Specific antibody for detecting human cardiac myosin binding protein C

Abstract

The present invention discloses specific antibodies against cardiac myosin binding protein C. Specifically, the present invention discloses monoclonal antibodies against cardiac myosin binding protein C, and the antibodies have significant binding activity to cardiac myosin binding protein C antigen.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to specific antibodies against cardiac myosin binding protein C and their applications. Background Art

[0002] Acute myocardial infarction (AMI) increases the risk of death and recurrent ischemia, and thus must be rapidly identified and distinguished from other causes of chest pain. Cardiac necrosis biomarkers are crucial for confirming or excluding AMI in suspected non-ST-segment elevation acute coronary syndrome (NSTE-ACS), and a diagnosis must be made in an appropriate clinical context.

[0003] Currently, cardiac troponin (cTn) has become the gold standard biomarker. However, cTn has potential drawbacks: 1) the concentration of cTn in the blood does not reach its peak until 16 - 18 hours after myocardial infarction; 2) the detectable concentration of cTn in the blood is not high; 3) cTn can be detected in the blood of most normal people, and the content difference between about 25% of myocardial infarction patients and normal patients cannot be detected. Therefore, the specificity of cTn diagnosis is not strong.

[0004] The incidence rate of AMI patients is fast and the mortality rate is high. Currently, classic detection markers such as troponin and myoglobin appear late, and more rapid and sensitive detection methods are needed.

[0005] Therefore, there is an urgent clinical need to find new myocardial infarction diagnostic biomarkers and develop specific detection kits. An ideal biomarker for AMI is to be rapidly and massively released from myocardial cells into the blood after myocardial infarction, rapidly shortening the diagnostic time and saving lives. Summary of the Invention

[0006] The purpose of the present invention is to provide a monoclonal antibody against cardiac myosin binding protein C and its applications to solve the problems existing in the above-mentioned prior art.

[0007] In the first aspect of the present invention, a heavy chain variable region of an antibody is provided, and the heavy chain variable region has complementarity-determining regions CDR selected from the following groups:

[0008] (i) V H -CDR1 shown in SEQ ID NO.2, V H -CDR2 shown in SEQ ID NO.3, and V H -CDR3 shown in SEQ ID NO.4; preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.8; or

[0009] (ii) V H-CDR1, the V shown in SEQ ID NO.11 H -CDR2, and the V shown in SEQ ID NO.12 H -CDR3; Preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.16.

[0010] In another preferred embodiment, the CDR region of the V H chain of the antibody comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to any one of SEQ ID NO:2, 3 and 4, SEQ ID NO:10, 11 and 12.

[0011] In another preferred embodiment, the CDR region of the V H chain of the antibody comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NO:2, 3 and 4, SEQ ID NO:10, 11 and 12.

[0012] In another preferred embodiment, the V H chain of the antibody comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to any one of SEQ ID NO:8 and 16.

[0013] In another preferred embodiment, the V H chain of the antibody comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NO:8 and 16.

[0014] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and capable of retaining the ability to specifically bind to cardiac myosin binding protein C.

[0015] In another preferred embodiment, the cardiac myosin binding protein C is human cardiac myosin binding protein C.

[0016] In the second aspect of the present invention, there is provided a heavy chain of an antibody, the heavy chain having the heavy chain variable region and heavy chain constant region described in the first aspect of the present invention.

[0017] In another preferred embodiment, the heavy chain constant region is human or murine.

[0018] In the third aspect of the present invention, there is provided a light chain variable region of an antibody, the light chain variable region having complementarity determining regions CDR selected from the group consisting of:

[0019] (i)V as shown in SEQ ID NO.5 L -CDR1, V as shown in SEQ ID NO.6 L -CDR2, V as shown in SEQ ID NO.7 L -CDR3. Preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO.9, or

[0020] (ii)V as shown in SEQ ID NO.13 L -CDR1, V as shown in SEQ ID NO.14 L -CDR2, V as shown in SEQ ID NO.15 L -CDR3. Preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO.17.

[0021] In another preferred embodiment, the CDR region of the V L chain of the antibody contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to any one of SEQ ID NO:5, 6 and 7, SEQ ID NO:13, 14 and 15.

[0022] In another preferred embodiment, the CDR region of the V L chain of the antibody contains one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NO:5, 6 and 7, SEQ ID NO:13, 14 and 15.

[0023] In another preferred embodiment, the V L chain of the antibody contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity to any one of SEQ ID NO:9 and 17.

[0024] In another preferred embodiment, the V L chain of the antibody contains one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NO:9 and 17.

[0025] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that optionally has at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid added, deleted, modified and / or substituted and can retain the ability to specifically bind to cardiac myosin binding protein C.

[0026] In a fourth aspect of the present invention, there is provided a light chain of an antibody, which light chain has the light chain variable region and the light chain constant region as described in the third aspect of the present invention.

[0027] In another preferred embodiment, the light chain constant region is human or murine.

[0028] In a fifth aspect of the present invention, there is provided an antibody, which antibody has: (1) a heavy chain variable region as described in the first aspect of the present invention; and / or (2) a light chain variable region as described in the third aspect of the present invention; alternatively, the antibody has: a heavy chain as described in the second aspect of the present invention; and / or a light chain as described in the fourth aspect of the present invention.

[0029] In another preferred embodiment, the antibody is a specific antibody against cardiac myosin binding protein C.

[0030] In another preferred embodiment, the antibody includes: single-chain antibody (scFv), diabody, monoclonal antibody, chimeric antibody (such as human-mouse chimeric antibody), murine antibody, or humanized antibody.

[0031] In another preferred embodiment, the antibody is a fully human antibody.

[0032] In another preferred embodiment, the antibody is a full-length antibody or an immunologically active antibody fragment thereof.

[0033] In another preferred embodiment, the immunologically active antibody fragment is Fab or (Fab”)2.

[0034] In another preferred embodiment, the antibody recognizes a linear epitope of cardiac myosin binding protein C.

[0035] In another preferred embodiment, the antibody recognizes a conformational epitope of cardiac myosin binding protein C.

[0036] In a sixth aspect of the present invention, there is provided a recombinant protein, which recombinant protein has: (i) a sequence of a heavy chain variable region as described in the first aspect of the present invention, a sequence of a heavy chain as described in the second aspect of the present invention, a sequence of a light chain variable region as described in the third aspect of the present invention, a sequence of a light chain as described in the fourth aspect of the present invention, or a sequence of an antibody as described in the fifth aspect of the present invention; and (ii) optionally, a tag sequence for assisting expression and / or purification.

[0037] In another preferred embodiment, the tag sequence is selected from the group consisting of: 6×His tag, GGGS sequence, FLAG tag.

[0038] In another preferred embodiment, the recombinant protein includes bispecific antibody, chimeric antibody.

[0039] In another preferred embodiment, the recombinant protein specifically binds to cardiac myosin binding protein C.

[0040] In a seventh aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of: (1) a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, a light chain variable region as described in the third aspect of the present invention, a light chain as described in the fourth aspect of the present invention, or an antibody as described in the fifth aspect of the present invention; or (2) a recombinant protein as described in the sixth aspect of the present invention.

[0041] In another preferred embodiment, the polynucleotide has a sequence shown in any one of SEQ ID NOs. 18, 19, 20, and 21.

[0042] In another preferred embodiment, the polynucleotide has a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to any one of SEQ ID NOs. 18, 19, 20, and 21.

[0043] In an eighth aspect of the present invention, there is provided a vector containing the polynucleotide as described in the seventh aspect of the present invention.

[0044] In another preferred embodiment, the vector includes: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0045] In a ninth aspect of the present invention, there is provided a genetically engineered host cell containing the vector as described in the eighth aspect of the present invention or having the polynucleotide as described in the seventh aspect of the present invention integrated into its genome.

[0046] In a tenth aspect of the present invention, there is provided an immunoconjugate containing: (a) a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, a light chain variable region as described in the third aspect of the present invention, a light chain as described in the fourth aspect of the present invention, or an antibody as described in the fifth aspect of the present invention, or a recombinant protein as described in the sixth aspect of the present invention; and (b) a conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0047] In another preferred example, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin), or any form of nanoparticles, etc.

[0048] In another preferred example, the detectable marker is selected from the group consisting of: biotin, fluorescein, chemiluminescent groups, chemofluorescent groups, fluorescent proteins, enzymes, colloidal gold, colored magnetic beads, latex particles, biotin labels, radionuclides, antibodies, ligands, antigens, receptors, nanoparticles, or combinations thereof.

[0049] In another preferred example, the nanoparticles are selected from the group consisting of: gold nanoparticles, silver nanoparticles, quantum dots, or combinations thereof.

[0050] In another preferred example, the enzyme is selected from the group consisting of: horseradish peroxidase, acid phosphatase, or combinations thereof.

[0051] In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, a light chain variable region as described in the third aspect of the present invention, a light chain as described in the fourth aspect of the present invention, or an antibody as described in the fifth aspect of the present invention, or a recombinant protein as described in the sixth aspect of the present invention, or an immunoconjugate as described in the tenth aspect of the present invention; and (ii) a pharmaceutically acceptable carrier.

[0052] In another preferred example, the pharmaceutical composition is in injectable form.

[0053] In another preferred example, the pharmaceutical composition is in the form of tablets or capsules.

[0054] In another preferred example, the pharmaceutical composition is used for preparing a medicament for treating heart diseases.

[0055] In another preferred example, the pharmaceutical composition is used for preparing a medicament for treating myocardial infarction or myocardial ischemia.

[0056] The twelfth aspect of the present invention provides the use of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or the immunoconjugate as described in the tenth aspect of the present invention, for preparing a medicament, a reagent, a test plate or a kit.

[0057] In another preferred embodiment, the reagent, test plate or kit is used for detecting myocardial ischemia.

[0058] In another preferred embodiment, the reagent, test plate or kit is used for detecting myocardial infarction.

[0059] In another preferred embodiment, the medicament is used for treating or preventing myocardial infarction.

[0060] In another preferred embodiment, the reagent includes a chip and immunoparticles coated with an antibody.

[0061] The thirteenth aspect of the present invention provides a detection article, which includes:

[0062] (1) The antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or the conjugate as described in the tenth aspect of the present invention; and

[0063] (2) Optionally, a buffer solution or a buffer agent.

[0064] In another preferred embodiment, the detection article is used for detecting cardiac myosin binding protein C.

[0065] In another preferred embodiment, the detection article includes a detection reagent, a lateral flow strip, a chip, a test strip, a test plate, a test piece.

[0066] In another preferred embodiment, the test piece includes a detection area, in which another antibody (antibody two) against cardiac myosin binding protein C is immobilized, and the antibody two is used for capturing the cardiac myosin binding protein C.

[0067] In another preferred embodiment, the antibody two is a capture antibody.

[0068] In another preferred embodiment, the antibody two is selected from the antibody as described in the fifth aspect of the present invention or a fragment thereof.

[0069] In another preferred embodiment, the test piece includes a quality control area, in which an antibody (secondary antibody) that binds to an antibody (primary antibody) against cardiac myosin binding protein C is immobilized, and the secondary antibody is used for capturing the antibody (primary antibody) against cardiac myosin binding protein C.

[0070] In another preferred example, the primary antibody is a detection antibody.

[0071] In another preferred example, the primary antibody is selected from the antibodies or their fragments as described in the fifth aspect of the present invention.

[0072] In another preferred example, the primary antibody is an antibody against cardiac myosin binding protein C labeled with biotin.

[0073] In another preferred example, the primary antibody is selected from the antibodies or their fragments as described in the fifth aspect of the present invention and is an antibody against cardiac myosin binding protein C labeled with biotin.

[0074] In another preferred example, both the secondary antibody and the primary antibody are selected from the antibodies or their fragments as described in the fifth aspect of the present invention.

[0075] In another preferred example, the detection piece is selected from: a porous plate, preferably a 96-well plate, and a PVDF membrane.

[0076] In another preferred example, the antibody (secondary antibody) that binds to the antibody against cardiac myosin binding protein C (primary antibody) is selected from: an HRP-labeled goat anti-mouse IgG secondary antibody.

[0077] The fourteenth aspect of the present invention provides a detection kit, which comprises the antibody described in the fifth aspect of the present invention, the recombinant protein described in the sixth aspect of the present invention, or the conjugate described in the tenth aspect of the present invention, or the detection product described in the thirteenth aspect of the present invention.

[0078] In another preferred example, the kit includes a detection product and an instruction manual.

[0079] The fifteenth aspect of the present invention provides a method for detecting cardiac myosin binding protein C in a sample, the method comprising the steps of:

[0080] (a) Providing a sample to be detected;

[0081] (b) Mixing the sample with the antibody described in the fifth aspect of the present invention, the recombinant protein described in the sixth aspect of the present invention, the conjugate described in the tenth aspect of the present invention, or the detection product described in the thirteenth aspect of the present invention to form a mixture;

[0082] (c) Detecting the presence or absence of the "antibody-cardiac myosin binding protein C complex" in the mixture, wherein if the complex exists, it indicates the presence of cardiac myosin binding protein C in the sample; if the complex does not exist, it indicates the absence of cardiac myosin binding protein C in the sample.

[0083] The sixteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, which method comprises:

[0084] (a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for expression;

[0085] (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.

[0086] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0087] Figure 1 Shows the schematic diagrams of the protein structures of Mybpc1, Mybpc2, and Mybpc3, where Mybpc1 is the slow skeletal muscle type, Mybpc2 is the fast skeletal muscle type, and Mybpc3 is the cardiac muscle type.

[0088] Figure 2 Shows the schematic diagram of the process for antibody detection. The double antibody sandwich method is used for sample detection, and finally color development is carried out by an enzymatic method.

[0089] Figure 3 Shows the non-reduced and reduced SDS-PAGE electrophoresis diagrams of the purified SEQ NO.1 supernatant. The results show that the protein is purified by an Ni column, and it can be seen that the purity after purification is relatively high, the SDS-PAGE electrophoresis purity is ≥90%, and the electrophoresis size is between 55 - 70KDa.

[0090] Figure 4 Shows the non-reduced and reduced SDS-PAGE electrophoresis diagrams of the recovered SEQ NO.1 after enzymatic digestion. It can be seen that the purity of the protein after enzymatic digestion is relatively high, the SDS-PAGE electrophoresis purity is ≥90%, and the electrophoresis size after removing the tag is between 40 - 55KDa.

[0091] Figure 5 Shows the results of the hybridoma cell fusion experiment. The concentration of the Marker is 0.1mg / ml, and the size is as marked in the figure. The mouse polyclonal antibody is diluted four times, and the antibody 2F112D7, 4C53C5, and 3C12A8 are loaded at the original concentration, and the loading amount is 5ul for each. The SDS results show that the purity after ascites purification is above 90%, the size of the heavy chain of the antibody is about 55KDa, and the size of the light chain is about 25KDa.

[0092] Figure 6 Shows the EC of the antibody of the present invention 50Values, fitting graphs of ELISA results, analyzed by GradPad Prism software to obtain the EC of 4C53C5 50 = 0.0067 μg / ml, and the EC of 2F112D7 50 = 0.0069 μg / ml.

[0093] Figure 7 Shows the results of ELISA detection using the antibodies 2F112D7 and 4C53C5 of the present invention after labeling. The diluted primary antibody 2F112D7 was coated equally on the ELISA detection plate as the capture antibody. After steps such as blocking, capturing gradient-diluted antigen standards, adding HRP-labeled secondary antibody for incubation, and TMB color development, the OD450 value was finally read. Detailed implementation mode

[0094] Through extensive and in-depth research and a large number of screenings, the present inventors unexpectedly obtained high-affinity monoclonal antibodies (such as murine monoclonal antibodies) that recognize and bind to cardiac myosin-binding protein C. Experimental results show that the antibodies of the present invention can specifically bind to two different antigenic epitopes of cardiac myosin-binding protein C (cMyBP-C) and do not interact with the related isomers of MyBP-C from fast and slow skeletal muscles. After modifying biotin at the C position of IgG, the antibodies of the present invention can bind cMyBP-C well. This antibody (especially the antibody pair) is suitable as a capture antibody and a detection antibody for ELISA (enzyme-linked immunosorbent assay) to quantitatively determine cMyBP-C in serum, plasma, whole blood or other body fluids, and is suitable for developing detection reagents, test kits, etc. for the early diagnosis of acute myocardial infarction (AMI). In addition, the antibodies of the present invention can also be applied to antibody chip reactions, immunoprecipitation, and Western Blotting for research on this protein.

[0095] Based on the characteristics of MYBPC3, the present invention prepared the MYBPC3-C0 antibody, a specific human anti-MYBPC3 antibody, for the capture antibody and the detection antibody to bind different antigenic epitopes, and for the development of a detection kit for human cMYBPC3 for detecting MYBPC3.

[0096] Specifically, the present invention uses the MYBPC3-C0 recombinant protein antigen to generate MYBPC3-C0 monoclonal antibodies, constructs a tag-MYBPC3-C0C1f recombinant protein vector, expresses the tag-C0C1f protein using bacteria, yeast, insect cells, animal or human cells, and purifies the tag-C0C1f protein as the MYBPC3 calibrator.

[0097] On this basis, the present invention was completed.

[0098] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0099] 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 invention belongs. As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0100] As used herein, the term "optionally" or "optionally" means that the subsequent described event or situation can occur but is not required to occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may or may not be present, and can be 1, 2, or 3.

[0101] Although any methods and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, preferred methods and materials are exemplified herein.

[0102] The term

[0103] MyBP-C

[0104] There are three isoforms of MyBP-C in adult muscle - fast skeletal and slow skeletal (encoded by the mybpc1 and mybpc2 genes on chromosomes 12q23.3 and 19q33.3, respectively) and the cardiac isoform (gene MYBPC3 on chromosome 11p11.2). The amino acid sequences of the three MyBP-C isoforms have a high degree of homology. Uniquely, the cardiac isoform contains an additional immunoglobulin-like domain (C0) at the N-terminus, a phosphorylation site (M-motif) between C1 and C2, and a 28-amino acid insertion in the C5 domain, and has specific antigenic epitopes, by which the cardiac-type MYBPC3 can be specifically detected by immunological methods. The whole protein consists of 12 domains, including 8 immunoglobulin (IgC2)-like domains, 3 fibronectin (FN3) domains, plus the above-mentioned M domain (see attached Figure 1 . Schematic diagrams of the protein structures of Mybpc1, Mybpc2, and Mybpc3).

[0105] MYBPC3 (Mybpc3 or mybpc-3) has 1,274 amino acids (UniprotKB: Q14896) and a molecular weight of 140 kDa. MYBPC3 belongs to the type III fibronectin superfamily and the immunoglobulin superfamily, containing three type III fibronectin domains (Fibronectin type 3, FN3), eight immunoglobulin-like domains (Immunoglobulin, Ig), a Pro-Ala domain (PA domain), and a Myosin (S2) binding region (M domain).

[0106] MYBPC3 is involved in the structure and function of sarcomeric proteins. The C10 domain of MYBPC3 binds to myosin filaments, while the C8 - C10 structural region binds to titin, playing a role in stabilizing the sarcomere structure. There is a specific region between the C1 and C2 domains of MYBPC3. The M domain (M domain) binds to the neck of myosin and is also the phosphorylation site (Phosphorylation sites) of MYBPC3. As the action site of cAMP-dependent protein kinase and calmodulin-dependent protein kinase, phosphorylation of specific motifs in the myocardium regulates myocardial contractility. MYBPC3 not only participates in the maintenance of myocardial structure but also in intracellular information transmission, affects the contraction and relaxation of myofilaments, and protects the myocardium from ischemic injury. These functions are mainly mediated by phosphorylation at sites such as Ser-273, Ser-282, and Ser-302. Dephosphorylation of these specific sites leads to the degradation of MYBPC3, releasing a 30 - 40 kDa MYBPC3-N-terminal fragment (C0C1f) into the bloodstream, increasing the level of C0C1f in the blood.

[0107] Antibody

[0108] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light and heavy chains.

[0109] As used herein, the term "variable" refers to the fact that certain portions of the variable regions in antibodies differ in sequence and that they form the binding and specificity of various particular antibodies for their particular antigens. However, variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions which generally assume a β-sheet conformation, connected by three CDRs which form loops and in some cases form part of the β-sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antibody's antigen-binding site (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit various effector functions, such as participating in antibody-dependent cytotoxicity.

[0110] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (designated κ and λ) based on the amino acid sequence of their constant regions. Immunoglobulins can be classified into different isotypes based on the amino acid sequence of their heavy chain constant regions. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known to those skilled in the art.

[0111] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, in contrast to conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a homogeneous population of antibodies and should not be construed as requiring any particular method for producing the antibody.

[0112] The present invention also includes monoclonal antibodies having the corresponding amino acid sequences of the anti-cardiac myosin binding protein C monoclonal antibodies, monoclonal antibodies having the variable region chains of the anti-cardiac myosin binding protein C monoclonal antibodies, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing hypervariable regions (complementary determining regions, CDRs), provided that the hypervariable regions are the same as or at least 90% homologous to the hypervariable regions of the light chain and heavy chain of the present invention, preferably at least 95% homologous.

[0113] As is known to those skilled in the art, immunoconjugates and fusion expression products include: conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the anti-cardiac myosin binding protein C monoclonal antibody or its fragments. The present invention also includes cell surface markers or antigens that bind to the anti-cardiac myosin binding protein C monoclonal antibody or its fragments.

[0114] The present invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments (antigen-binding fragments), such as Fab or (Fab”)2 fragments; antibody heavy chains; and antibody light chains.

[0115] As used herein, the term “antigen-binding fragment” refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment having antigen-binding activity. An Fv antibody contains the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment having all antigen-binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding.

[0116] As used herein, the term “epitope” refers to a discontinuous three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0117] As used herein, the term “heavy chain variable region” is interchangeable with “V H ”.

[0118] As used herein, the terms “variable region” and “complementarity determining region (CDR)” are interchangeable.

[0119] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody has a complementarity determining region CDR selected from the group consisting of:

[0120] V H-CDR1, with an amino acid sequence of SEQ ID NO.2;

[0121] V H -CDR2, with an amino acid sequence of SEQ ID NO.3;

[0122] V H -CDR3, with an amino acid sequence of SEQ ID NO.4.

[0123] In another preferred embodiment, the amino acid sequence of the heavy chain variable region (V H ) is SEQ ID NO.8.

[0124] In another preferred embodiment of the present invention, the heavy chain variable region of the antibody has complementarity-determining regions CDR selected from the following group:

[0125] V H -CDR1, with an amino acid sequence of SEQ ID NO.10;

[0126] V H -CDR2, with an amino acid sequence of SEQ ID NO.11;

[0127] V H -CDR3, with an amino acid sequence of SEQ ID NO.12.

[0128] In another preferred embodiment, the amino acid sequence of the heavy chain variable region (V H ) is SEQ ID NO.16.

[0129] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the above heavy chain variable region and a heavy chain constant region, and the heavy chain constant region can be murine or human.

[0130] As used herein, the term "light chain variable region" is interchangeable with "V L ".

[0131] In a preferred embodiment of the present invention, the light chain variable region of the antibody has complementarity-determining regions CDR selected from the following group:

[0132] V L -CDR1, with an amino acid sequence of SEQ ID NO.5;

[0133] V L -CDR2, with an amino acid sequence of SEQ ID NO.6;

[0134] V L -CDR3, with an amino acid sequence of SEQ ID NO.7;

[0135] In another preferred embodiment, the amino acid sequence of the light chain variable region (V L ) is SEQ ID NO.9.

[0136] In another preferred embodiment of the present invention, the light chain variable region of the antibody has complementarity determining regions CDR selected from the group consisting of:

[0137] V L -CDR1, whose amino acid sequence is SEQ ID NO.13;

[0138] V L -CDR2, whose amino acid sequence is SEQ ID NO.14;

[0139] V L -CDR3, whose amino acid sequence is SEQ ID NO.15;

[0140] In another preferred embodiment, the amino acid sequence of the light chain variable region (V L ) is SEQ ID NO.17.

[0141] In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and a light chain constant region, and the light chain constant region can be murine or human.

[0142] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and all refer to an antibody that specifically binds to cardiac myosin binding protein C, for example, a protein or polypeptide having a heavy chain variable region (amino acid sequence shown in SEQ ID NO.8 or SEQ ID NO.16) and / or a light chain variable region (amino acid sequence shown in SEQ ID NO.9 or SEQ ID NO.17). They may or may not contain a starting methionine.

[0143] In another preferred embodiment, the antibody is a murine or human-mouse chimeric monoclonal antibody against cardiac myosin binding protein C, and its heavy chain constant region and / or light chain constant region can be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is the heavy chain constant region or light chain constant region of human IgG1, IgG2, etc.

[0144] The present invention also provides other proteins or fusion expression products having the antibody of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain and a light chain containing variable regions, as long as the variable regions are the same as or at least about 90% homologous, preferably at least about 95% homologous, to the variable regions of the heavy chain and light chain of the antibody of the present invention.

[0145] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called complementarity-determining regions (CDRs), which divide the segments into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures that are brought close to each other in the spatial structure by the β-sheets formed by the intervening FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acids that make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.

[0146] Particularly interesting are the variable regions of the heavy and / or light chains of the antibodies of the present invention because at least part of them is involved in binding the antigen. Accordingly, the present invention includes molecules having monoclonal antibody light and heavy chain variable regions with CDRs, provided that the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0147] The present invention includes not only intact monoclonal antibodies but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives, and analogs of the said antibodies.

[0148] As used herein, the terms "fragment", "derivative", and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention can be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0149] The antibody of the present invention refers to a polypeptide having cardiac myosin binding protein C binding activity and comprising the above CDR regions. This term also includes variant forms of the polypeptide comprising the above CDR regions that have the same function as the antibody of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar or close properties, the function of the protein is usually not changed. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. This term also includes active fragments and active derivatives of the antibody of the present invention.

[0150] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using the antiserum against the antibody of the present invention.

[0151] The present invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of the antibody of the present invention. Generally, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, most preferably at least about 100 consecutive amino acids.

[0152] In the present invention, the "conservative variant of the antibody of the present invention" refers to a polypeptide in which, compared with the amino acid sequence of the antibody of the present invention, at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0153] Table 1

[0154]

[0155]

[0156] The present invention also provides polynucleotide molecules encoding the above antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0157] The coding region sequence encoding the mature polypeptide may be the same as or a degenerate variant of the coding region sequences shown in SEQ ID NOs. 18, 19, 20 and 21. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes an amino acid sequence identical to the polypeptide of the present invention, but is different from the coding region sequences shown in SEQ ID NOs. 18, 19, 20 and 21.

[0158] The polynucleotides encoding the mature polypeptide of the present invention include: the coding sequence encoding only the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0159] The term "polynucleotide encoding a polypeptide" may be a polynucleotide including the polynucleotide encoding this polypeptide, or may also be a polynucleotide further including additional coding and / or non-coding sequences.

[0160] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at a lower ionic strength and a higher temperature, such as 0.2×SSC, 0.1% SDS, 60 °C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 °C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least above 90%, preferably above 95%. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 16, and SEQ ID NO. 17.

[0161] The full-length nucleotide sequence or fragment of the antibody of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis methods. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is short. Usually, very long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0162] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually to clone them into a vector, then transfer them into cells, and then isolate the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0163] At present, it is already possible to obtain the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) entirely by chemical synthesis. Then, this DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0164] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0165] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0166] The transformation of host cells with recombinant DNA can be carried out by conventional techniques well-known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase, treated with the CaCl2 method, and the steps used are well-known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0167] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. According to the host cell used, the culture medium used in the culture can be selected from various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0168] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical, and other properties. These methods are well-known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0169] The antibodies of the present invention can be used alone or conjugated or coupled with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the foregoing substances.

[0170] Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of generating a detectable product.

[0171] Detectable labels for detection purposes include, but are not limited to: biotin, fluorescein, chemiluminescent groups, chemifluorescent groups, fluorescent proteins, enzymes, colloidal gold, colored magnetic beads, latex particles, biotinylation, radionuclides, antibodies, ligands, antigens, receptors, nanoparticles, or combinations thereof.

[0172] Typically, the nanoparticles are selected from the group consisting of: gold nanoparticles, silver nanoparticles, quantum dots, or combinations thereof. Typically, the enzymes are selected from the group consisting of: horseradish peroxidase, acid phosphatase, or combinations thereof.

[0173] Couplable therapeutic agents include, but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptides, enteropeptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0174] Therapeutic agents that can also be bound or conjugated to the antibodies of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biological toxins (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc. (Gillies et al., 1992, PNAS 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202); 4. Gold nanoparticles / nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 5. Virus particles (Peng et al., 2004, Gene therapy 11, 1234); 6. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 7. Nanomagnetic particles; 8. Prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0175] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substances being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

[0176] The pharmaceutical composition of the present invention can be directly used to bind to the cardiac myosin binding protein C molecule, thus can be used to extend the half-life of the drug. In addition, other therapeutic agents can also be used simultaneously.

[0177] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or its conjugate) of the present invention as described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present invention can be made into an injectable form, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectables and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight - about 10 milligrams / kg body weight per day. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0178] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 8 milligrams / kg body weight. Preferably, the dose is about 10 micrograms / kg body weight - about 1 milligram / kg body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health status, which are within the scope of the skills of a skilled physician.

[0179] Hybridoma cell line

[0180] The present invention also provides a hybridoma cell line capable of producing the monoclonal antibody against cardiac myosin binding protein C of the present invention; preferably, the present invention provides a hybridoma cell line with a high titer of monoclonal antibody against cardiac myosin binding protein C.

[0181] After obtaining the hybridoma for producing the monoclonal antibody against cardiac myosin binding protein C of the present invention, those skilled in the art can conveniently use this hybridoma cell line to prepare the antibody. In addition, those skilled in the art can also easily know the structure of the antibody of the present invention (such as the variable region of the heavy chain and the variable region of the light chain of the antibody), and then can prepare the monoclonal antibody of the present invention by recombinant methods.

[0182] Preparation of monoclonal antibody

[0183] The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, the antigen of the present invention can be administered to an animal to induce the production of monoclonal antibodies. For monoclonal antibodies, hybridoma technology can be utilized for preparation (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by recombinant DNA methods (U.S. Patent No. 4,816,567), obtained through phage display systems; obtained from immunotransgenic humanized mice.

[0184] Representative myeloma cells are those that fuse efficiently, support the stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to the culture medium (HAT medium substrate), including myeloma cell lines, such as murine myeloma cell lines, including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, California, USA) and SP-2, NZ0 or X63-Ag8-653 cells (available from American Type Culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibodies Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)].

[0185] The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies with the desired specificity, such as, by in vitro binding analysis, for example, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). The location of cells expressing the antibody can be detected by FACS. Then, the hybridoma clones can be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59-103 pages). Suitable culture media used to achieve this purpose include, for example, DMEM or RPMI-1640 culture media. In addition, hybridoma cells can be grown as ascites tumors in animals.

[0186] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0187] The present invention provides a monoclonal antibody against cardiac myosin binding protein, in particular a monoclonal antibody against cardiac myosin binding protein C. In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. The supernatant of the hybridoma cell culture is taken, IgG is crudely extracted by saturated ammonium sulfate precipitation, and the crudely extracted antibody is purified by affinity chromatography column (Protein G-Sephrose).

[0188] In a preferred embodiment of the present invention, the monoclonal antibody is prepared by the method of producing monoclonal antibodies using the ascites of Balb / C mice. The hybridoma cells are inoculated into the abdominal cavity of sensitized mice, and the abdomen is obviously swollen in about 10 days. The ascites is extracted, and the crude antibody is purified by affinity chromatography column (Protein G-Sephrose) after crude extraction by saturated ammonium sulfate precipitation method.

[0189] The main advantages of the present invention are:

[0190] (1) cMyBPC is rapidly released into the blood after a myocardial infarction occurs. It has a high concentration and a fast rate, making it suitable as a marker for diagnosing acute myocardial infarction.

[0191] (2) The present invention provides a pair of cMyBPC detection antibodies with better sensitivity, which can buy time for diagnosis and treatment for patients.

[0192] (3) The antibody can be used in other detection methods based on the double antibody sandwich method.

[0193] (4) It has a better binding degree to the target antigen of human samples.

[0194] The present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out according to the conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual" by Sambrook. J et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0195] Materials and Methods

[0196] The experimental materials used in the embodiments of the present invention can be obtained from commercial channels without special instructions.

[0197] The present invention mainly includes 1. Expression of recombinant proteins; 2. Immunization and antibody screening; 3. ELISA for detecting affinity (the schematic diagram of the detection process is shown in Figure 2 ); 4. Human blood sample testing.

[0198] 1. Design and expression of in vitro recombinant antigens:

[0199] >MYBPC3-C0C1f (SEQ NO.1):

[0200] MPEPGKKPVSAFSKKPRSVEVAAGSPAVFEAETERAGVKVRWQRGGSDISASNKYGLATEGTRHTLTVREVGPADQGSYAVIAGSSKVKFDLKVIEAEKAEPMLAPAPAPAEATGAPGEAPAPAAELGESAPSPKGSSSAALNGPTPGAPDDPIGLFVMRPQDGEVTVGGSITFSARVAGASLLKPPVVKWFKGKWVDLSSKVGQHLQLHDSYDRASKVYLFELHITDAQPAFTGSYRCEVSTKDKFDCSNFNLTVHEAMGTGDLDLLSAFRRTSLAGGG(1-280aa)

[0201] The codon-optimized amino acid sequence was constructed onto the modified pCDNA3.4 vector (gift from Fudan University). The N-terminus of this vector contains a His-sumom tag. The corresponding plasmid was constructed, transfected into Expi293F cells (A14527, Gbico). After 3 days of expression, the supernatant was collected by centrifugation, and Ni Smart Beads (SA035005, Smart-Lifescience) were used for the purification of the recombinant protein. The purified protein was added with ULPM for digestion (HE001, Oritop Biotech), and the purity of the protein obtained could be analyzed by SDS-PAGE electrophoresis. The antigen band obtained after purification is shown in Figure 3 and Figure 4 .

[0202] 2. Mouse Immunization

[0203] 1) Antigen Emulsification: The antigen was diluted with PBS and then mixed with adjuvant at a volume ratio of 1:1 and emulsified thoroughly.

[0204] 2) Complete Freund's adjuvant was used for the primary immunization, and incomplete Freund's adjuvant was used for the remaining immunizations.

[0205] 3) After emulsification, the mice were immunized by subcutaneous multi-point injection, with a volume of 0.05 - 0.1 ml injected at each point.

[0206] 4) After immunization, write down the immunization time and dosage on the mouse cage and make good experimental records.

[0207] The detailed steps are shown in Table 2.

[0208] Table 2

[0209] List of Mouse Immunization

[0210]

[0211] 3. Serum Titer Detection - ELISA Method

[0212] 1) Antigen Coating: Coated with C0C1f antigen, diluted to a concentration of 10 μg / ml with 1X CBS. The diluted antigen was added to each well with 100 μl using a multichannel pipette (the amount of antigen per plate is 10 μg), and reacted at 37°C for 2 h.

[0213] 2) Washing: After discarding the liquid, pat dry on the absorbent paper. Add 200 μl of washing solution PBST to each well using a multichannel pipette, shake gently, discard the washing solution and pat dry on the absorbent paper, and repeat the washing 2 times.

[0214] 3) Blocking: Add 200 μl of blocking solution (PBST + 5% skim milk powder) to each reaction well and block at 37°C for 1 h.

[0215] 4) Washing: After shaking off the liquid, pat dry on absorbent paper. Add 200 μl of washing solution PBST to each well using a multichannel pipette, gently shake, shake off the washing solution, and pat dry on absorbent paper. Repeat the washing 3 times and store at 4°C for later use. 5) Serum collection: Collect the blood from the corners of the mouse's eyes using a capillary blood collection tube, and blow the blood into a 1.5 ml EP tube using a rubber bulb. Label the EP tube and the corresponding mouse. After all the blood has been collected, let it stand at 4°C for half an hour.

[0216] 6) Sample preparation: Take out after standing for half an hour and centrifuge at 8000 rpm for 15 min.

[0217] 7) Primary antibody incubation: Add the centrifuged serum to the reaction wells after dilution. Dilute with the blocking solution, 100 μl per well, cover with tin foil, and incubate at 37°C for 1 h.

[0218] 8) Washing: After shaking off the liquid, pat dry on absorbent paper. Add 200 μl of washing solution PBST to each well using a multichannel pipette, gently shake, shake off the washing solution, and pat dry on absorbent paper. Repeat the washing 3 times.

[0219] 9) Secondary antibody incubation: Dilute goat anti-mouse IgG-HRP at a ratio of 1:10000 with the blocking solution, add 100 μl per well using a multichannel pipette, cover with tin foil, and incubate at 37°C for 1 h.

[0220] 10) Washing: After shaking off the liquid, pat dry on absorbent paper. Add 200 μl of washing solution PBST to each well using a multichannel pipette, gently shake, shake off the washing solution, and pat dry on absorbent paper. Repeat the washing 4 times.

[0221] 11) Color development: Add 100 μl of TMB color development solution to each well using a multichannel pipette, cover with tin foil, and react at room temperature for 5 min.

[0222] 12) Termination: Add the termination solution, 50 μl per well using a multichannel pipette.

[0223] After the mouse was immunized five times, the animal immunization experiment was completed, the serum titer was qualified, and the OD values shown on the ELISA plate are as follows in Table 3:

[0224] Table 3 Serum titer of the mouse after five immunizations

[0225] Dilution factor 2 3 4 5 800 3.1058 2.7575 2.8522 2.8741 1600 2.8876 2.2836 2.6034 2.6203 3200 2.8296 2.0861 2.4322 2.3965 6400 2.6598 1.7449 2.1811 1.9665 12800 2.4074 1.3151 1.8085 1.5082 25600 2.0334 0.933 1.3424 1.0151 51200 1.4905 0.6282 0.9596 0.6716 102400 1.1644 0.4729 0.7138 0.5147 204800 0.6031 0.2587 0.4294 0.2901 409600 0.3009 0.1393 0.2082 0.1466 819200 0.1763 0.09 0.1194 0.0917 1638400 0.1113 0.0633 0.0795 0.0656 3276800 0.0764 0.0508 0.0564 0.0507 6553600 0.0577 0.0444 0.0482 0.0446 Negative 0.0411 0.0428 0.041 0.043

[0226] 4. Hybridoma cell fusion experiment

[0227] After immunization, the spleen of the immunized animal was prepared and fused with myeloma cells (Ag8). The resulting hybridoma cells were cultured, and the reactivity of the cell supernatant after fusion to mybpc-3 was tested by ELISA. Cells with higher titer and affinity were selected for subcloning. After culture, the reactivity of the subcloned cell culture supernatant to mybpc-3 was tested again. Cells with higher titer and affinity were selected for the second subcloning. The cell culture supernatant of the second subcloning was detected by indirect ELISA, and the cell lines with all positive results were expanded in culture. The supernatant was collected and subjected to subtype identification. After collecting the cells, mice were injected to prepare ascites. After purification, the purity of the ascites was verified by SDS to be above 90%. The results are shown in Figure 5 。

[0228] 5. Antibody titer and sensitivity test

[0229] Antigen: After digestion with C0C1f, concentration: 0.92 mg / ml, 300 ng / well, 32 wells;

[0230] Primary antibody:

[0231] C0C1f-2F112D7, batch number: D240408C05, concentration: 0.91 mg / ml, starting from 5 μg in the first well (5.49 μl of antibody was made up to 100 μl with PBST), diluted 5-fold from the second well until the 15th well, and the 16th well was the negative control;

[0232] HM134-4C53C5, batch number: D240408C03, concentration: 2.85 mg / ml, starting from 5 μg in the first well (1.75 μl of antibody was made up to 100 μl with PBST), diluted 5-fold from the second well until the 15th well, and the 16th well was the negative control;

[0233] Secondary antibody: Goat anti-mouse (1:5000)

[0234] The results are shown in Figure 6 :EC 50 :

[0235] C0C1f-4C53C5 was 0.0067 μg / ml, and C0C1f-2F112D7 was 0.0069 μg / ml.

[0236] 6. The above 2F112D7 and 4C53C5 were labeled and used for ELISA detection.

[0237] Primary antibody (coating antibody):

[0238] 2F112D7, batch number: D240408C05, concentration: 0.91 mg / ml. Start with 5 μg of the coated antibody in the first well (make up 5.49 μl of the antibody to 100 μl with PBST), and perform 5-fold dilution starting from the second well until the 15th well. The 16th well is the negative control;

[0239] Secondary antibody (labeled antibody):

[0240] 4C53C5, batch number: D240408C03, concentration: 2.85 mg / ml. The labeled antibody is directly labeled with horseradish peroxidase (HRP, Suzhou Yake). The labeling method is sodium periodate oxidation method. After the aldehyde group-modified HRP binds to the antibody, it is blocked with ethanolamine and the buffer is replaced by ultrafiltration. After recovery, the concentration is determined to be 2 - 3 mg / ml. The results of antibody recovery rate are as follows:

[0241] Experimental procedure:

[0242] 1) Coating with primary antibody: Coat with 2F112D7 antibody, dilute it to a concentration of 2 μg / ml with 1X CBS, and add 100 μl of the diluted antibody to each well using a multi-channel pipette (the amount of antibody per plate is 2 μg), and incubate at 37°C for 2 h. After incubation, discard the liquid in the wells and pat dry on absorbent paper.

[0243] 2) Blocking: Add 200 μl of the blocking solution (1.5% BSA solution) to each reaction well and block at 37°C for 2 h.

[0244] 3) Washing: Discard the liquid and pat dry on absorbent paper. Add 200 μl of the washing solution TBST to each well using a multi-channel pipette, gently shake, discard the washing solution, and pat dry on absorbent paper. Repeat the washing 3 times and store at 4°C for later use. 4) Preparation of antigen calibrator: Dilute the antigen concentration to 20,000 pg / ml with 1X PBS, and then perform 2-fold serial dilution to obtain 8 gradient concentrations of antigen calibrators.

[0245] 5) Loading sample: Add 100 μl of the diluted antigen calibrator to each well using a pipette and incubate at 37°C for 2 h. After incubation, discard the liquid in the wells and pat dry on absorbent paper.

[0246] 6) Incubation with secondary antibody: Dilute the labeled antibody 1:2000 with 1X PBS, add 100 μl to each well using a multi-channel pipette, cover with tin foil, and incubate at 37°C for 1 h.

[0247] 7) Washing: Discard the liquid and pat dry on absorbent paper. Add 200 μl of the washing solution PBST to each well using a multi-channel pipette, gently shake, discard the washing solution, and pat dry on absorbent paper. Repeat the washing 4 - 5 times.

[0248] 8) Color development: Add 100 μl of TMB color development solution to each well using a multi-channel pipette, cover with tin foil, and react at room temperature for 5 min.

[0249] 9) Termination: Add the termination solution, 50 μl per well with a multi-channel pipette.

[0250] 10) Reading and data processing: Place the ELISA plate at the designated position on the microplate reader, select the reading area and a wavelength of 450 nm, and read the OD 450 data of each well. Make a fitting curve based on the data and calculate the correlation coefficient.

[0251] The experimental results are as Figure 7 shown, indicating that there is a good correlation between the concentration of the antigen standard and the final colorimetric degree, proving that the antibody used in our method can meet the detection requirements of the antigen.

[0252] Discussion

[0253] In the existing technical solutions, due to the time lag in the appearance of troponin after the onset of AMI in patients, the low concentration in the patient's body, and the presence in healthy people, etc., the occurrence of AMI cannot be accurately judged. The existing cmybp-c antibody has poor sensitivity and specificity in detecting the presence of human proteins, and cannot solve the early diagnosis of acute myocardial infarction patients.

[0254] Cardiac myosin-binding protein C (Myosin-Binding Protein C, Cardiac-Type, MYBPC3 or cMyBP-C) better meets this standard. The reasons are as follows: 1) MYBPC3 is rapidly released into the blood after myocardial infarction, which helps to shorten the diagnostic time. Three hours after the occurrence of AMI, the level of MYBPC3 in the blood has increased, reached the peak at 6 hours, and continued until 12 hours after the occurrence of AMI, and the MYBPC3 level returned to the baseline level; while the level of cTn in the blood will increase only after 6 - 12 hours. Some studies have shown that when 3 - 9 mg of myocardial necrosis occurs, the increase in cTn level can be detected; while when only 0.07 mg of myocardial tissue necrosis occurs, the increase in MYBPC3 level can be detected. 2) The concentration of MYBPC3 in the blood of AMI patients is relatively high. cMyC is mainly a thick filament-related protein and is one of the most abundant proteins in myocardial proteins, ranking 19th among more than 2300 quantified proteins. In the ultra-early stage of AMI, intact MYBPC3 and its hydrolysis products are continuously and massively released into the blood, which can be several times or even dozens of times higher than cTnT. It is relatively easier to measure the concentration of MYBPC3 in the blood. Therefore, MYBPC3 can be a sensitive and specific indicator for the ultra-early diagnosis of AMI.

[0255] Cmbpc can be used as an alternative detection index, but currently there is no well-developed detection antibody to develop an ELISA detection kit. At present, there is no reliable kit for detecting cMyBPC in acute myocardial infarction patients at the clinical end. The possible reason is the lack of antibodies with high affinity and good specificity, and new antibodies labeled as specific ones need to be developed.

[0256] The present invention is based on the following points: (1) the specificity of the recombinant protein antigen sequence and the antigen-antibody binding epitope; (2) the good affinity of the antibody for the natural human protein; (3) the specificity of the antigen-antibody binding epitope, forming a pair of specific binding antibodies for sandwich detection with high affinity; a pair of specific detection antibodies have been developed, with high sensitivity and specificity, and are suitable for the development of detection kits.

[0257] All the documents mentioned in the present invention are cited in this application as references, just as each document is cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0258] Sequence information of the present invention

[0259] Sequences related to the first monoclonal antibody 2F112D7:

[0260] V H -CDR1: Its amino acid sequence is ANTFH (SEQ ID NO.2)

[0261] V H -CDR2: Its amino acid sequence is GFNPYNGVSNINQNFKG (SEQ ID NO.3)

[0262] V H -CDR3: Its amino acid sequence is NYDPFDY (SEQ ID NO.4)

[0263] V L -CDR1: Its amino acid sequence is RSSQNIVHSNGNTYLE (SEQ ID NO.5)

[0264] V L -CDR2: Its amino acid sequence is KVSNRFS (SEQ ID NO.6)

[0265] V L -CDR3: Its amino acid sequence is FQGSHVPLT (SEQ ID NO.7)

[0266] The variable region of the heavy chain of 2F112D7 (VH ) Amino acid sequence

[0267] EVQLQQSGPELVKPGASVKISCKTSGFTFTANTFHWMKQSHGKSLEWIGGFNPYNGVSNINQNFKGKATLTVDKSSSTAYMELRSLTSDDSAVYYCARNYDPFDYWGQGTTLTVSS(SEQ ID NO.8)

[0268] Encoding the variable region of the heavy chain of 2F112D7 (V H ) Nucleotide sequence encoding the amino acid

[0269] GAGGTTCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATTCACATTCACTGCAAACACCTTTCACTGGATGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTTTTAATCCTTACAATGGTGTTTCTAACATCAACCAGAATTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAACTCCGCAGCCTGACATCTGACGATTCTGCAGTCTATTACTGTGCAAGGAATTACGACCCTTTTGACTACTGGGGCCAAGGCACCACTCTCACCGTCTCGAGC(SEQ ID NO.18)

[0270] Amino acid sequence of the variable region of the light chain of 2F112D7 (VL)

[0271] DVVMTQSPLSLTVSLGDHASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK(SEQ ID NO.9)

[0272] Encoding the variable region of the light chain of 2F112D7 (V L ) Nucleotide sequence encoding the amino acid

[0273] GATGTTGTGATGACCCAAAGTCCACTCTCCCTGACTGTCAGTCTTGGAGATCACGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTGCATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTTGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO.19)

[0274] Sequences related to the second monoclonal antibody 4C53C5:

[0275] V H -CDR1: Its amino acid sequence is DYYIH (SEQ ID NO.10)

[0276] V H -CDR2: Its amino acid sequence is WIDPENGDTEYVPKFQG (SEQ ID NO.11)

[0277] V H -CDR3: Its amino acid sequence is RGVVAHYYAMDY (SEQ ID NO.12)

[0278] V L -CDR1: Its amino acid sequence is KSSQSLLNSGDQKNKLA (SEQ ID NO.13)

[0279] V L -CDR2: Its amino acid sequence is GASTRES (SEQ ID NO.14)

[0280] V L -CDR3: Its amino acid sequence is QNDHSYPLT (SEQ ID NO.15)

[0281] Amino acid sequence of the variable region of the heavy chain of 4C53C5 (V H )

[0282] QVQLQQSGAELVRSGASVKLSCTASGFNIKDYYIHWVKQRPEQGLEWIGWIDPENGDTEYVPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNTRGVVAHYYAMDYWGQGTSVTVSS(SEQ ID NO.16)

[0283] Nucleotide sequence encoding the variable region of the 4C53C5 heavy chain (V H ) amino acids

[0284] CAGGTTCAACTGCAGCAGTCTGGGGCAGAGCTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTATATACACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTGATACTGAATATGTCCCGAAATTCCAGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTATTGTAATACAAGGGGGGTAGTAGCTCATTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCGAGC(SEQ IDNO.20)

[0285] Amino acid sequence of the variable region of the 4C53C5 light chain (V L )

[0286] DVVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGDQKNKLAWYQQKPGQSPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLELK(SEQ ID NO.17)

[0287] Nucleotide sequence encoding the variable region of the 4C53C5 light chain (V L ) amino acids

[0288] GATGTTGTGATGACCCAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAGATCAAAAAAACAAGTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGTTGATCTACGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATCATAGTTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO.21)

Claims

1. An anti-MYBPC3 antibody, characterized in that, The heavy chain variable region of the anti-MYBPC3 antibody comprises the following three complementarity determining regions (CDRs): (i)V as shown in SEQ ID NO.2 H -CDR1, V as shown in SEQ ID NO.3 H -CDR2, and V as shown in SEQ ID NO.4 H -CDR3; and, The light chain variable region of the anti-MYBPC3 antibody comprises the following three complementarity determining regions (CDRs): (ii) V as shown in SEQ ID NO.5 L - CDR1, V as shown in SEQ ID NO.6 L - CDR2, and V as shown in SEQ ID NO.7 L - CDR3.

2. An anti-MYBPC3 antibody, characterized in that, The heavy chain variable region of the anti-MYBPC3 antibody comprises the following three complementarity determining regions (CDRs): (iii) V as shown in SEQ ID NO. 10 H -CDR1, V as shown in SEQ ID NO. 11 H -CDR2, and V as shown in SEQ ID NO. 12 H -CDR3; and, The light chain variable region of the anti-MYBPC3 antibody comprises the following three complementarity determining regions (CDRs): (iv) V shown in SEQ ID NO. 13 L -CDR1, V shown in SEQ ID NO. 14 L -CDR2, and V shown in SEQ ID NO. 15 L -CDR3.

3. The anti-MYBPC3 antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.8; and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO.

9.

4. The anti-MYBPC3 antibody according to claim 2, wherein The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.16; and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO.

17.

5. A recombinant protein, characterized in that, The recombinant protein has: (i) an antibody as described in any one of claims 1-4.

6. The recombinant protein according to claim 5, wherein The recombinant protein further has: (ii) a tag sequence for assisting expression and / or purification.

7. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) an antibody as described in any one of claims 1-4, or a recombinant protein as described in claim 5; and (b) a conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

8. Use of an antibody as described in any one of claims 1-4, a recombinant protein as described in claim 5, or an immunoconjugate as described in claim 7 for the preparation of a detection reagent, a detection plate or a kit.

9. A detection article, characterized in that, The detection article comprises: (1) an antibody as described in any one of claims 1-4, a recombinant protein as described in claim 5, or a conjugate as described in claim 7.

10. The test article according to claim 9, wherein, The detection article further comprises: (2) a buffer solution or a buffering agent.

11. The test article according to claim 9, wherein, The detection article is a detection reagent, a lateral flow strip, a chip, a test strip, a detection plate or a test piece.

12. A detection kit, characterized in that, The detection kit contains an antibody as described in any one of claims 1-4, a recombinant protein as described in claim 5, or a conjugate as described in claim 7 or a detection article as described in claim 9.

Images