each of solution A and solution B, incubate in the dark for 10 min, add 50 μL of stop solution, and read the results using an ELISA reader. The results are shown in Table 3. The results show that the antibodies Anti-cTnI-5H13-Rmb and Anti-cTnI-6G17 bind to the three forms of cardiac troponin I (CTNI, ITC, and IC) but do not bind to CTNC and CTNT, showing good specificity; Anti-cTnI-11F13-Rmb binds to the CTNI form of cardiac troponin I but does not bind to CTNC and CTNT, showing good specificity.

[0161] Table 3: Cell supernatant binding activity data by indirect ELISA

[0162]

[0163]

[0164] 3. Antibody supernatant purification

[0165] The supernatant of the recombinant expressed antibody was centrifuged and purified using a protein A affinity chromatography column to obtain the purified antibody.

[0166] The resulting antibodies were named Anti-cTnI-11F13-Rmb, Anti-cTnI-6G17-Rmb, and Anti-cTnI-5H13-Rmb.

[0167] The heavy chain amino acid sequence of the antibody Anti-cTnI-11F13-Rmb is shown in SEQ ID NO:33, and the light chain amino acid sequence is shown in SEQ ID NO:36;

[0168] The heavy chain amino acid sequence of the antibody Anti-cTnI-6G17-Rmb is shown in SEQ ID NO:34, and the light chain amino acid sequence is shown in SEQ ID NO:37.

[0169] The heavy chain amino acid sequence of the antibody Anti-cTnI-5H13-Rmb is shown in SEQ ID NO:35, and the light chain amino acid sequence is shown in SEQ ID NO:38.

[0170] Example 3: Antibody Performance Detection

[0171] 1. Performance of antibodies in magnetic microparticle chemiluminescence detection (exemplary performance data of coating and labeling combinations are shown below)

[0172] 1.1 Antibody Coating Process

[0173] 10 mg / mL carboxyl magnetic beads were washed three times with MES buffer. The beads were resuspended in MES buffer, and EDC was added to a final concentration of 1 mg / mL. The mixture was shaken at 25°C and reacted for 30 min. The beads were then resuspended in MES buffer again, and Anti-cTnI-A (from Phytobio) and Anti-cTnI-5H13-Rmb were added to a final concentration of 0.2 mg / mL. This solution was used as the working solution for the magnetic microparticles, and the mixture was shaken at 25°C and reacted for 120 min. The mixture was stored in Tris buffer at 2–8°C.

[0174] 1.2 Antibody labeling process

[0175] 3 mg / ml of the antibodies Anti-cTnI-B (from Phytobio) and Anti-cTnI-11F13-Rmb were replaced with PBS (100 mM PB, 50 mM sodium chloride, pH 8.0) using a Zeba desalting column (10K MWCO). Acridinium ester was prepared into a 4 mM solution using DMSO. 10 eq of acridinium ester was added to both the Anti-cTnI-B and Anti-cTnI-11F13-Rmb antibody solutions, and the reactions were carried out at 25°C for 2 hours. Excess reagents were removed by desalting, and the acridinium ester-modified antibodies were used as the acridinium ester working solution and stored at 4°C for later use.

[0176] 1.3 Testing Process

[0177] The assay was performed on the Shine i2910 fully automated chemiluminescence immunoassay analyzer using a double-antibody sandwich method. The instrument was prepared by sequentially adding 100 μL of sample, 50 μL of magnetic microparticle working solution, and 50 μL of acridinium ester working solution, mixing, and incubating for 10 minutes. After incubation, the reaction mixture was rinsed, and pre-excitation and excitation solutions were added. The relative luminescence intensity (RLU) was then measured.

[0178] Remark:

[0179] 1) Samples: refers to samples containing different concentrations of cTnI calibrators, clinical samples containing cTnI, samples containing 1000 ng / mL of cTnT and low concentrations of cTnI, samples containing 1000 ng / mL of cTnC and low concentrations of cTnI, and samples containing 1000 ng / mL of sTnI and low concentrations of cTnI.

[0180] 2) Antibody pairing detection was divided into 3 groups: Group 1: Coating: Anti-cTnI-A, Label: Anti-cTnI-B

[0181] Group 2: Coating: Anti-cTnI-A, Label: Anti-cTnI-11F13-Rmb

[0182] Group 3: Coating: Anti-cTnI-5H13-Rmb, Label: Anti-cTnI-B

[0183] 1.4 Test Results

[0184] 1.4.1 Test results of calibrators

[0185] The detection data of cTnI calibrators are shown in Table 4. The results show that the detection sensitivity of groups 2 and 3 is significantly better than that of group 1. The detection using antibodies Anti-cTnI-5H13-Rmb or Anti-cTnI-11F13-Rmb has high activity and sensitivity.

[0186] Table 4: Calibration Test Results

[0187] Calibrator (ng / mL) Group 1 Group 2 Group 3 0 364 310 571 0.002 392 584 1718 0.02 497 1454 4886 0.2 1914 10716 33495 5 66860 190484 429910 50 875388 2042486 5074150

[0188] 1.4.2 Specificity Detection

[0189] In low-concentration cTnI samples, 1000 ng / mL of cross-reactants cTnT, cTnC, and sTnI were added respectively. Three sets of paired antibodies were used for detection. The concentrations of cTnT, cTnC, and sTnI were tested, and the cross-reactivity was calculated as (test concentration / cross-reactant concentration) × 100%. The results are shown in Table 5. The results show that the cross-reactivity rates of the three paired antibodies for detecting different cross-reactants are all ≤0.1%, and the cross-reactivity rates of groups 2 and 3 are significantly lower than that of group 1. Detection using antibodies Anti-cTnI-5H13-Rmb or Anti-cTnI-11F13-Rmb has good specificity.

[0190] Table 5: Results of Cross-Reactant Detection

[0191]

[0192] 1.4.3 Clinical Sample Test Results

[0193] The clinical sample test results are shown in Table 6, and the standard curve is shown in Table 6. Figure 1 As shown. The results indicate that the correlation R between clinical samples using magnetic microparticle chemiluminescence reagents composed of antibodies Anti-cTnI-5H13-Rmb or Anti-cTnI-11F13-Rmb and those using imported reagents is significantly higher. 2 >0.98, which is superior to the paired antibody in group 1.

[0194] Table 6: Clinical Sample Test Results

[0195]

[0196]

[0197] 2. Performance of antibodies in fluorescence chromatography detection (example data of coating and labeling combinations are shown below)

[0198] 2.1 Antibody labeling:

[0199] T-labeled material: Add 100 μL of 1% solids fluorescent microspheres to 900 μL of activation buffer and mix well. After centrifugation and removal of supernatant, add 1 mL of activation buffer and sonicate to mix well. Then add activator, mix well in the dark by shaking for 20 min, centrifuge to remove supernatant, add a coupling buffer (MES buffer, pH=6.0) of equal volume to the microspheres, mix well by sonication, add 0.1–0.2 mg of Anti-cTnI-6G17-Rmb labeled antibody, mix well in the dark by shaking for 3 h, and finally add blocking buffer for blocking. After mixing well in the dark by shaking for 45 min, stop labeling, centrifuge to remove supernatant, reconstitute the microspheres with microsphere preservation solution, mix well by sonication, and store at 4 °C for use.

[0200] C-labeled material: Add 100 μL of 1% solids fluorescent microspheres to 900 μL of activation buffer and mix well. After centrifugation and removal of supernatant, add 1 mL of activation buffer and sonicate to mix well. Then add activator, mix well in the dark by shaking for 20 min, centrifuge to remove supernatant, add a coupling buffer (MES buffer, pH=6.5) of equal volume to the microspheres, mix well by sonication, add 0.4–0.8 mg of goat anti-chicken IgY labeled antibody, mix well in the dark by shaking for 3 h, and finally add blocking buffer for blocking. After mixing well in the dark by shaking for 45 min, stop labeling, centrifuge to remove supernatant, reconstitute the microspheres with microsphere preservation solution, mix well by sonication, and store at 4 °C for use.

[0201] 2.2 Preparation of microsphere working solution: Dilute Anti-cTnI-6G17-Rmb marker to 10-20% with microsphere diluent, and dilute sheep anti-chicken IgY marker to 1-2%. Mix them together and use a spray pad device to spray the markers onto glass fiber to make a fluorescent pad.

[0202] 2.3 Preparation of dried fluorescent pads: Place the sprayed fluorescent pads in a 50℃ oven and dry for more than 2 hours.

[0203] 2.4 Sample pad treatment: Dilute the blocking agent to 0.4 mg / ml with sample pad diluent, spread it on glass fiber, and dry it in a 50°C oven overnight.

[0204] 2.5NC membrane coating:

[0205] T-line: Anti-cTnI-C was diluted to 1.0 mg / ml with coating diluent before coating;

[0206] C line: Chicken IgY coated antibody was diluted to 1.0 mg / ml with coating diluent before coating;

[0207] After completing the T and C line scribing, place the film in a 50℃ oven to dry overnight.

[0208] 2.6 Preparation of fluorescence chromatography strips: Use a strip cutter to cut the fluorescence chromatography strips to the required width, assemble them, add samples, and perform detection.

[0209] 2.7 Detection method: Add the sample to the sample diluent, where the sample:sample diluent ratio = 1:1. After thorough mixing, load the sample (e.g., load 75uL onto a 3.5mm wide strip). Allow the test card to react for 15 minutes, then immediately insert the test card into the instrument to take the reading.

[0210] Note: Samples refer to internal reference materials and clinical samples containing different concentrations of cTnI.

[0211] 2.8 Test Results

[0212] 2.8.1 Internal reference sample test results

[0213] The results of the internal reference test are shown in Table 7. The results show that the detection using the antibody Anti-cTnI-6G17-Rmb has good binding activity and sensitivity.

[0214] Table 7: Results of Internal Reference Item Testing Table 7: Results of Internal Reference Item Testing

[0215] Sample number Sample concentration (ng / ml) T / C S1 0.015 0.0048 S2 0.031 0.0120 S3 0.063 0.0260 S4 0.250 0.0940 S5 1.000 0.4362 S6 10.000 1.3937 S7 25.000 3.6220

[0216] 2.8.2 Clinical Sample Test Results

[0217] The clinical sample test results are shown in Table 8, and the standard curve is shown in Table 8. Figure 2 As shown, the results indicate that the correlation R between the fluorescent detection reagent composed of antibody Anti-cTnI-6G17-Rmb and the clinical samples assigned by the imported reagent is significant. 2 >0.98.

[0218] Table 8: Clinical Sample Test Results

[0219]

[0220]

[0221] The partial amino acid sequences involved in this application are shown in Table 9:

[0222]

[0223]

[0224]

[0225]

[0226] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody against cardiac troponin I, characterized in that, The antibody comprises three complementary determining regions having a heavy chain variable region having an amino acid sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:3 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:4 to SEQ ID NO:6; Optionally, the complementary determination region of the variable region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody against cardiac troponin I, characterized in that, The antibody contains the following complementarity-determining regions: HCDR1, which contains or consists of any of the following amino acid sequences: amino acids 30 to 34 of SEQ ID NO:1, amino acids 30 to 34 of SEQ ID NO:2 and amino acids 30 to 34 of SEQ ID NO:3; HCDR2, which contains or consists of any of the following amino acid sequences: amino acids 49 to 63 of SEQ ID NO:1, amino acids 49 to 65 of SEQ ID NO:2, and amino acids 49 to 64 of SEQ ID NO:3; HCDR3, which contains or consists of any of the following amino acid sequences: amino acids 94 to 103 of SEQ ID NO:1, amino acids 97 to 106 of SEQ ID NO:2, and amino acids 97 to 108 of SEQ ID NO:3; LCDR1, which contains or consists of any of the following amino acid sequences: amino acids 24 to 36 of SEQ ID NO:4, amino acids 24 to 34 of SEQ ID NO:5 and amino acids 24 to 34 of SEQ ID NO:6; LCDR2, which contains or consists of any of the following amino acid sequences: amino acids 52 to 58 of SEQ ID NO:4, amino acids 50 to 56 of SEQ ID NO:5 and amino acids 50 to 56 of SEQ ID NO:6; LCDR3 comprises, or is composed of, any of the following amino acid sequences: amino acids 91-100 of SEQ ID NO:4, amino acids 89-100 of SEQ ID NO:5, and amino acids 89-102 of SEQ ID NO:

6.

3. The antibody according to claim 1 or 2, characterized in that, The complementarity-determining region of the antibody includes any one of (a) to (c): (a) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are as follows: amino acids 30-34, 49-63, and 94-103 of SEQ ID NO:1; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as follows: amino acids 24-36, 52-58, and 91-100 of SEQ ID NO:

4. (b) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are as follows: amino acids 30–34, 49–65, and 97–106 of SEQ ID NO:2; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as follows: amino acids 24–34, 50–56, and 89–100 of SEQ ID NO:5; and (c) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are as follows: amino acids 30-34, 49-64, and 97-108 of SEQ ID NO:3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as follows: amino acids 24-34, 50-56, and 89-102 of SEQ ID NO:

6.

4. The antibody according to any one of claims 1 to 3, characterized in that, The antibody includes HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4, wherein HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are selected from any one of (a') to (c'): (a') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:7 to SEQ ID NO:10, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:11 to SEQ ID NO:14; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions. (b') The amino acid sequences are as shown in SEQ ID NO:15 to SEQ ID NO:18, HFR1, HFR2, HFR3, HFR4, and the amino acid sequences are as shown in SEQ ID NO:19 to SEQ ID NO:22, LFR1, LFR2, LFR3, LFR4; or amino acid sequences having at least 80% identity with the sequences of each of the frame regions; and (c') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:23 to SEQ ID NO:26, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:27 to SEQ ID NO:30; or the amino acid sequences have at least 80% identity with the sequences of each of the frame regions.

5. An antibody against cardiac troponin I, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID NO:1 to SEQ ID NO:3; and the amino acid sequence of the light chain variable region is shown as any one of SEQ ID NO:4 to SEQ ID NO:6; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any combination of (A) to (C): (A) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:4; (B) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:5; and (C) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:6; Optionally, the antibody comprises any one of F(ab')2, Fab', Fab, Fv, and scFv.

6. The antibody according to any one of claims 1 to 5, characterized in that, The antibody also includes a constant region; Optionally, the constant region includes a heavy chain constant region and 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, the hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM and / or the tail peptide of IgM. Optionally, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Optionally, the species source of the constant region is rabbit; Optionally, the antibody includes the following constant region: The amino acid sequence CH as shown in SEQ ID NO:31; and the amino acid sequence CL as shown in SEQ ID NO:32; or an amino acid sequence having at least 80% identity with each of the constant regions.

7. An antibody against cardiac troponin I, comprising a heavy chain and a light chain, characterized in that, The heavy chain amino acid sequence of the antibody is shown in any one of SEQ ID NO:33 to SEQ ID NO:35; the light chain amino acid sequence is shown in any one of SEQ ID NO:36 to SEQ ID NO:

38.

8. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody according to any one of claims 1 to 7; Optionally, the antibody conjugate further includes biotin or a biotin derivative conjugated with the antibody; Optionally, the antibody conjugate further includes a marker conjugated to the antibody; Optionally, the marker is selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers; Optionally, the antibody conjugate further includes a solid-phase support conjugated to the antibody; Optionally, the solid support is selected from microspheres, plates, and membranes.

9. A reagent or kit, characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1 to 7 or the antibody conjugate as described in claim 8.

10. Use of the antibody according to any one of claims 1 to 7, the antibody conjugate according to claim 8, or the reagent or kit according to claim 9 in the preparation of a product for detecting cardiac troponin I; Optionally, the uses include: a) Under conditions sufficient to induce antibody / antigen binding, the test sample is bound to cardiac troponin I antibody to form a complex; and b) Detect the presence of the complex, the presence of which indicates the presence of cardiac troponin I in the test sample; Optionally, the immune complex further includes a second antibody, which binds to the antibody; Optionally, the immune complex further includes a second antibody that binds to cardiac troponin I.

11. A nucleic acid molecule, a vector, a cell, or a method for preparing an antibody according to any one of claims 1 to 7, wherein the nucleic acid molecule encodes an antibody according to any one of claims 1 to 7; the vector contains a nucleic acid molecule encoding an antibody according to any one of claims 1 to 7; the cell contains the aforementioned nucleic acid molecule or vector; and the method comprises culturing the aforementioned cell.

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