Monoclonal antibodies against human thrombomodulin and their applications

Abstract

The present invention provides a monoclonal antibody against human thrombin modulin and its application. The provided antibody comprises the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 1, 2, and 3 and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 4, 5, and 6; or comprises the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 7, 8, and 9 and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 10, 11, and 12. The provided antibody can specifically bind to thrombin modulin and can be used to prepare a kit with high specificity and sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies, and in particular relates to a monoclonal antibody against human thrombin modulin and its application. Background Art

[0002] Human thrombomodulin (TM) is a single-chain transmembrane glycoprotein with a relative molecular mass of 75,000 Da. Upon binding to thrombin, human thrombomodulin reduces thrombin's coagulation activity and enhances its ability to activate protein C. Because activated protein C has an anticoagulant effect, human thrombomodulin is an important intravascular coagulation inhibitor that shifts thrombin's activity from procoagulant to anticoagulant.

[0003] Human thrombomodulin is located on the surface of endothelial cell membranes and has multiple functions such as clearing thrombin, activating anticoagulant factor protein C (PC) and promoting fibrinolysis. It plays an important role in regulating thrombus formation and dissolution.

[0004] When endothelial cells become diseased or damaged, they often cause abnormal expression and secretion of thrombomodulin, which is then released into the bloodstream, leading to changes in thrombomodulin levels. Therefore, thrombomodulin can be used as a marker for endothelial cell damage. The normal human thrombomodulin level ranges from 3.82 to 13.35 TU / mL. As an indicator of endothelial damage, elevated levels of thrombomodulin indicate endothelial system damage and can aid in the diagnosis of DC and atherosclerosis.

[0005] Therefore, it is very necessary to develop a diagnostic kit for human thrombin modulin with excellent performance. Summary of the Invention

[0006] The present invention aims to solve one of the technical problems in the related art to at least a certain extent. In order to solve the problems of sensitivity and specificity, the present invention provides a monoclonal antibody against human thrombin regulatory protein and its application. The provided anti-human thrombin regulatory protein monoclonal antibody has excellent performance, can specifically bind to human thrombin regulatory protein, and has high sensitivity, accuracy and strong specificity. The provided anti-human thrombin regulatory protein monoclonal antibody can be used to develop a diagnostic kit with excellent performance, which is used to diagnose the content of human thrombin regulatory protein in various samples and to assist clinical diagnosis.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention provides an anti-human thrombin modulin monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment thereof is selected from at least one of the following:

[0009] (1) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 1, 2 and 3;

[0010] and LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NOs: 4, 5, and 6, or sequences having one or two conservative amino acid mutations compared to the sequences as shown in SEQ ID NOs: 4, 5, and 6;

[0011] (2) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 7, 8 and 9, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 7, 8 and 9;

[0012] and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences having one or two conservative amino acid mutations compared to the sequences shown in SEQ ID NOs: 10, 11 and 12.

[0013] The second aspect of the present invention provides an anti-human thrombomodulin monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment thereof is selected from at least one of the following:

[0014] (a) the HCDR1, HCDR, and HCDR3 sequences of the heavy chain variable region set forth in SEQ ID NO: 13, and the LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region set forth in SEQ ID NO: 14; or

[0015] (b) The HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 15, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown in SEQ ID NO: 16.

[0016] According to an embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof provided above, the sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 13 or 15;

[0017] The sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO:14 or 16.

[0018] According to a specific embodiment of the present invention, the sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the sequence of the light chain variable region is shown in SEQ ID NO: 14;

[0019] Alternatively, the sequence of the heavy chain variable region is shown as SEQ ID NO: 15, and the sequence of the light chain variable region is shown as SEQ ID NO: 16.

[0020] The third aspect of the present invention provides a polynucleotide encoding the anti-human thrombomodulin monoclonal antibody or antigen-binding fragment thereof according to the first aspect.

[0021] According to an embodiment of the present invention, the polynucleotide comprises the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18;

[0022] Or include the sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20.

[0023] The fourth aspect of the present invention provides a construct comprising the polynucleotide described in the third aspect.

[0024] The fifth aspect of the present invention provides a host cell containing the polynucleotide described in the third aspect or the construct described in the fourth aspect.

[0025] The sixth aspect of the present invention provides a kit comprising the anti-human thrombomodulin monoclonal antibody or antigen-binding fragment thereof according to the first aspect or the second aspect.

[0026] The present invention provides a kit, which can be used for in vitro quantitative analysis of the content of human thrombomodulin in human serum, plasma, cell lysate, cell culture supernatant and urine.

[0027] According to an embodiment of the present invention, the kit includes a first antibody and a second antibody, wherein the first antibody includes the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 4, 5, and 6; and the second antibody includes the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 10, 11, and 12. The provided kit can include both the first and second antibodies, and, for example, a double-antibody sandwich enzyme-linked immunosorbent assay can be used to detect a sample. One of the antibodies is used as a coating antibody, and the other is used as a detection antibody.

[0028] A seventh aspect of the present invention provides a method for detecting the content of anti-human thrombin modulin in a biological sample, comprising:

[0029] Incubating the anti-human thrombomodulin monoclonal antibody or the antigen-binding fragment thereof according to the first aspect or the second aspect with a biological sample, and measuring the OD value of the reaction product at a predetermined wavelength;

[0030] Based on the OD value, the content of thrombin modulin in the biological sample is determined.

[0031] The eighth aspect of the present invention provides a pharmaceutical composition comprising the anti-human thrombomodulin monoclonal antibody or antigen-binding fragment thereof according to the first aspect or the second aspect, and a pharmaceutically acceptable carrier.

[0032] A ninth aspect of the present invention provides a use of the antibody or antigen-binding fragment thereof described in the first or second aspect in the preparation of a human thrombin modulin detection kit or a drug.

[0033] The beneficial effects achieved by the present invention are at least:

[0034] The present invention provides monoclonal antibodies against human thrombomodulin, which can be used to prepare diagnostic kits. The present invention provides a monoclonal antibody composition against human thrombomodulin, comprising anti-human thrombomodulin Clone 1 and anti-human thrombomodulin Clone 2. This antibody combination exhibits specificity and high affinity. A method for preparing a diagnostic test kit based on these antibodies is also provided, thereby improving the specificity of the diagnostic reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The following are the ELISA test results of the antibodies provided in the embodiments of the present invention.

[0036] Figure 2 This is the specific detection result of the antibody provided according to the embodiment of the present invention.

[0037] Figure 3 The figure is a standard curve diagram of the kit provided according to an embodiment of the present invention.

[0038] Figure 4 3 is a reference value performance analysis diagram of the kit provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0039] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0040] As used in this specification and the claims, "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0041] As used herein, the term "antibody" is used in the broadest sense to refer to a protein or polypeptide containing an antigen-binding site, antigen-binding fragment, or antigen-binding portion, encompassing natural and artificial antibodies of various structures, including but not limited to intact antibody forms or antigen-binding fragments of antibodies. According to a specific embodiment, the antibody referred to is a monoclonal antibody. A "complete antibody" is generally a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region includes the heavy chain constant domain CH1, the heavy chain constant domain CH2, and the heavy chain constant domain CH3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant domain CL. The VH and VL regions can be further divided into complementarity determining regions (also called hypervariable regions or hypervariable regions, abbreviated as CDRs), which are separated by conserved framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) in the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2, and HCDR3, respectively, starting from the amino terminus, and the CDRs of the light chain variable region are referred to as LCDR1, LCDR2, and LCDR3, respectively, starting from the amino terminus.

[0042] An "antigen-binding fragment" refers to a portion of a full-length antibody that exhibits the property or function of binding to an antigen. The term "variable region" or "variable domain" refers to the domain involved in antigen binding to the heavy or light chain of an antibody. As mentioned above, the hypervariable or hypervariable regions of the heavy and light chains of an intact antibody exhibit specific binding to an antigen. Examples of antigen-binding fragments or antigen-binding portions include, but are not limited to, Fab, Fab', F(ab')2, bispecific Fab' and Fv fragments (variable regions), linear antibodies, single-chain antibodies, single-domain antibodies, and the like. Papain digestion of an intact antibody produces two identical antigen-binding fragments, called Fab fragments, each containing the heavy and light chain variable regions and the light chain constant domain and heavy chain constant domain CH1. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxyl terminus of the heavy chain constant domain CH1, including one or more cysteines from the antibody hinge region. Pepsin digestion of an intact antibody produces F(ab')2 fragments. F(ab')2 fragments contain two antigen-binding F(ab) moieties linked by disulfide bonds, making them bivalent antibodies. Single-chain antibodies are fusion proteins composed of the variable regions of the heavy and light chains of antibodies, linked by a flexible peptide of approximately 10-25 amino acids. Single-domain antibodies are antibody fragments composed of the variable regions of a single monomer. Because single-domain antibodies are typically derived from the variable regions of the heavy chains of camelid or shark antibodies, they are often referred to as nanobodies. Nanobodies contain only the heavy chain CDR regions and are the smallest fully functional antigen-binding fragments. Nanobodies consist of three CDR regions (CDR1-CDR3) and four framework regions (FR1-FR4). The framework regions FR1, FR2, FR3, and FR4 are separated by complementarity determining regions (CDR1, CDR2, and CDR3), respectively. Antigen-binding fragments can be obtained recombinantly. For example, a nucleic acid encoding the desired antigen-binding fragment can be constructed, introduced into an expression vector, and expressed in a suitable host to produce the antigen-binding fragment.

[0043] The term "comprising" or "including" means including the elements or steps mentioned, but does not exclude other elements or steps. Of course, unless otherwise specified, "comprising" or "including" also covers the situation where it consists of the elements or steps mentioned. For example, when referring to an antibody variable region comprising a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0044] The "affinity" or "binding affinity" mentioned herein is understood according to the common meaning in the art, and is used to reflect the strength and / or stability of the binding sites between an antigen and an antibody or antigen-binding fragment.

[0045] "Specific binding" or "specifically binds to", "binds to", "specifically targets" a specific antigen or epitope, or "has specificity" or "can bind to" a specific antigen or epitope means to distinguish it from non-specific interactions, and this specific binding can be measured by some methods commonly used in the art. The ability of an antibody to bind to an antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, cells carrying an antigen can be detected by flow cytometry, and the competitive binding between the test antibody and the labeled antibody can be detected by measuring the positive rate index of the cells. Since the spatial structure of the antigen on the cell surface is closer to the form existing in the body, this method can better reflect the real situation.

[0046] Whether provided herein as an antibody or antigen-binding fragment, or a polynucleotide, it is generally separable or recombinant. "Separable" refers to being able to identify and separate and / or recycle from cells or cell cultures expressing a polypeptide or protein. Typically, the isolated polypeptide will be prepared by at least one purification step. "Isolated antibody" refers to being substantially free of other antibodies or their antigen-binding fragments that have different antigenic specificities. "Recombinant" means that the antibody can be produced in an exogenous host cell using genetic recombination techniques.

[0047] The CDR sequences of the antibodies shown herein can be obtained by combining existing database analysis. The CDR sequences of the antibodies provided are obtained in combination with the Kabat definition scheme (for example, see US Patent No. 1983, “Sequences of Proteins of Immunological Interest”). They can also be obtained by definition schemes such as IMGT (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38: D301-D307 (2010); Ehrenmann, F., Lefranc, M.-P. Cold Spring Harbor Protoc., 6: 737-749 (2011)), Chothia (for example, see J. Mol. Biol. 196: 901-917 (1987)). It is understood by those skilled in the art that differences in CDRs due to differences in definition methods are also included in the scope of protection of the present invention.

[0048] Antibodies or antigen-binding fragments thereof

[0049] The present invention provides an anti-human thrombin modulin antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment thereof is selected from at least one of the following:

[0050] (1) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 1, 2 and 3;

[0051] and LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NOs: 4, 5, and 6, or sequences having one or two conservative amino acid mutations compared to the sequences as shown in SEQ ID NOs: 4, 5, and 6;

[0052] (2) comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 7, 8 and 9, or sequences having one or two conservative amino acid mutations compared to the sequences of SEQ ID NOs: 7, 8 and 9;

[0053] and the LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences having one or two conservative amino acid mutations compared to the sequences shown in SEQ ID NOs: 10, 11 and 12.

[0054] Herein, "conservative amino acid mutation" means that the amino acid mutation does not cause a significant change in the conformational structure of the protein or polypeptide and retains its biological activity. The conservative amino acid mutation mentioned can be a mutation between different amino acids with similar physiological and biochemical properties. For example, after conservative amino acid mutation, the provided antibody still retains specific binding activity to the CD235a protein. According to an embodiment, the conservative amino acid mutation mentioned can be a substitution, deletion or addition of conservative amino acids. According to a specific embodiment, the deletion of conservative amino acids is based on the listed CDR sequences, deleting one amino acid, two amino acids or three amino acids. According to a specific embodiment, the addition of conservative amino acids is based on the listed CDR sequences, adding one amino acid, two amino acids or three amino acids. According to a specific embodiment, "conservative amino acid substitution" means replacing another amino acid residue with a different amino acid residue having a side chain with similar physiological and chemical properties. For example, conservative amino acid substitutions can occur between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative amino acid substitutions generally do not cause significant changes in the conformational structure of the protein, and thus can retain the biological activity of the protein. The conservative amino acid substitutions mentioned can be one conservative amino acid substitution, two conservative amino acid substitutions, three conservative amino acid substitutions, etc. The names of the amino acids used herein are represented by standard single-letter or three-letter codes commonly used in the art. In some embodiments, the HCDR sequences provided have one conservative amino acid substitution compared to the HCDR sequences shown. In some embodiments, the HCDR sequences provided have one conservative amino acid deletion from the HCDR sequences shown. In some embodiments, the HCDR sequences provided have one conservative amino acid addition from the HCDR sequences shown. In some embodiments, the LCDR sequences provided have one conservative amino acid substitution from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have one conservative amino acid deletion from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have one conservative amino acid addition from the LCDR sequences shown.

[0055] The present invention also provides a monoclonal antibody or antigen-binding fragment thereof, comprising the HCDR1, HCDR2, and HCDR3 sequences from the heavy chain variable region of SEQ ID NO: 13, and the LCDR1, LCDR2, and LCDR3 sequences from the light chain variable region of SEQ ID NO: 14; or comprising the HCDR1, HCDR2, and HCDR3 sequences from the heavy chain variable region of SEQ ID NO: 15, and the LCDR1, LCDR2, and LCDR3 sequences from the light chain variable region of SEQ ID NO: 16. The HCDR sequences and LCDR sequences from these heavy chain variable regions and light chain variable regions may differ slightly according to different definition schemes, but are all included within the scope of protection of the present invention.

[0056]

[0057]

[0058]

[0059]

[0060] According to a specific embodiment, the present invention provides an anti-human thrombin modulin monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 13, and the sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 14. According to a specific embodiment, the present invention provides an anti-human thrombin modulin monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 15, and the sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 16.

[0061] The "sequence identity", "sequence homology" and "sequence similarity" mentioned herein refer to the degree of sequence identity when two polypeptides, proteins or nucleotide sequences are compared. In order to determine the ratio of sequence identity, it can be achieved in a variety of ways known in the art. For example, it can be obtained using public software such as BLAST, ALIGN, BLAST-2 and the like. In some specific embodiments, the sequence identity is due to conservative amino acid mutations. According to a specific embodiment, the sequence having sequence identity with the above-mentioned heavy chain variable region or light chain variable region may be due to 1 conservative amino acid mutation, 2 conservative amino acid mutations, 3 conservative amino acid mutations, 4 conservative amino acid mutations, 5 conservative amino acid mutations, 6 conservative amino acid mutations, 7 conservative amino acid mutations, 8 conservative amino acid mutations, 9 conservative amino acid mutations or 10 conservative amino acid mutations. According to a specific embodiment, the sequence having sequence identity with the above-mentioned heavy chain variable region or light chain variable region differs in the framework region.

[0062] According to a preferred embodiment, the present invention provides an anti-human thrombomodulin monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the sequence of the light chain variable region is shown in SEQ ID NO: 14. According to a preferred embodiment, the present invention provides an anti-human thrombomodulin monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0063] According to a more preferred embodiment, the provided anti-human thrombomodulin monoclonal antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 22 and the light chain shown in SEQ ID NO: 23. According to a more preferred embodiment, the provided anti-human thrombomodulin monoclonal antibody or its antigen-binding fragment comprises the heavy chain shown in SEQ ID NO: 24 and the light chain shown in SEQ ID NO: 25.

[0064] polynucleotides

[0065] The present invention also provides a polynucleotide encoding the anti-human thrombin modulator antibody or its antigen-binding fragment described above. The polynucleotide mentioned is separable, including but not limited to DNA, RNA or cDNA, etc. Conventional methods in the art can be used to obtain isolated polynucleotide sequences to encode the monoclonal antibody or antibody or antigen-binding fragment mentioned above. According to a specific embodiment, the polynucleotide provided encodes the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18, and these polynucleotide sequences encode the heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 14. According to a specific embodiment, the polynucleotide provided encodes the sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20, and these polynucleotide sequences encode the heavy chain variable region shown in SEQ ID NO: 15 and the light chain variable region shown in SEQ ID NO: 16.

[0066] The term "polynucleotide" as used herein refers to a polynucleotide that is not naturally present in nature, including polynucleotides isolated from nature (including organisms) by biological techniques, and also includes artificially synthesized polynucleotides. The isolated polynucleotide can be genomic DNA, cDNA, mRNA or other synthetic RNA, or a combination thereof. Provided herein are multiple nucleotide sequences for encoding the heavy chain variable region and light chain variable region of anti-human thrombin modulator monoclonal antibodies. It should be noted that those skilled in the art can, based on the amino acid sequences of the heavy chain variable region and light chain variable region provided herein and based on codon degeneracy, design nucleotide sequences that are not completely identical to the nucleotide sequences provided above, but all encode the same amino acid sequence. These modified nucleotide sequences are also included within the scope of the present invention.

[0067] Construct

[0068] To prepare the antibodies described herein, the polynucleotide sequence encoding the antibody can be inserted into a replicable expression vector and expressed in a host cell or cell-free expression system. The present invention also provides a construct comprising the polynucleotide described above. Constructs can be obtained using a variety of methods commonly used in the art, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. For example, the polynucleotide can be inserted into the multiple cloning site of an expression vector to form a construct. The construct can contain various operators, such as promoters, terminators, and marker genes, as needed, operably linked to the polynucleotide. Promoters typically provide a signal to initiate transcription; promoters can include the lactose promoter (Lac), Trp promoter, Tac promoter, or phage PL and PR promoters. Terminators provide a signal to terminate transcription during the transcription process, and marker genes in the construct are often used for screening. Enhancers can also be included as needed to enhance protein expression. The expression vector is not particularly limited and can be commercially available or artificially modified, such as plasmids, phages, or viruses. Viruses can include plant cell viruses and mammalian cell viruses. The construct can express antibodies or proteins in vitro or be transferred into cells to express antibodies or proteins.

[0069] host cells

[0070] The present invention also provides a host cell containing the aforementioned polynucleotide or construct. Any cell suitable for expressing an antibody or protein using a polynucleotide or construct can be used as a host cell. The host cell can be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a yeast cell or a mammalian cell. Commonly used host cells include yeast cells, CHO cells, HEK-293 cells, COS cells, and Drosophila S2 or Sf9 insect cells. Host cells containing the polynucleotide or construct can be obtained using methods commonly used in the art, such as microinjection, electroporation, chemical transfection, and viral-mediated transformation.

[0071] Pharmaceutical composition

[0072] The present invention also provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier. The prepared pharmaceutical composition can be used to inhibit the content of thrombin modulin, thereby reducing endothelial damage and alleviating arteriosclerosis.

[0073] The pharmaceutical composition also includes a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt (e.g., as described in Berge, S. M. et al (1977) J. Pharma. Sci. 66: 1-19). The pharmaceutically acceptable carrier mentioned is acceptable to the subject at the dose or concentration used. Pharmaceutically acceptable carriers include, but are not limited to, buffers or salts, such as disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium acetate, citric acid, sodium citrate, citrate, Tris; carbohydrates, such as trehalose, polysorbate, sucrose, mannitol; surfactants such as polysorbate; preservatives such as hexamethonium chloride, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine, etc. Sterile pharmaceutical preparations can be obtained by methods commonly used in the art, such as by filtration through a sterile filtration membrane. Those skilled in the art can select different pharmaceutically acceptable carriers for the pharmaceutical composition as needed to prepare different dosage forms, such as lyophilized dosage forms, injections, and other dosage forms. The prepared different pharmaceutical dosage forms can be formulated and administered to a subject by any suitable route of administration, including but not limited to intravenous, dermal, intramuscular, peritoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, and the like.

[0074] Reagent test kit

[0075] The present invention also provides a kit comprising the above-mentioned monoclonal antibody or antigen-binding fragment. The antibody or antigen-binding fragment can be present in the kit as a reagent.

[0076] The provided kit includes at least one of the above-mentioned antibodies. When it contains only one antibody, the biological sample can be directly detected (for example, the antibody and sample are directly incubated, and then the labeled secondary antibody is used for detection to determine the content of human thrombin regulatory protein in the biological sample); when it contains two antibodies, the biological sample can be detected by the double antibody sandwich method (that is, the target antigen in the biological sample is captured and detected in a sandwich manner using two specific antibodies). The double antibody sandwich method is used for detection because the use of two specific antibodies can effectively capture and detect the target antigen, avoiding false positives and false negatives; although two incubations are required, the operation is simple and can reduce the impact of human errors.

[0077] According to a specific embodiment, the provided kit includes a first antibody and a second antibody, wherein the first antibody includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6; and the second antibody includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12. According to a specific embodiment, the first antibody includes a heavy chain variable region set forth in SEQ ID NO: 13 and a light chain variable region set forth in SEQ ID NO: 14. According to a specific embodiment, the second antibody includes a heavy chain variable region set forth in SEQ ID NO: 15 and a light chain variable region set forth in SEQ ID NO: 16.

[0078] The first antibody mentioned above can be used as a coating antibody, and the second antibody can be used as a detection antibody; or the first antibody can be used as a detection antibody, and the second antibody can be used as a coating antibody.

[0079] According to a specific embodiment, the kit comprises a first antibody composition and a second antibody composition,

[0080] The first antibody composition includes a first antibody and a 0.1 M PBS buffer;

[0081] The second antibody composition comprises a biotin-labeled second antibody, 0.1 M MES buffer, 0.1% to 0.5% v / v Tween-20, 0.5% to 1.0% w / v NaCl, 1% to 3% w / v BSA, and 0.1% to 0.5% v / v Proclin-300. According to a preferred embodiment, the second antibody composition comprises a biotin-labeled second antibody, 0.1 M MES buffer, 0.2% Tween-20, 0.85% NaCl, 2% BSA, and 0.2% Proclin-300.

[0082] The kits mentioned above may also include a human thrombomodulin standard, which includes human thrombomodulin, the sequence of which is shown in SEQ ID NO: 21. The purity of human thrombomodulin meets certain requirements, such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc. The kits may also include containers, buffers, etc. as needed. For example, PBS and 1% bovine serum albumin may also be included. These kits can be used in a variety of experimental techniques, including but not limited to flow cytometry, immunofluorescence, immunohistochemistry, and Western blot. Accordingly, the kits may also include instructions for use to facilitate operation and use by those skilled in the art.

[0083] To this end, the present invention also provides a method for detecting the content of human thrombin modulin in a biological sample, comprising:

[0084] Incubating the above-mentioned anti-human thrombin modulin monoclonal antibody or antigen-binding fragment thereof with a biological sample, and measuring the OD value of the reaction product at a predetermined wavelength;

[0085] Based on the OD value, the content of thrombin modulin in the biological sample is determined.

[0086] According to a specific embodiment, the provided method includes:

[0087] 1) Coat the plate with the primary antibody and block with blocking solution;

[0088] 2) Add human thrombin modulin standard or biological sample, incubate at 37°C, and rinse the plate;

[0089] 3) Continue adding biotin-labeled secondary antibody, incubate at 37°C, and then rinse the plate;

[0090] 4) Add horseradish peroxidase-labeled streptavidin, incubate at 37°C, and rinse the plate.

[0091] 5) Continue adding colorimetric reagent to develop color, then add stop solution to terminate the reaction. The reaction product is detected at wavelengths of 450 nm and 630 nm, and the OD value is determined based on the difference between the two wavelengths.

[0092] 6) preparing a standard curve based on the OD value of the human thrombomodulin standard, and determining the content of thrombomodulin in the biological sample based on the standard curve.

[0093] The provided kit can be used to detect human thrombin modulin with reference to this method.

[0094] In this article, the biological samples mentioned include but are not limited to serum, plasma, cell lysate, cell culture supernatant, urine, etc.

[0095] Methods for producing antibodies

[0096] The present invention further provides a method for producing the above-mentioned antibody or antigen-binding fragment, comprising: culturing the above-mentioned host cell, and collecting the antibody or antigen-binding fragment from the culture.

[0097] The antibodies or antigen-binding fragments collected from the culture can be purified to obtain a substantially pure product. "Substantially pure" means that the purity of the antibody or antigen-binding fragment is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or even greater than 99.5%, 99.6%, 99.7%, or 99.8%.

[0098] The technical scheme of the present invention is described in detail below with reference to Examples. It should be noted that these Examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention. The embodiments of the present invention are described in detail below. The described embodiments are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention. Unless otherwise stated, the methods or reagents used in the examples are methods or reagents commonly used in the art.

[0099] Example 1

[0100] Example 1 Human thrombin modulin was identified by the following method, and was produced and purified to obtain the corresponding protein.

[0101] First, the amino acid sequence of human thrombin regulatory protein (as shown in SEQ ID NO: 21) was searched on the NCBI official website, a histidine tag was added to the C-terminus of the protein amino acid, codon optimization software was used to optimize the codons in a mammalian cell expression system, the expression plasmid was synthesized, and the gene sequence was verified by sequencing.

[0102] Then, human thrombin modulator protein was expressed and produced:

[0103] One day before transfection, cells were cultured at 0.8 × 10 6Inoculate 100 mL of the thrombin-modulin protein vector into a cell culture flask and culture in OPM-293CD03 Medium at 37°C, 5% CO2 in a 37°C, 5% CO2 incubator until the cells reach 80%-90% confluency and greater than 95% viability before transfection. Mix the thrombin-modulin protein expression vector with the transfection reagent PEI and incubate at room temperature for 15-30 minutes. Then slowly add the vector to the HEK293 cells, add the enhancer and excipients, and continue culturing at 37°C, 5% CO2 in a 37°C, 5% CO2 incubator. When cell viability is <70%, harvest the cell culture, centrifuge at 8000g for 10 minutes, collect the supernatant, and then centrifuge at 12000g at 4°C for 30 minutes.

[0104] The collected cell supernatant was then affinity purified:

[0105] The cell supernatant was filtered through a 0.22 μm filter and affinity purified using a Ni TED-4FF bead-prepacked gravity column. Washing was performed with 20 mM Pyridoxine (PBB), 300 mM NaCl, 10% glycerol, 50 mM imidazole, pH 8.0. Elution was performed with 20 mM Pyridoxine (PBB), 300 mM NaCl, 10% glycerol, 250 mM imidazole, pH 8.0. Protein purity was assessed by SDS-PAGE, which showed >85%. The protein was dialyzed into 20 mM Pyridoxine (PBB), 300 mM NaCl, 10% glycerol, pH 8.0, sterile filtered through a 0.22 μm filter, and the protein concentration was determined by BCA assay. The protein was then stored at -20°C at 1 mg / mL until use. The purified antigen protein was at least 80% pure.

[0106] Example 2

[0107] Example 2: A monoclonal antibody against human thrombomodulin was prepared by the following method, comprising:

[0108] After affinity-purified human thrombin modulin was mixed and emulsified with Freund's adjuvant in equal volumes, mice were immunized at a dose of 100 μg / mouse. Three weeks later, the mice were boosted with a dose of 50 μg / mouse, and the boosted immunizations were repeated twice, with an interval of two weeks between each. Blood was collected from the mice to separate serum, and the serum antibody titer was determined by enzyme-linked immunosorbent assay. Mice with better immunity were selected for hybridoma fusion experiments. After screening and culture in HAT screening medium, human thrombin modulin was used as the coating antigen on an ELISA plate, and the hybridoma monoclonal antibody supernatant was subjected to enzyme-linked immunosorbent assay to screen high-quality positive cell lines. Two rounds of cell subcloning were performed to clone monoclonal cell lines, named Clone 1 and Clone 2, and the antibodies produced were denoted as Clone 1 antibody and Clone 2 antibody.

[0109] Total RNA from B lymphocytes was extracted using the TRIzol method and reverse transcribed into cDNA by RT-PCR. The variable regions of the antibody heavy and light chains were amplified by nested PCR and sequenced. The nucleic acid sequences encoding the heavy and light chains were then ligated into the pCDNA3.4 vector and transiently transfected into HEK293F cells. The cell culture supernatant was purified using a Protein A column to obtain an antibody that recognizes human thrombin modulin.

[0110] Sequencing revealed that the heavy chain variable region sequence of Clone 1 is shown in SEQ ID NO: 13, and the light chain variable region sequence is shown in SEQ ID NO: 14; the heavy chain variable region sequence of Clone 2 is shown in SEQ ID NO: 15, and the light chain variable region sequence is shown in SEQ ID NO: 16. SDS-PAGE analysis revealed that the purity of Clone 1 and Clone 2 was >95%; and that of Clone 1 and Clone 2 was >95%.

[0111] Example 3

[0112] Example 3 studied the affinity of the Clone 1 antibody and the Clone 2 antibody prepared in Example 2 for the antigen, including:

[0113] The human thrombin modulin prepared in Example 1 was used as the coating antigen and incubated at 4°C overnight. The titers of the two antibodies were then tested as follows:

[0114] Antigen was diluted to 1 μg / mL and coated onto an ELISA plate at 50 μL / well, followed by overnight incubation at 4°C. Different dilutions of clone 1 and clone 2 antibodies were added, with PBS added to the control group, and the plate was incubated at 37°C for 30 minutes. After washing, horseradish peroxidase (HRP)-labeled antibody was added at 50 μL / well, with GAM-HRP added to the control group, and the plate was incubated at 37°C for 30 minutes. The plate was then washed, displayed, and read.

[0115] like Figure 1 As shown: the EC50 of clone 1 antibody is 393 ng / mL, and the EC50 of clone 2 antibody is 406 ng / mL.

[0116] The human thrombin modulin prepared in Example 1 was used as the coating antigen and incubated at 4°C overnight. The specificity of the two antibodies was then detected separately as follows:

[0117] Dilute the antigen to 1 μg / mL and add 50 μL / well to the coated ELISA plate, incubating overnight at 4°C. Dilute clone 1 and clone 2 antibodies to the concentration sensitivity range according to the titer of the previous step, mix with the serially diluted antigen protein, and incubate at 37°C for 30 min. After washing the plate, add 50 μL / well of HRP-labeled secondary antibody of the corresponding species (mouse) and incubate at 37°C for 30 min. Wash the plate, display, and read the results.

[0118] The results are as follows Figure 2 As shown: the IC50 of clone 1 antibody is 302.7 ng / mL, and the IC50 of clone 2 antibody is 261.8 ng / mL.

[0119] The results are shown above. The results show that both Clone1 antibody and Clone2 antibody have good specific binding to the antigen, high affinity and strong specificity.

[0120] Example 4 Monoclonal Antibody Performance Analysis

[0121] Example 3: Using the Clone 1 antibody and Clone 2 antibody obtained in Example 2, a kit was prepared based on the double antibody sandwich method. The kit includes:

[0122] (1) Using Clone1 antibody as coating antibody

[0123] The Clone 1 antibody prepared in Example 2 was transferred to a 14 kD dialysis bag and dialyzed against PBS buffer, and then the concentration was adjusted to 2 mg / mL for use. The antibody storage solution was 0.1 M PBS.

[0124] (2) Using biotin-labeled Clone2 antibody as the detection antibody

[0125] Labeling reaction: 10mM biotin and Clone2 antibody were mixed at a molecular molar ratio of 20:1 at room temperature for 1-2 hours. After the reaction, the labeled solution was transferred to a 14kD dialysis bag and dialyzed against 1× PBS by rotation, changing the solution 6 times for 1-2 hours each time. After overnight dialysis, the labeled solution was removed and the protein content in the labeled solution was measured using a spectrophotometer. The protein content was calculated and protective solution was added. After thorough mixing, the solution was stored at 18-20°C. For testing, the protein concentration was diluted to 0.5ug / mL using the labeled preservation solution.

[0126] The labeling preservation solution used had a formulation of 0.1 M MES buffer, 0.2% v / v Tween-20, 0.85% w / v NaCl, 2% w / v BSA, and 0.2% v / v Proclin-300.

[0127] (3) Preparation of standard products

[0128] Human thrombin modulin antigen was diluted to 0, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 pg / mL using antigen diluent and dispensed into calibrator tubes.

[0129] The diluent used for antigen dilution was 1×PBS and 1% w / v BSA.

[0130] (4) ELISA test method evaluation

[0131] Coating: Dilute the coating antibody to the working concentration (2 μg / mL) with PBS buffer (pH 9.6), add 100 μL / well to the ELISA plate, and incubate overnight at 4°C.

[0132] Blocking: The next day, wash the coated microplate 4 times, pat dry on absorbent paper, add 10% newborn calf serum, 400 μL / well, incubate at 37°C for 1 hour, and then pat dry on absorbent paper.

[0133] To add standards: Add 100 μL of protein or serum sample (diluted serially with 1% bovine serum albumin at the specified concentration). Add 100 μL of 2% newborn calf serum to the blank wells. Incubate at 37°C for 1 hour, then pat dry on absorbent paper. Wash the plate four times with TBST, patting dry on absorbent paper after each wash.

[0134] Add detection antibody: Add 100 μL of detection antibody diluted with 1% bovine serum albumin to each well and incubate at 37°C for 1 hour. Discard the liquid and wash the plate twice by adding 300 μL of TBST wash buffer to each well. Pat the plate dry on absorbent paper after each wash.

[0135] Add enzyme: Add 100 μL of diluted horseradish peroxidase-conjugated streptavidin (1:4000 dilution) to each well and incubate at 37°C for 45 minutes. Discard the liquid and wash the plate six times with 300 μL of TBST per well. Pat the plate dry on absorbent paper after each wash. For optimal assay performance, remove any residual liquid completely.

[0136] Add substrate for color development: Add 50 μL of Solution A and 50 μL of Solution B to each well. Incubate at room temperature for 40 minutes in the dark. Then, add 50 μL of Stop Solution to each well. The color should change from blue to yellow. If the color changes from green to yellow or if the color change is noticeably uneven, gently tap the plate frame to mix thoroughly.

[0137] Reading: Within 30 minutes, measure the OD value at either the 450nm maximum absorbance wavelength or the 630nm reference wavelength using a microplate reader. The calibrated OD value is the 450nm value minus the 630nm value. Using only the 450nm wavelength will result in an inflated OD value and reduced accuracy.

[0138] Finally, the concentration of human thrombomodulin in the serum sample was calculated as follows:

[0139] The mean value of the standard is calculated through the test results of the standard, and then the concentration is used as the horizontal axis and the actual detection OD value is used as the vertical axis to make a standard curve; the concentration of human thrombin modulin in the serum sample is determined by the detection OD value of the serum sample.

[0140] Example 5

[0141] Example 5 investigated the kit prepared in Example 4 from different perspectives. Samples 1, 2, and 3 were all quality-controlled samples, with corresponding concentrations of human thrombomodulin at 25.00 ng / mL, 50.00 ng / mL, and 100.00 ng / mL, respectively. The samples were then tested using the provided kit.

[0142] (1) Intra-batch precision

[0143] The kit of the present invention was subjected to intra-batch precision studies. Intra-batch precision indicates the reproducibility between wells within a test plate, helping to ensure comparable results when testing samples in different wells of an ELISA plate. Within each assay, samples of known concentration were evaluated 20 times. The CV (coefficient of variation, %) of the samples can be used to determine whether the test samples exhibit good reproducibility.

[0144] Table 1 Intra-batch precision results

[0145]

[0146]

[0147] The results showed that the CV (%) of sample 1, sample 2, and sample 3 were 3.05%, 3.95%, and 3.05%, respectively, and the intra-batch precision was less than 10%, proving that the detection method had good reproducibility in the same test.

[0148] (2) Inter-batch precision

[0149] Inter-batch precision studies were performed on the test kits of the present invention. Inter-batch precision indicates the reproducibility between tests performed on different days and is generally required to be less than 10%. This helps ensure that test results are not limited by time. Within each test, samples of known concentration were evaluated 16 times, each replicated on three separate days.

[0150] Table 2 Inter-batch precision

[0151]

[0152]

[0153] The results showed that the CV (%) of sample 1, sample 2 and sample 3 repeated for 3 days were all less than 10%, proving that the detection method had good reproducibility at different detection times.

[0154] (3) Dilution linearity study

[0155] Dilution linearity studies are performed on the test kits of this invention: Dilution linearity validation experiments provide information about the precision of sample results tested at different dilution levels, which is important for accurate measurement of analyte concentrations within the dynamic range of the assay. Dilution linearity testing is performed on each validated sample type and is considered good if the results are between 70% and 130% of the expected concentration at each dilution.

[0156] Based on clinical testing needs, clinical serum was selected as the validation sample for dilution linearity testing in this evaluation. Within the dynamic range of the test, clinical serum was diluted 2, 4, 8, and 16 times with diluent and tested.

[0157] Table 3 Dilution linearity results

[0158]

[0159] The results showed that the linearity of clinical serum dilution was good, and the expected % was within the range of 70%-130%. At the same time, the correlation coefficient generated by the linear regression test of the expected concentration and the detection concentration was 0.9802, indicating that the kit was not significantly interfered with by clinical serum.

[0160] (4) Linear range

[0161] The linear range of the kit of the present invention was studied: 6 concentrations within the linear range were randomly selected, each concentration was tested 3 times, and the mean and R were calculated. 2 .

[0162] Table 4 Linear range results

[0163]

[0164]

[0165] Figure 3 Corresponding linear curve. Combined Figure 3 It can be seen that R 2 =0.9965, meeting the detection requirements (R 2 ≥0.990). Therefore, the linear range was determined to have passed the performance verification.

[0166] (5) Recovery rate

[0167] Recovery studies were performed on the kits of the present invention. Recovery is used to determine whether analyte detection is affected by differences in the sample matrix. The matrix (e.g., serum) may contain interfering factors that affect the ELISA's ability to accurately quantify the analyte. Recovery was calculated by spiking known amounts of analyte into clinical serum. Generally, an average recovery of 80%-120% is considered to minimize the impact of the sample matrix on the ability to accurately quantify the analyte.

[0168] Table 5 Recovery results

[0169]

[0170] The results showed that the recovery rates of the kit were in the range of 80%-120%, indicating that clinical serum had little influence on the accurate quantification of the analyte.

[0171] (6) Sensitivity

[0172] The kits of the present invention were subjected to sensitivity studies: sensitivity (LOD) was defined as the lowest level of analyte that could be distinguished from background. This is commonly referred to as analytical sensitivity or limit of detection. Sensitivity was determined by adding 2 standard deviations to the mean OD value obtained from the zero standard replicates.

[0173] Table 6 Sensitivity results

[0174]

[0175]

[0176] The results showed that the sensitivity of the kit was 1.46 ng / mL.

[0177] (7) Reference value

[0178] Reference value research was conducted on the kit of the present invention: In qualitative immunoassays, the basis for determining the positive or negative result is the reference value. Determining an appropriate reference value is crucial for accurate interpretation of the results. A large number of negative control samples were randomly tested, and the mean value plus two standard deviations was used as the reference value for the kit (95% confidence level). Serum from 177 clinically normal individuals was randomly collected and tested, and the data were statistically analyzed using SPSS.

[0179] like Figure 4 The results showed that the test data were normally distributed and the reference value of the kit was 18.68 ng / mL (with a 95% confidence interval).

[0180] In the description of this specification, the reference terms "one embodiment", "some embodiments", "specific implementation methods", etc. mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and are not to be construed as limiting the present invention. Those skilled in the art may make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-human thrombin modulin monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that: The antibody or antigen-binding fragment thereof is selected from one of the following: (1) HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 1, 2, and 3; and LCDR1, LCDR2, and LCDR3 sequences as shown in SEQ ID NOs: 4, 5, and 6; (2) HCDR1, HCDR2, and HCDR3 sequences as shown in SEQ ID NOs: 7, 8, and 9; and the LCDR1, LCDR2 and LCDR3 sequences shown as SEQ ID NOs: 10, 11 and 12.

2. The anti-human thrombomodulin monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The sequence of the heavy chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 13 or 15; The sequence of the light chain variable region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 14 or 16.

3. The anti-human thrombomodulin monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the sequence of the light chain variable region is shown in SEQ ID NO: 14; Alternatively, the sequence of the heavy chain variable region is shown as SEQ ID NO: 15, and the sequence of the light chain variable region is shown as SEQ ID NO:

16.

4. The anti-human thrombomodulin monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises the heavy chain shown in SEQ ID NO: 22 and the light chain shown in SEQ ID NO: 23; or comprises the heavy chain shown in SEQ ID NO: 24 and the light chain shown in SEQ ID NO:

25.

5. A polynucleotide, characterized in that The polynucleotide encodes the anti-human thrombomodulin monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The polynucleotide according to claim 5, wherein The polynucleotide comprises the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18; Or include the sequences shown in SEQ ID NO: 19 and SEQ ID NO:

20.

7. A construct, characterized in that Comprising the polynucleotide according to claim 5 or 6.

8. A host cell, characterized in that Containing the polynucleotide according to claim 5 or 6 or the construct according to claim 7.

9. A kit, characterized in that The invention comprises the anti-human thrombin modulin monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

10. The kit according to claim 9, characterized in that The kit comprises a first antibody and a second antibody, wherein the HCDR1, HCDR2 and HCDR3 sequences of the first antibody are shown as SEQ ID NOs: 1, 2 and 3, and the LCDR1, LCDR2 and LCDR3 sequences of the first antibody are shown as SEQ ID NOs: 4, 5 and 6; The HCDR1, HCDR2 and HCDR3 sequences of the second antibody are shown in SEQ ID NOs: 7, 8 and 9, and the LCDR1, LCDR2 and LCDR3 sequences of the second antibody are shown in SEQ ID NOs: 10, 11 and 12.

11. The kit according to claim 10, characterized in that The first antibody is a coating antibody, and the second antibody is a detection antibody.

12. The kit according to claim 10, characterized in that The kit comprises a first antibody composition and a second antibody composition, The first antibody composition includes a first antibody and a 0.1 M PBS buffer; The second antibody composition includes a biotin-labeled second antibody, 0.1M MES buffer, 0.1%-0.5% v / v Tween-20, 0.5%-1.0% w / v NaCl, 1%-3% w / v BSA and 0.1%-0.5% v / v Proclin-300.

13. The kit according to claim 10, characterized in that The kit further comprises a human thrombomodulin standard product, wherein the human thrombomodulin standard product comprises human thrombomodulin, and the sequence of the human thrombomodulin is shown in SEQ ID NO:

21.

14. The kit according to claim 13, characterized in that The purity of the human thrombomodulin is at least 80%.

15. The kit according to claim 13, characterized in that The kit also includes PBS and 1% bovine serum albumin.

16. A pharmaceutical composition, characterized in that The invention comprises the anti-human thrombin modulin monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

17. Use of the anti-human thrombomodulin monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in preparing a human thrombomodulin detection kit.

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