Monoclonal antibody against human thrombin regulatory protein and application

Abstract

The invention provides a monoclonal antibody for resisting human thrombin regulatory protein and application of the monoclonal antibody. The provided antibody comprises an HCDR1 sequence, an HCDR2 sequence and an HCDR3 sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and an LCDR1 sequence, an LCDR2 sequence and an LCDR3 sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; or comprises HCDR1, HCDR2 and HCDR3 sequences as shown in SEQ ID NO: 7, 8 and 9 and LCDR1, LCDR2 and LCDR3 sequences as shown in SEQ ID NO: 10, 11 and 12. The provided antibody can be specifically combined with thrombin regulatory protein, can be used for preparing a kit, and is high in specificity and sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of antibodies, and specifically relates to a monoclonal antibody against human thrombin modulator protein and its application. Background Art

[0002] Human thrombomodulin (TM) is a single-chain transmembrane glycoprotein with a relative molecular mass of 75,000 Da. After binding to thrombin, human thrombomodulin can reduce the coagulation activity of thrombin and enhance its activity of activating protein C. Since activated protein C has an anticoagulant effect, human thrombomodulin is an important intravascular coagulation inhibitor that turns thrombin from a procoagulant to an anticoagulant.

[0003] Human thrombomodulin is located on the surface of endothelial cell membranes and has multiple functions, including 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 are diseased or damaged, the expression and secretion of thrombomodulin are often abnormal, and the protein is released into the blood, which causes changes in the content of thrombomodulin. Therefore, thrombomodulin can be used as a marker for endothelial cell damage. The content of human thrombomodulin in a normal human body is 3.82-13.35TU / mL. As an indicator of endothelial damage, its increase indicates damage to the endothelial system and assists in the diagnosis of DC and atherosclerosis.

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

[0006] The present invention aims to solve one of the technical problems in the related art at least to 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 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 a variety of 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 the antigen-binding fragment thereof is selected from at least one of the following:

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

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

[0011] (2) comprising the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences having one or two conservative amino acid mutations compared to the sequences shown in 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 with the sequences shown in SEQ ID NOs: 10, 11 and 12.

[0013] The second 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 the 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 shown in SEQ ID NO: 13, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown 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 as shown in SEQ ID NO:15, and the sequence of the light chain variable region is as shown in SEQ ID NO:16.

[0020] The third aspect of the present invention provides a polynucleotide encoding the anti-human thrombin modulin monoclonal antibody or antigen-binding fragment thereof as described in 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 comprising 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 thrombin modulin monoclonal antibody or antigen-binding fragment thereof according to the first aspect or the second aspect.

[0026] The 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 HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6; the second antibody includes HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 7, 8 and 9, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 10, 11 and 12. The kit provided may include the first antibody and the second antibody at the same time, for example, the sample may be detected by double antibody sandwich enzyme-linked immunosorbent assay. One of the antibodies is used as a coating antibody, and the other antibody is used as a detection antibody.

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

[0029] Incubate the anti-human thrombin modulin monoclonal antibody or the antigen-binding fragment thereof described in the first aspect or the second aspect with a biological sample, and measure the OD value of the reaction product at a predetermined wavelength;

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

[0031] The eighth aspect of the present invention provides a pharmaceutical composition comprising the anti-human thrombin modulin 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 aspect or the 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 a monoclonal antibody against human thrombin modulator protein, which can be used to prepare a diagnostic kit. The monoclonal antibody composition against human thrombin modulator protein of the present invention comprises anti-human thrombin modulator protein-Clone 1 and anti-human thrombin modulator protein-Clone 2, and the antibody combination has the characteristics of specificity and high affinity. A method for preparing a diagnostic test kit based on the above antibodies is also provided, thereby improving the specificity of the diagnostic reagent. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0039] 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 construed 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] Herein, the term "antibody" is used in the broadest sense, referring to a protein or polypeptide comprising an antigen binding site or antigen binding fragment or antigen binding portion, covering natural antibodies and artificial antibodies of various structures, including but not limited to complete antibody forms or antigen binding fragments of antibodies. According to a specific embodiment, the antibody mentioned is a monoclonal antibody. The so-called "complete antibody" is a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region includes a heavy chain constant domain CH1, a heavy chain constant domain CH2, and a heavy chain constant domain CH3. Each light chain consists 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 complementary determining regions (also called hypervariable regions or hypervariable regions, abbreviated as CDR), which are separated by a conserved framework region (FR). 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 called HCDR1, HCDR2 and HCDR3 respectively from the amino terminus, and the CDRs of the light chain variable region are called LCDR1, LCDR2 and LCDR3 respectively from the amino terminus.

[0042] "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 the binding of an antigen to an antibody heavy chain or light chain. As mentioned above, the hypervariable or hypervariable regions of the heavy and light chains of a complete 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 a complete antibody produces two identical antigen binding fragments, called Fab fragments, each of which contains a heavy and light chain variable region and a light chain constant domain and a heavy chain constant domain CH1. The Fab' fragment differs from the Fab fragment by having a few additional residues at the carboxyl terminus of the heavy chain constant domain CH1, including one or more cysteines from the hinge region of the antibody. Pepsin digestion of a complete antibody yields a F(ab')2 fragment. The F(ab')2 fragment has two antigen-binding F(ab) parts linked together by a disulfide bond, and the F(ab')2 fragment is a bivalent antibody. A single-chain antibody is a fusion protein formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through a flexible short peptide consisting of about 10-25 amino acids. A single-domain antibody is an antibody fragment consisting of the variable region of a single monomer. Since single-domain antibodies are usually derived from the variable region of the heavy chain of camelid antibodies or shark antibodies, they are often called nanobodies. Nanobodies contain only the heavy chain CDR region and are the smallest antigen-binding fragment with complete functions. Nanobodies include three CDR regions (CDR1-CDR3) and four framework regions FR (FR1-FR4). Framework regions FR1, FR2, FR3 and FR4 are separated by complementary determining regions CDR1, CDR2 and CDR3, respectively. Antigen-binding fragments can be obtained by recombinant means. For example, a nucleic acid encoding a target antigen-binding fragment can be constructed, introduced into an expression vector, and expressed in a suitable host to produce an antigen-binding fragment.

[0043] The term "comprising" or "including" means including the mentioned elements or steps, but not excluding other elements or steps. Of course, unless otherwise stated, comprising or including also covers the situation consisting of the mentioned elements or steps. 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 an 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 antigens can be detected by flow cytometry, and the competitive binding between the antibody to be tested 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, antibody or antigen binding fragment, or polynucleotide, is usually separable or recombinant. "Separable" refers to being able to identify and separate and / or recover from cells or cell cultures expressing polypeptides or proteins. Usually, the separated polypeptide will be prepared by at least one purification step. "Separated antibody" refers to being substantially free of other antibodies or their antigen binding fragments having different antigen specificities. "Recombinant" means that antibodies can be produced in exogenous host cells 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 by combining the Kabat definition scheme (for example, see USDept. of Health and Human Servies, "Sequences of Proteins of Immunological Interest" (1983)). It 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 known to those skilled in the art that the differences in CDRs caused by 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 the antigen-binding fragment thereof is selected from at least one of the following:

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

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

[0052] (2) comprising the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences having one or two conservative amino acid mutations compared to the sequences shown in 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 with 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 with the CD235a protein. According to an embodiment, the conservative amino acid mutation mentioned can be a substitution, deletion or increase of conservative amino acids. According to a specific embodiment, the deletion of conservative amino acids is based on the listed CDR sequences, and one amino acid, two amino acids or three amino acids are deleted. According to a specific embodiment, the increase of conservative amino acids is based on the listed CDR sequences, and one amino acid, two amino acids or three amino acids are added. 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 may 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 the biological activity of the protein can be retained. The conservative amino acid substitutions mentioned may be 1 conservative amino acid substitution, 2 conservative amino acid substitutions, 3 conservative amino acid substitutions, and the like. 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 sequence provided has a conservative amino acid substitution with the HCDR sequence shown. In some embodiments, the HCDR sequences provided have a conservative amino acid deletion from the HCDR sequences shown. In some embodiments, the HCDR sequences provided have a conservative amino acid addition from the HCDR sequences shown. In some embodiments, the LCDR sequences provided have a conservative amino acid substitution from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have a conservative amino acid deletion from the LCDR sequences shown. In some embodiments, the LCDR sequences provided have a conservative amino acid addition from the LCDR sequences shown.

[0055] The present invention also provides a monoclonal antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2 and HCDR3 sequences from the heavy chain variable region shown in SEQ ID NO: 13, and LCDR1, LCDR2 and LCDR3 sequences from the light chain variable region shown in SEQ ID NO: 14; or comprising HCDR1, HCDR2 and HCDR3 sequences from the heavy chain variable region shown in SEQ ID NO: 15, and LCDR1, LCDR2 and LCDR3 sequences from the light chain variable region shown in SEQ ID NO: 16. The HCDR sequences and LCDR sequences from these heavy chain variable regions and light chain variable regions may be slightly different in combination with different definition schemes, but are all included in the protection scope 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 other software. 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 thrombin modulin 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 thrombin modulin 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 anti-human thrombin modulin monoclonal antibody or its antigen-binding fragment provided 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 anti-human thrombin modulin monoclonal antibody or its antigen-binding fragment provided comprises the heavy chain shown in SEQ ID NO: 24 and the light chain shown in SEQ ID NO: 25.

[0064] Polynucleotide

[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. The isolated polynucleotide sequence can be obtained by conventional methods in the art to encode the monoclonal antibody or antibody or antigen-binding fragment mentioned above. According to a specific embodiment, the provided polynucleotide 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 provided polynucleotide 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" used herein refers to a polynucleotide in a non-natural state, 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. A plurality of nucleotide sequences for encoding the heavy chain variable region and light chain variable region of anti-human thrombin modulator monoclonal antibody are provided herein. It should be noted that those skilled in the art can design nucleotide sequences that are not completely identical to the nucleotide sequences provided above based on the amino acid sequences of the heavy chain variable region and light chain variable region provided herein, but all encode the same amino acid sequence. These modified nucleotide sequences are also included in the scope of the present invention.

[0067] Constructs

[0068] In order to prepare the antibodies mentioned in the present invention, the polynucleotide sequence encoding the antibody can be inserted into a replicable expression vector and expressed in a host cell or a cell-free expression system. The present invention also provides a construct comprising the polynucleotide described above. A variety of methods commonly used in the art can be used to obtain the construct, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. For example, the polynucleotide can be inserted into the multiple cloning site of the expression vector to form a construct. The construct can contain a variety of operating factors such as promoters, terminators, marker genes, etc. as needed, and these operating factors can be operably connected to the polynucleotide. The promoter is usually used to provide a signal to start transcription. The promoter can select lactose promoter (Lac), Trp promoter, Tac promoter, phage PL and PR promoter as needed; the terminator provides a signal for transcription termination during the transcription process, and the marker gene on the construct is often used for screening. Of course, there can also be enhancers as needed to enhance protein expression. The expression vector is not particularly limited, and can be some commercially available expression vectors, or it can be an expression vector artificially modified as needed, such as a plasmid, a phage, a virus, etc. The virus can be a plant cell virus, a mammalian cell virus, etc. The construct can express antibodies or proteins in vitro or be introduced into cells to express antibodies or proteins.

[0069] Host cells

[0070] The present invention also provides a host cell containing the above-mentioned polynucleotide or the above-mentioned construct. Any cell suitable for expressing antibodies or proteins with polynucleotides or constructs 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, a mammalian cell, etc. Commonly used host cells can be yeast cells, CHO, HEK-293 cells, COS cells, insect cells of Drosophila S2 or Sf9. Host cells containing polynucleotides or constructs can be obtained by methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, etc.

[0071] Pharmaceutical composition

[0072] The present invention also provides a pharmaceutical composition, comprising: the above antibody or antigen-binding fragment, and a pharmaceutically acceptable carrier. The prepared pharmaceutical composition can be used to inhibit the content of thrombin modulator protein, thereby reducing endothelial system damage and relieving 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; sugars, 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 filtering with a sterile filter 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 freeze-dried dosage forms, injections, etc. The prepared different pharmaceutical dosage forms can be formulated into any suitable administration route for administration to the subject, including but not limited to intravenous, dermal, intramuscular, peritoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, etc.

[0074] Reagent test kit

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

[0076] The kit provided includes at least one of the above antibodies. When only one antibody is contained, the biological sample can be directly detected (for example, the antibody and the 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 two antibodies are contained, 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 and avoid false positives and false negatives; although two incubations are required, the operation is simple and the influence of human errors can be reduced.

[0077] According to a specific embodiment, the kit provided includes a first antibody and a second antibody, the first antibody includes HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6; the second antibody includes HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 7, 8 and 9, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 10, 11 and 12. According to a specific embodiment, the first antibody includes a heavy chain variable region shown in SEQ ID NO: 13 and a light chain variable region shown in SEQ ID NO: 14. According to a specific embodiment, the second antibody includes a heavy chain variable region shown in SEQ ID NO: 15 and a light chain variable region shown 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 comprises a first antibody and a 0.1 M PBS buffer;

[0081] The second antibody composition comprises a biotin-labeled second antibody, 0.1M 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, 0.1M MES buffer, 0.2% Tween-20, 0.85% NaCl, 2% BSA and 0.2% Proclin-300.

[0082] The kit mentioned may also include a human thrombin regulatory protein standard, which includes human thrombin regulatory protein, and the sequence of the human thrombin regulatory protein is shown in SEQ ID NO: 21. The purity of human thrombin regulatory protein meets certain requirements, such as at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc. The kit may also include containers, buffer reagents, 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 kit may also include instructions for use to facilitate the operation and use of those skilled in the art.

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

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

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

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

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

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

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

[0090] 4) Continue to add horseradish peroxidase-labeled streptavidin, incubate at 37°C, and rinse the plate;

[0091] 5) Continue to add colorimetric reagent to develop color, then add stop solution to terminate the reaction, and detect the reaction product at wavelengths of 450 nm and 630 nm, and determine the OD value based on the difference between the two;

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

[0093] The provided kit can be used to detect human thrombin modulin by referring 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 antibody or antigen-binding fragment 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 the present invention. The embodiments of the present invention are described in detail below, and the described embodiments are exemplary and are intended to be used to explain the present invention, and 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 modulator protein was identified by the following method, and 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 official website of NCBI, a histidine tag was added to the C-terminus of the protein amino acid, codon optimization software was used to optimize codons in a mammalian cell expression system, expression plasmids were synthesized, and the gene sequence was verified by sequencing.

[0102] Then the expression and production of human thrombin regulatory protein was carried out:

[0103] One day before transfection, cells were cultured at 0.8 × 10 6 / mL was inoculated into cell culture flasks and cultured with OPM-293CD03 Medium at 37°C and 5% CO 2 Incubate in a culture box until the cell confluency reaches 80%-90% and the viability is greater than 95% for transfection. After the thrombin regulatory protein expression vector is mixed with the transfection reagent PEI, incubate at room temperature for 15-30 minutes, slowly add it to the HEK293 cells, add the enhancer and excipients, and incubate at 37°C, 5% CO 2Continue to culture in the incubator. When the cell viability is less than 70%, collect the cell culture, centrifuge at 8000g for 10 min, collect the supernatant, and then centrifuge at 12000g, 4°C for 30 min to collect the cell supernatant.

[0104] The collected cell supernatant was then subjected to affinity purification:

[0105] The cell supernatant was filtered with a 0.22 μm filter membrane, and Ni TED-4FF beads were pre-loaded with a gravity column for affinity purification. The protein was washed with 20 mM Pb, 300 mM NaCl, 10% glycerol, 50 mM imidazole, pH 8.0 solution; 20 mM Pb, 300 mM NaCl, 10% glycerol, 250 mM imidazole, pH 8.0 solution was used for elution; SDS-PAGE was used to detect the purity of the protein, and the results showed that the purity was > 85%; the protein was dialyzed into 20 mM Pb, 300 mM NaCl, 10% glycerol, pH 8.0 buffer, and sterile filtered with a 0.22 μm filter. The protein concentration was determined by BCA and stored at -20°C at a concentration of 1 mg / mL for later use. The purity of the purified antigen protein was at least 80%.

[0106] Example 2

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

[0108] After affinity-purified human thrombin modulin and Freund's adjuvant were mixed and emulsified in equal volumes, mice were immunized with a dose of 100 μg / mouse, and immunized with a dose of 50 μg / mouse 3 weeks later, and the immunization was continuously strengthened twice, each time with an interval of 2 weeks; the blood of the mice was collected to separate the serum, and the serum antibody titer was determined by enzyme-linked immunosorbent assay, and the mice with better immunity were screened for hybridoma fusion experiments; after screening and culturing with HAT screening medium, human thrombin modulin was used as the ELISA plate coating antigen, and the hybridoma monoclonal antibody supernatant was taken for enzyme-linked immunosorbent assay to screen out high-quality positive cell lines, and two rounds of cell subcloning were performed to clone monoclonal cell lines, which were named Clone1 and Clone2, and the antibodies produced were represented as Clone1 antibody and Clone2 antibody.

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

[0110] After sequencing, the heavy chain variable region sequence of Clone1 antibody 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 Clone2 antibody is shown in SEQ ID NO: 15, and the light chain variable region sequence is shown in SEQ ID NO: 16. The purity of Clone1 antibody on SDS-PAGE showed >95%; the purity of Clone2 antibody on SDS-PAGE showed >95%.

[0111] Example 3

[0112] Example 3 studied the affinity of the Clone 1 antibody and the Clone 2 antibody prepared in Example 2 to the antigen. It included:

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

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

[0115] like Figure 1 As shown: the EC50 of clone1 antibody is 393 ng / mL, and the EC50 of clone2 antibody is 406 ng / mL.

[0116] The human thrombin modulin prepared in Example 1 was used as the coating antigen, incubated at 4° C. overnight, and then the two antibodies were specifically detected respectively, the steps were as follows:

[0117] The antigen was diluted to 1 μg / mL, added to the ELISA plate at 50 μL / well, and incubated overnight at 4°C; clone 1 antibody and clone 2 antibody were diluted to the concentration sensitive range according to the titer of the previous step, mixed with the gradient diluted antigen protein, and incubated at 37°C for 30 min; after washing the plate, HRP-labeled secondary antibody of the corresponding species (mouse) was added at 50 μl / well, and incubated at 37°C for 30 min; the plate was washed, displayed and read.

[0118] The results are as follows Figure 2 As shown: the IC50 of clone1 antibody is 302.7 ng / mL, and the IC50 of clone2 antibody is 261.8 ng / mL.

[0119] The results are shown above, and 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 principle of double antibody sandwich method. It includes:

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

[0123] The Clone1 antibody prepared in Example 2 was transferred to a 14 kD dialysis bag, and after sufficient dialysis in PBS buffer, the concentration was adjusted to 2 mg / mL for later use. The above antibody storage solution was formulated as 0.1 M PBS.

[0124] (2) Biotin-labeled Clone2 antibody as detection antibody

[0125] 10mM biotin and Clone2 antibody were labeled 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 in 1×PBS for 6 times, each time for 1-2 hours. After dialysis overnight, the labeled solution was taken out and the protein content in the labeled solution was tested using a spectrophotometer. The protective solution was calculated and added. After being fully mixed, it can be stored at 18-20℃. When testing, the protein concentration was diluted to 0.5ug / mL using the labeled preservation solution.

[0126] The formulation of the labeling preservation solution used was 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] Use antigen diluent to dilute human thrombin modulin antigen to 0, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 pg / mL, respectively, and dispense into calibrator tubes.

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

[0130] (4) Evaluation of ELISA detection method

[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] Add standard: add 100 μL protein or serum sample (diluted with 1% bovine serum albumin at a certain concentration). Add 100 μL 2% newborn calf serum to the blank well. After incubation at 37°C for 1 hour, pat the microplate dry on absorbent paper and wash the plate with TBST solution for 4 times. Pat dry on absorbent paper each time.

[0134] Add detection antibody: Add 100 μL detection antibody diluted with 1% bovine serum albumin to each well and incubate at 37°C for 1 hour. Discard the liquid and add 300 μL TBST wash solution to each well to wash the plate twice, patting the plate dry on absorbent paper each time.

[0135] Add enzyme: Add 100 μL diluted horseradish peroxidase-labeled streptavidin (1:4000 dilution) to each well and incubate at 37°C for 45 minutes. Discard the liquid and add 300 μL TBST wash solution to each well to wash the plate 6 times. After each wash, pat dry on absorbent paper. To obtain ideal experimental performance, the residual liquid must be completely removed.

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

[0137] Test reading: Within 30 minutes, use a microplate reader for dual wavelength detection and measure the OD value at the maximum absorption wavelength of 450nm or the reference wavelength of 630nm. The calibrated OD value is the measured value at 450nm minus the measured value at 630nm. Using only 450nm for measurement will result in a higher OD value and reduced accuracy.

[0138] Finally, the concentration of human thrombin modulin 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 a standard curve is prepared with the concentration as the horizontal axis and the actual detection OD value as the vertical axis; 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 studied the kit prepared in Example 4 from different perspectives. Samples 1, 2, and 3 used were all quality-controlled samples, and their corresponding concentrations were 25.00 ng / mL, 50.00 ng / mL, and 100.00 ng / mL of human thrombin modulin, 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 an intra-batch precision study: intra-batch precision shows the reproducibility between wells in the test plate, which helps to ensure that the samples tested in different wells of the ELISA plate give comparable results. In the same test, samples of known concentrations were evaluated 20 times each. The CV (coefficient of variation, %) of the sample can determine whether the test sample has 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 detection.

[0148] (2) Batch-to-batch precision

[0149] The kit of the present invention was subjected to an inter-batch precision study: inter-batch precision shows the reproducibility between tests performed on different days, which is generally required to be less than 10%, helping to ensure that the test results are not limited by time. In the same test, samples of known concentration were evaluated 16 times each and repeated on 3 independent 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 study of the kit of the present invention: Dilution linearity validation experiments provide information about the accuracy of sample results tested at different dilution levels, which is very important for accurate measurement of analyte concentrations within the dynamic range of the test. Dilution linearity tests are performed on each validated sample type and are considered good if the results are 70%-130% of the expected concentration for each dilution.

[0156] Based on the needs of clinical testing, clinical serum was selected as the validation sample for dilution linearity testing in this evaluation. Within the dynamic range of the test, the 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 % met 70%-130%. At the same time, the correlation coefficient produced by the linear regression test of expected concentration and detection concentration was 0.9802, indicating that the kit was not significantly interfered 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. Figure 3 It can be seen that R 2 =0.9965, meeting the testing requirements (R 2 ≥0.990). Therefore, it was determined that the linear range 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 sample matrix. The matrix (such as serum) may have interfering factors that affect the ability of the ELISA to accurately quantify the analyte. The analyte is tested by spiking a known amount into clinical serum and the recovery is calculated. Generally, if the average recovery is 80%-120%, it is considered that the sample matrix has minimal impact on the ability to detect an accurate quantitative analyte.

[0168] Table 5 Recovery results

[0169]

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

[0171] (6) Sensitivity

[0172] The kit of the present invention was subjected to sensitivity studies: Sensitivity (LOD) is defined as the lowest analyte level that can be distinguished from the background. This is often referred to as analytical sensitivity or limit of detection. Sensitivity is 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] The reference value of the kit of the present invention is studied: In qualitative immunoassay, the basis for determining the positive or negative result is the reference value, and determining the appropriate reference value is of great significance for accurately determining the result. A large number of negative control samples were randomly tested, and the test mean value plus 2 standard deviations was the reference value of the kit (95% confidence). 177 clinical normal human sera were randomly collected and tested, and the data were statistically analyzed using SPSS.

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

[0180] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementation methods", etc. means 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 representation of the above terms does not necessarily refer to the same embodiment or example. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Those of ordinary skill in the art can change, modify, replace and modify 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 at least one of the following: (1) comprising the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences having one or two conservative amino acid mutations compared with the sequences shown in SEQ ID NOs: 1, 2 and 3; and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences having one or two conservative amino acid mutations compared to the sequences shown in SEQ ID NOs: 4, 5 and 6; (2) comprising the HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences having one or two conservative amino acid mutations compared to the 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, or sequences having one or two conservative amino acid mutations compared with the sequences shown in SEQ ID NOs: 10, 11 and 12.

2. An anti-human thrombin modulator 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 at least one of the following: (a) the HCDR1, HCDR and HCDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 13, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown in SEQ ID NO: 14; or (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.

3. The anti-human thrombin modulin monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, 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 with 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; Optionally, the sequence of the heavy chain variable region is as shown in SEQ ID NO: 13, and the sequence of the light chain variable region is as shown in SEQ ID NO: 14; Or the sequence of the heavy chain variable region is as shown in SEQ ID NO: 15, and the sequence of the light chain variable region is as shown in SEQ ID NO: 16; Optionally, 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.

4. 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 3; Optionally, 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.

5. A construct, characterized in that Comprising the polynucleotide of claim 4.

6. A host cell, characterized in that Containing the polynucleotide according to claim 4 or the construct according to claim 5.

7. A kit, characterized in that: An anti-human thrombin modulin monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3; Optionally, the kit comprises a first antibody and a second antibody, wherein the first 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; The second antibody comprises HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 7, 8 and 9, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 10, 11 and 12; Optionally, the first antibody is a coating antibody and the second antibody is a detection antibody; Optionally, the kit comprises a first antibody composition and a second antibody composition, The first antibody composition comprises a first antibody and a 0.1 M PBS buffer; 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; Optionally, the kit further comprises a human thrombin modulator standard, wherein the human thrombin modulator standard comprises human thrombin modulator, and the sequence of the human thrombin modulator is shown in SEQ ID NO: 21; Optionally, the purity of human thrombin modulin is at least 80%; Optionally, the kit further comprises PBS and 1% bovine serum albumin.

8. A method for detecting the content of human thrombin regulatory protein in a biological sample, characterized in that: include: Incubate the anti-human thrombin modulin monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 with a biological sample, and measure the OD value of the reaction product at a predetermined wavelength; Based on the OD value, determining the content of thrombin modulating protein in the biological sample; Optionally, include: 1) Coat the plate with the primary antibody and block with blocking solution; 2) Add human thrombin modulator standard or biological sample, incubate at 37°C, and then rinse the plate; 3) Continue to add biotin-labeled secondary antibody, incubate at 37°C, and then rinse the plate; 4) Continue to add horseradish peroxidase-labeled streptavidin, incubate at 37°C, and rinse the plate; 5) Continue to add colorimetric reagent to develop color, then add stop solution to terminate the reaction, and detect the reaction product at wavelengths of 450 nm and 630 nm, and determine the OD value based on the difference between the two; 6) preparing a standard curve based on the OD value of the human thrombin modulin standard, and determining the content of the thrombin modulin in the biological sample based on the standard curve; Optionally, the biological sample is selected from at least one of serum, plasma, cell lysate, cell culture supernatant, and urine.

9. 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 3, and a pharmaceutically acceptable carrier.

10. Use of the anti-human thrombomodulin monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a human thrombomodulin kit or a drug.

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