D-dimeric antibodies and uses thereof

Abstract

The invention provides an anti-D-dimer antibody and a preparation method thereof. The antibody comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises a variant of SEQ ID NO: 1, and compared with an amino acid sequence shown in SEQ ID NO: 1, the variant of SEQ ID NO: 1 comprises at least one of the 52nd mutation, the 59th mutation, the 102nd mutation, the 107th mutation and the 110th mutation; the light chain variable region comprises a variant of SEQ ID NO: 2, and compared with an amino acid sequence shown in SEQ ID NO: 2, the variant of SEQ ID NO: 2 comprises at least one of 30 , 60 , 61 and 98 mutations. The anti-D-dimer antibody disclosed by the invention can be specifically combined with the D-dimer in a targeting manner, and can be used for qualitative or quantitative detection of the D-dimer.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of a Chinese patent application with the application number 202311821205.1 and the title "D - dimer antibody and its uses" filed with the Chinese Patent Office on December 26, 2023, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention belongs to the technical field of antibodies. Specifically, the present invention relates to a D - dimer antibody and its uses. More specifically, the present invention relates to an antibody, nucleic acid molecule, vector, cell or host, method for preparing an antibody, conjugate, reagent or kit and its uses, method for detecting D - dimer, method for screening D - dimer antibody, and mutant library of D - dimer. Background art

[0004] D - dimer is the end - product of cross - linked fibrin after the action of plasmin. During the coagulation process, after thrombin hydrolyzes fibrinogen, fibrinopeptide A (FPA) and peptide B (FPB) are successively released, and the remaining part is soluble fibrin monomer (SFM). Under the action of transamidase, SFM is transformed into fibrin, and then blood coagulation occurs. This process is completed after a series of cross - linkings. The formed fibrin is stable in nature and generally insoluble, but can be degraded by plasmin. During the degradation of cross - linked fibrin by plasmin, several kinds of polymers are gradually generated, and D - dimer is one of its specific products, with a molecular weight of 184,000 - 202,000. Under pathological conditions, the dynamic balance between coagulation and fibrinolysis is disrupted, the tendency of coagulation is enhanced, the fibrin degradation products increase, and the content of D - dimer increases. The increase in D - dimer level indicates the formation of fibrin thrombus and fibrinolysis in the body. Therefore, clinically, it can be used as a molecular marker for hypercoagulable state and fibrinolysis hyperactivity in the body.

[0005] The detection of D - dimer has important value for the diagnosis and treatment of various diseases. In particular, an increase in the content of D - dimer can detect the course of various diseases, such as deep vein thrombosis (DVT), disseminated intravascular coagulation (DIC), myocardial infarction, severe hepatitis, pulmonary embolism (PE), etc.; it also has a certain monitoring effect on the possible complications of pregnant women with pre - eclampsia and high - risk pregnancy. Moreover, the change in D - dimer level can be used as an indicator for monitoring thrombolytic therapy and guiding the dosage of thrombolytic drugs. Thus, the detection of D - dimer has important clinical significance in aspects such as the early diagnosis of thrombotic diseases, course monitoring, and treatment monitoring of thrombolytic drugs.

[0006] Currently, the detection of D-dimer includes three batches of tests, latex agglutination method (LATEX), ELISA method, immunofiltration colloidal gold color reaction method, etc. These methods all require antibodies against D-dimer. Therefore, it is urgent to develop an antibody that can effectively bind to D-dimer. Summary of the Invention

[0007] The present invention aims to provide an antibody against D-dimer, a reagent or a kit for detecting D-dimer.

[0008] In the first aspect of the present invention, an anti-D-dimer antibody is proposed. The antibody includes: a heavy chain variable region and / or a light chain variable region; wherein, the heavy chain variable region includes or is the amino acid sequence shown in SEQ ID NO:1 or a variant of SEQ ID NO:1. Compared with the amino acid sequence shown in SEQ ID NO:1, the variant of SEQ ID NO:1 includes mutations at at least one of the following sites: the 52nd, 59th, 102nd, 107th, and 110th positions; the light chain variable region includes or is the amino acid sequence shown in SEQ ID NO:2 or a variant of SEQ ID NO:2. Compared with the amino acid sequence shown in SEQ ID NO:2, the variant of SEQ ID NO:2 includes mutations at at least one of the following sites: the 30th, 60th, 61st, and 98th positions; the heavy chain variable region including or being the amino acid sequence shown in SEQ ID NO:1 and the light chain variable region including or being the amino acid sequence shown in SEQ ID NO:2 do not exist simultaneously.

[0009] In the second aspect of the present invention, an anti-D-dimer antibody is proposed. The antibody includes HCDRs and LCDRs. The HCDRs include or are the HCDRs that are identical to the HCDRs of the heavy chain variable region defined by the antibody in the first aspect, and the LCDRs include or are the LCDRs that are identical to the LCDRs of the light chain variable region defined by the antibody in the first aspect.

[0010] In the third aspect of the present invention, a nucleic acid molecule, a vector, a cell or a host, or a method for preparing the above-mentioned antibody is proposed. The nucleic acid molecule encodes the antibody in the first aspect or the second aspect; the vector includes the aforementioned nucleic acid molecule; the cell or the host includes the aforementioned nucleic acid molecule or vector or expresses the aforementioned antibody; the method includes culturing the aforementioned cell or host.

[0011] In the fourth aspect of the present invention, a conjugate is proposed. The conjugate includes: the antibody in the first aspect or the second aspect and a conjugate part conjugated thereto.

[0012] In a fifth aspect of the present invention, there is provided a reagent or a kit, which comprises: the antibody according to the first aspect or the second aspect, or the conjugate according to the fourth aspect.

[0013] In a sixth aspect of the present invention, there is provided the use of the antibody according to the first aspect or the second aspect, the conjugate according to the fourth aspect, or the reagent or the kit according to the fifth aspect in detecting D-dimer or in preparing a product for diagnosing D-dimer related diseases.

[0014] In a seventh aspect of the present invention, there is provided a method for detecting D-dimer, which comprises: contacting a sample to be detected with the antibody according to the first aspect or the second aspect, the conjugate according to the fourth aspect, or the reagent or the kit according to the fifth aspect to form an immune complex.

[0015] In an eighth aspect of the present invention, there is provided a method for screening D-dimer antibodies, which comprises: a) designing primers for amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8 or 9 sites among X1, X2, X3, X4, X5, X6, X7, X8 and X9 defined in the antibody according to the second aspect; b) using the nucleic acid molecule according to the fourth aspect, the vector according to the fifth aspect, or the cell according to the sixth aspect as a template to construct a mutant library with the primers in a); c) screening D-dimer antibodies from the mutant library.

[0016] In a ninth aspect of the present invention, there is provided a mutant library, which comprises variants of SEQ ID NO:1 and variants of SEQ ID NO:2 defined in the antibody according to the first aspect, the second aspect or the third aspect.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Detailed Description of the Invention

[0018] Embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0019] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, "a plurality of" means two or more.

[0020] The endpoints and any values disclosed in this document are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.

[0021] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0022] In this document, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0023] In this document, the terms "optionally", "optional", "option", "optionally", "optional", or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0024] As used herein, the terms "identity", "homology", or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and refer to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional methods, such as, for example, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.)). There are many algorithms for aligning sequences and determining sequence identity, including, for example, the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0025] Without substantially affecting the antibody activity (retaining at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody sequences. They are all considered to be included within the scope of protection of the present invention. Amino acids with similar properties are substituted in the variable region. The variant sequences of the present invention can have at least 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequences. The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in the N-terminal to C-terminal manner.

[0026] It should be noted that in the claims and the specification herein, the variants of the antibody sequences are obtained by adding and / or deleting one or more amino acids based on the heavy chain, light chain, heavy chain variable region, or light chain variable region. The positions or numbers of the mutation sites defined in the specification and claims of the present invention also need to be adjusted according to the number and position of the added and / or deleted amino acids. For example, a variant of SEQ ID NO:1 is obtained by adding one amino acid before the 52nd amino acid (e.g., the 10th or 27th amino acid). Those skilled in the art can understand that S52D should be adjusted to S53D; or, a variant of SEQ ID NO:1 is obtained by deleting two amino acids before the 52nd amino acid (e.g., the 10th and 27th amino acids). Those skilled in the art can understand that S52D should be adjusted to S50D.

[0027] In this article, the term "at least 80% homology" means at least 80% with each reference sequence, and can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology. The term "at least 90% homology" means at least 90% with each reference sequence, and can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology.

[0028] In this article, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule containing one or more nucleotide or amino acid mutations.

[0029] In this text, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host for self - replication, which transfers the inserted nucleic acid molecule into cells or hosts and / or between cells or hosts. The vector may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and expression vectors mainly used for the transcription and / or translation of DNA or RNA. The vector also includes vectors with multiple of the above - mentioned functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Generally, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

[0030] In this text, the term "cell" generally refers to a cell that has been modified or recombined with respect to the genetic material of a host cell using genetic engineering techniques or cell fusion techniques to obtain cells with unique traits that are stably inherited. Among them, the term "host cell" refers to a prokaryotic cell or a eukaryotic cell that can introduce a recombinant vector. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acid (such as a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and can be used for the expression and / or secretion of the target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0031] The present invention provides an antibody, a nucleic acid molecule, a vector, a cell or a host, a method for preparing an antibody, a conjugate, a reagent or a kit and their uses, a method for detecting D - dimer, a method for screening D - dimer antibodies, and a mutant library, which will be described in detail below respectively.

[0032] Antibody

[0033] In a first aspect of the present invention, the present invention provides an anti-D-dimer antibody. According to an embodiment of the present invention, the antibody comprises: a heavy chain variable region and / or a light chain variable region; wherein, the heavy chain variable region comprises or is an amino acid sequence as shown in SEQ ID NO:1 or a variant of SEQ ID NO:1, and compared with the amino acid sequence shown in SEQ ID NO:1, the variant of SEQ ID NO:1 comprises mutations at at least one of the following positions: position 52, position 59, position 102, position 107, and position 110; the light chain variable region comprises or is an amino acid sequence as shown in SEQ ID NO:2 or a variant of SEQ ID NO:2, and compared with the amino acid sequence shown in SEQ ID NO:2, the variant of SEQ ID NO:2 comprises mutations at at least one of the following positions: position 30, position 60, position 61, and position 98; the heavy chain variable region does not exist simultaneously with the amino acid sequence as shown in SEQ ID NO:1 and the light chain variable region does not exist simultaneously with the amino acid sequence as shown in SEQ ID NO:2.

[0034] As used herein, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, multispecific antibodies, as well as chimeric antibodies or functional fragments of antibodies, and the specific structure is not limited as long as they exhibit the desired biological activity. As used herein, the terms "full-length antibody", "full-length monoclonal antibody" or "full-length monoclonal antibody" are all composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE). It generally includes a light chain with a relatively low molecular weight and a heavy chain with a relatively high molecular weight, and the antibody molecule formed by connecting the heavy chain (H chain) and the light chain (L chain) by a disulfide bond. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly and is called the variable region (V region); the carboxyl-terminal (C-terminal) is relatively stable and varies little, and is called the constant region (C region). The V regions of the L chain and the H chain are respectively called VL and VH.

[0035] Functional fragments of antibodies generally have the same binding specificity as the antibodies from which they are derived. Those skilled in the art can easily understand from the content recorded in the present disclosure that the above-mentioned functional fragments of antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the structure of the intact antibody in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.

[0036] Functional fragments of antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0037] Functional fragments of antibodies are fragments that contain part or all of an antibody and lack at least some of the amino acids present in the full-length chain but still retain the functional activity of specifically binding to an antigen. For example, such a fragment may contain part or all of the CDRs of an antibody. Such fragments are biologically active because they bind to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include at least one of Fv fragments, disulfide-stabilized Fv fragments (dsFv), F(ab’)2 fragments, Fab’ fragments, Fab fragments, F(ab)2 fragments, scFv fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, Fv-Fc fusion proteins, multispecific antibodies formed from functional fragments, single-domain antibodies, VHH nanobodies, domain antibodies, bivalent domain antibodies, or minimal recognition units. Such fragments can be produced by recombinant nucleic acid techniques or can be produced by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0038] As used herein, the terms "polyclonal antibody" and "multispecific antibody" are synonymous and both refer to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that can recognize three antigenic epitopes, or antibodies that can recognize four antigenic epitopes. This is a broad understanding and the specific structure is not limited as long as it can recognize multiple antigenic epitopes. In the present invention, at least one of the multiple antigenic epitopes is derived from D-dimer.

[0039] According to embodiments of the present invention, the above-mentioned antibody may further include at least one of the following additional technical features:

[0040] According to embodiments of the present invention, compared with the amino acid sequence shown in SEQ ID NO:1, the variant of SEQ ID NO:1 includes at least one of the following mutations: S52D, D59L / M / W, Y102F, F107W, and H110V.

[0041] It should be noted that the numbering of the above sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO:1 from the N-terminus to the C-terminus. For example, the 52nd position refers to the 52nd position of the amino acid sequence shown in SEQ ID NO:1 starting from the N-terminus; the "S52D" means that the serine at the 52nd position of the amino acid sequence shown in SEQ ID NO:1 is replaced by aspartic acid; the "D59L / M / W" means that the aspartic acid at the 59th position of the amino acid sequence shown in SEQ ID NO:1 can be replaced by one of leucine, methionine, and tryptophan.

[0042] In some alternative embodiments of the present invention, the mutation at the 52nd position is S52D.

[0043] In some alternative embodiments of the present invention, the mutation at the 59th position is D59L, D59M, or D59W.

[0044] In some alternative embodiments of the present invention, the mutation at the 59th position is D59L.

[0045] In some alternative embodiments of the present invention, the mutation at the 59th position is D59M.

[0046] In some alternative embodiments of the present invention, the mutation at the 59th position is D59W.

[0047] In some alternative embodiments of the present invention, the mutation at the 102nd position is Y102F.

[0048] In some alternative embodiments of the present invention, the mutation at the 107th position is F107W.

[0049] In some alternative embodiments of the present invention, the mutation at the 110th position is H110V.

[0050] According to an embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO:1, the variant of SEQ ID NO:1 includes mutations at the following sites:

[0051]

[0052] According to an embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO:2, the variant of SEQ ID NO:2 includes at least one of the following mutations: V30K, F60W, S61W / D / E, and H98G / L / A / M.

[0053] It should be noted that the numbering of the above sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO:2 from the N-terminus to the C-terminus. For example, the 32nd position refers to the 30th position starting from the N-terminus of the amino acid sequence shown in SEQ ID NO:2; the "V30K" means that the valine at the 30th position of the amino acid sequence shown in SEQ ID NO:2 is replaced by lysine; the "S61W / D / E" means that the serine at the 61st position of the amino acid sequence shown in SEQ ID NO:2 can be replaced by one of tryptophan, aspartic acid, and glutamic acid; the "H98G / L / A / M" means that the histidine at the 98th position of the amino acid sequence shown in SEQ ID NO:2 can be replaced by one of glutamic acid, leucine, alanine, and methionine.

[0054] In some alternative embodiments of the present invention, the mutation at the 33rd position is V30K.

[0055] In some alternative embodiments of the present invention, the mutation at the 60th position is F60W.

[0056] In some alternative embodiments of the present invention, the mutation at the 61st position is S61W, S61D, or S61E.

[0057] In some alternative embodiments of the present invention, the mutation at the 61st position is S61W.

[0058] In some alternative embodiments of the present invention, the mutation at the 61st position is S61D.

[0059] In some alternative embodiments of the present invention, the mutation at the 61st position is S61E.

[0060] In some alternative embodiments of the present invention, the mutation at the 98th position is H98G, H98L, H98A, or H98M.

[0061] In some alternative embodiments of the present invention, the mutation at the 98th position is H98G.

[0062] In some alternative embodiments of the present invention, the mutation at the 98th position is H98L.

[0063] In some alternative embodiments of the present invention, the mutation at the 98th position is H98A.

[0064] In some alternative embodiments of the present invention, the mutation at the 98th position is H98M.

[0065] According to the embodiments of the present invention, compared with the amino acid sequence shown in SEQ ID NO:2, the variant of SEQ ID NO:2 includes mutations at the following sites:

[0066] Mutation site Mutation site VL variant 1 H98L VL variant 26 V30K, S61E, H98G VL variant 2 F60W, S61W, H98A VL variant 27 V30K, F60W, H98G VL variant 3 S61E, H98M VL variant 28 S61W, H98L VL variant 4 V30K, S61E, H98L VL variant 29 F60W, S61E, H98G VL variant 5 V30K, F60W, S61E, H98L VL variant 30 S61W, H98G VL variant 6 F60W, S61W, H98M VL variant 31 V30K, F60W, S61W, H98L VL variant 7 F60W, H98L VL variant 32 V30K, F60W, S61W, H98A VL variant 8 F60W, S61W, H98L VL variant 33 F60W, S61D, H98G VL variant 9 V30K, S61E, H98A VL variant 34 F60W, S61E, H98M VL variant 10 V30K, F60W, S61W, H98G VL variant 35 H98A VL variant 11 F60W, S61D, H98M VL variant 36 V30K, S61E VL variant 12 S61W, H98M VL variant 37 V30K, F60W, S61E, H98G VL variant 13 V30K, F60W, S61D, H98M VL variant 38 S61E VL variant 14 S61E, H98A VL variant 39 H98M VL variant 15 V30K, H98M VL variant 40 V30K, F60W, S61D VL variant 16 S61E, H98L VL variant 41 F60W, S61D VL variant 17 V30K, F60W, H98L VL variant 42 F60W, S61W, H98G VL variant 18 V30K, S61W, H98M VL variant 43 V30K, F60W, S61W, H98M VL variant 19 V30K, S61E, H98M VL variant 44 V30K, H98L VL variant 20 V30K, F60W, S61D, H98L VL variant 45 V30K VL variant 21 V30K, S61W, H98L VL variant 46 F60W VL variant 22 F60W, S61E, H98L VL variant 47 S61W VL variant 23 F60W, H98G VL variant 48 S61D VL variant 24 S61W, H98A VL variant 49 H98G VL variant 25 F60W, H98A 。

[0067] According to an embodiment of the present invention, the antibody comprises the heavy chain variable region and the light chain variable region as shown in the following table:

[0068]

[0069]

[0070]

[0071] Wherein, WT means no mutation site.

[0072] In a second aspect of the present invention, the present invention provides an anti-D-dimer antibody. According to an embodiment of the present invention, the antibody comprises a heavy chain and / or a light chain, the heavy chain comprises the heavy chain variable region defined in the antibody of the first aspect; the light chain comprises the light chain variable region defined in the antibody of the first aspect.

[0073] According to an embodiment of the present invention, the antibody comprises the heavy chain and the light chain as shown in the following table:

[0074]

[0075] Those skilled in the art can understand that the features and advantages described above for the antibody of the first aspect (such as the features of the heavy chain variable region and / or the light chain variable region defined in the first aspect) also apply to the antibody of the second aspect, and will not be elaborated herein.

[0076] In a third aspect of the present invention, the present invention provides an anti-D-dimer antibody. According to an embodiment of the present invention, the antibody comprises HCDRs and LCDRs, the HCDRs comprise or are the HCDRs identical to those of the heavy chain variable region defined in the antibody of the first aspect or the second aspect, and the LCDRs comprise or are the LCDRs identical to those of the light chain variable region defined in the antibody of the first aspect or the second aspect.

[0077] As used herein, the terms "complementary determining region", "CDR" or "CDRs" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, and refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody for the antigen or epitope it recognizes. In the specific embodiments of the present disclosure, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0078] In this text, the heavy chain complementarity determining regions (CDRs of the heavy chain variable region) are denoted by "HCDRs" or "HCDR", which include HCDR1, HCDR2, and HCDR3; the light chain complementarity determining regions (CDRs of the light chain variable region) are denoted by "LCDRs" or "LCDR", which include LCDR1, LCDR2, and LCDR3. Commonly used CDR numbering schemes in the art include: Kabat numbering, Chothia numbering, IMGT numbering, ChothiaMartin numbering, and AHoLesk numbering. CDR definition schemes include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As used herein, "Kabat numbering" and "Kabat definition" refer to the numbering and definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For "Chothia definition", see Chothia et al., J Mol Biol 196: 901-917 (1987). Exemplary defined CDRs are listed in Table 1 below. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues contain a particular CDR.

[0079] Table 1: CDR Definitions 1

[0080] CDR Kabat <![CDATA[AbM 2 > IMGT HCDR1 31～35 26-35 26-35 HCDR2 50～65 50-58 51-56 HCDR3 95～102 95-102 93-102 LCDR1 24～34 24-34 27-32 LCDR2 50～56 50-56 50-51 LCDR3 89～97 89-97 89-97

[0081] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below).

[0082] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0083] Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. A person of ordinary skill in the art can clearly map the Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). It should be noted that the polypeptide sequences in the sequence listing and Table 2 of the present invention are not numbered according to the Kabat numbering system. However, a person of ordinary skill in the art is fully capable of converting the sequence numbers in the sequence listing into Kabat numbers.

[0084] According to an embodiment of the present invention, the HCDRs and / or LCDRs are defined by the Kabat, Chothia, AbM, Contact or IMGT system.

[0085] According to an embodiment of the present invention, the HCDR1, HCDR2, and HCDR3 sequentially include or are the amino acid sequences at positions 26-35, 50-65, and 95-102 of the Kabat-numbered heavy chain variable region; the LCDR1, LCDR2, and LCDR3 sequentially include or are the amino acid sequences at positions 24-34, 50-56, and 89-97 of the Kabat-numbered light chain variable region.

[0086] It should be noted that the CDRs defined by other methods not limited to those in Table 1 also fall within the protection scope of the present disclosure.

[0087] According to an embodiment of the present invention, the HCDRs and / or LCDRs are defined by the Kabat system, and the HCDRs and LCDRs include or are the amino acid sequences shown below:

[0088] HCDR 1: GYRFTDYSIH;

[0089] HCDR 2: VIX1TYSGNPX2YNQKFKG, where X1 is S or D, and X2 is D, L, M or W;

[0090] HCDR 3: MNDX3YGDYX4FDX5, where X3 is Y or F, X4 is F or W, and X5 is H or V;

[0091] LCDR1: RSSQSLX6HTNGNTYLH, where X6 is V or K;

[0092] LCDR 2: KVSNRX7X8, where X7 is F or W, and X8 is S, W, D or E;

[0093] LCDR 3: SQSRX9VPLT, where X9 is H, G, L, A, or M;

[0094] Wherein, X1 being S, X2 being D, X3 being Y, X4 being F, X5 being H, X6 being V, X7 being F, X8 being S, and X9 being H do not exist simultaneously.

[0095] In some alternative embodiments of the present invention, X1 is S.

[0096] In some alternative embodiments of the present invention, X1 is D.

[0097] In some alternative embodiments of the present invention, X2 is D.

[0098] In some alternative embodiments of the present invention, X2 is L.

[0099] In some alternative embodiments of the present invention, X2 is M.

[0100] In some alternative embodiments of the present invention, X2 is W.

[0101] In some alternative embodiments of the present invention, X3 is Y.

[0102] In some alternative embodiments of the present invention, X3 is F.

[0103] In some alternative embodiments of the present invention, X4 is F.

[0104] In some alternative embodiments of the present invention, X4 is W.

[0105] In some alternative embodiments of the present invention, X5 is H.

[0106] In some alternative embodiments of the present invention, X5 is V.

[0107] In some alternative embodiments of the present invention, X6 is V.

[0108] In some alternative embodiments of the present invention, X6 is K.

[0109] In some alternative embodiments of the present invention, X7 is F.

[0110] In some alternative embodiments of the present invention, X7 is W.

[0111] In some alternative embodiments of the present invention, X8 is S.

[0112] In some alternative embodiments of the present invention, X8 is W.

[0113] In some alternative embodiments of the present invention, X8 is D.

[0114] In some alternative embodiments of the present invention, X8 is E.

[0115] In some alternative embodiments of the present invention, X9 is H.

[0116] In some alternative embodiments of the present invention, X9 is G.

[0117] In some alternative embodiments of the present invention, X9 is L.

[0118] In some alternative embodiments of the present invention, X9 is A.

[0119] In some alternative embodiments of the present invention, X9 is M.

[0120] According to an embodiment of the present invention, the antibody comprises or is HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 as shown in the following table,

[0121]

[0122]

[0123] According to an embodiment of the present invention, the antibody further comprises at least one of a heavy chain framework region and a light chain framework region. Among them, the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4; the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4.

[0124] According to an embodiment of the present invention, at least a part of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting chicken antibody or a mutant thereof.

[0125] As used herein, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the regions other than the CDRs in the heavy chain variable region (which can be denoted as VH) and the light chain variable region (which can be denoted as VL) of the antibody; among them, the heavy chain framework region is denoted as "HFR" and can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region is denoted as "LFR" and can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3 and LFR4 framework regions.

[0126] As used herein, the heavy chain variable region is obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0127] In an alternative embodiment of the present invention, the HFR1 comprises the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto;

[0128] The HFR2 comprises the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology thereto;

[0129] The HFR3 comprises the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 80% homology thereto;

[0130] The HFR4 comprises the amino acid sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 80% homology thereto;

[0131] The LFR1 comprises the amino acid sequence shown in SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto;

[0132] The LFR2 comprises the amino acid sequence shown in SEQ ID NO: 45 or an amino acid sequence having at least 80% homology thereto;

[0133] The LFR3 comprises the amino acid sequence shown in SEQ ID NO: 46 or an amino acid sequence having at least 80% homology thereto;

[0134] The LFR4 comprises the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 80% homology thereto.

[0135] According to an embodiment of the present invention, the antibody comprises the heavy chain variable region and / or the light chain variable region described in the first aspect or the second aspect of the claims.

[0136] Those skilled in the art can understand that the features and advantages described above for the antibodies in the first aspect and the second aspect (such as the features of the heavy chain variable region and / or the light chain variable region defined in the first aspect, and the features of the heavy chain and / or the light chain defined in the second aspect) also apply to the antibodies in this third aspect, and will not be elaborated herein.

[0137] According to an embodiment of the present invention, the antibodies described in the above first aspect, second aspect, and third aspect may further include at least one of the following technical features:

[0138] According to an embodiment of the present invention, the antibody further includes a constant region; wherein, the constant region includes at least one of a heavy chain constant region and a light chain constant region.

[0139] According to an embodiment of the present invention, at least a part of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a pig antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting chicken antibody or a mutant thereof.

[0140] According to an embodiment of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or, the light chain constant region includes a light chain constant region selected from κ type or λ type.

[0141] In an alternative embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from a murine antibody or a mutant thereof.

[0142] According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the N-terminus of the light chain variable region.

[0143] In this article, the division of variable region and constant region sequences refers to the IMGT division method, see Lefranc, the international ImMunoGeneTics database. Nucl. Acids Res., 29(1):207 - 209(2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: housemouse (Mus musculus) IGHC, IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. Or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020.. There will be some amino acid differences between the variable regions divided by different methods and the C-terminus of the variable region or the N-terminus of the constant region divided by IMGT. The variable regions or constant regions divided by other methods well-known in the art are also within the protection scope of the present invention.

[0144] In an alternative embodiment of the present invention, the heavy chain constant region comprises or is the heavy chain constant region shown by the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 80% identity therewith; or, the light chain constant region comprises or is the light chain constant region shown by the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 80% identity therewith.

[0145] According to an embodiment of the present invention, the antibody comprises the heavy chain and / or light chain described in the second aspect.

[0146] According to an embodiment of the present invention, the antibody comprises at least one selected from polyclonal antibody, full-length monoclonal antibody, Fab antibody, Fab' antibody, F(ab')2 antibody, Fv antibody, single-chain antibody, single-domain antibody and minimum recognition unit; or, the functional fragment comprises at least one selected from F(ab')2 fragment, Fab' fragment, Fab fragment, F(ab)2 fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, scFv-Fv fusion protein and minimum recognition unit.

[0147] It should be noted that a "functional fragment" refers to a fragment that retains the ability to specifically bind to D-dimer, including but not limited to at least one of an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), an F(ab’)2 fragment, a Fab’ fragment, a Fab fragment, an F(ab)2 fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, an Fv-Fc fusion protein, a multispecific antibody formed by functional fragments, a single-domain antibody, a VHH nanobody, a domain antibody, a bivalent domain antibody, or a minimum recognition unit.

[0148] In this article, the terms "single-domain antibody", "nanobody", and "VHH antibody" are used interchangeably and were originally described as the antigen-binding immunoglobulin (variable) domain of a "heavy-chain antibody" (i.e., an "antibody lacking a light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), which contains a heavy-chain variable region (VH) and conventional CH2 and CH3 regions and specifically binds to an antigen protein (such as D-dimer) through the heavy-chain variable region.

[0149] In this article, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment that contains only Fab molecules, which are composed of VH and CH1 of the heavy chain and a complete light chain, and the light chain and the heavy chain are connected by a disulfide bond.

[0150] In this article, the term "F(ab’)2 antibody" or "F(ab’)2 fragment" has two antigen-binding F(ab’) parts connected together by disulfide bonds.

[0151] In this article, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment formed only by the non-covalent connection of a light-chain variable region (VL) and a heavy-chain variable region (VH), and it is the smallest functional fragment of an antibody molecule that retains the complete antigen-binding site.

[0152] In this article, the terms "single-chain antibody" and "scFv fragment" are antibodies or fragments formed by connecting the heavy-chain variable region and the light-chain variable region of an antibody with a short peptide.

[0153] In this article, the terms "minimum recognition unit" and "MRU" both refer to an antibody or fragment composed of only one CDR, and its molecular weight is very small, accounting for only about 1% of a complete antibody.

[0154] According to an embodiment of the present invention, the antibody includes a light chain and / or a heavy chain. The heavy chain includes the heavy chain variable region and the heavy chain constant region defined above; the light chain includes the light chain variable region and the light chain constant region defined above.

[0155] Nucleic acid molecule, vector, cell or host, method for preparing antibody

[0156] In the process of preparing or obtaining the antibody described in the first, second or third aspect, nucleic acid molecules expressing these antibodies can be utilized, ligated to different vectors, and then expressed in different cells to obtain the corresponding antibodies.

[0157] In the fourth aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody described in the first, second or third aspect. The nucleic acid molecule according to the embodiment of the present invention can encode and obtain the above-mentioned antibody.

[0158] According to an embodiment of the present invention, the nucleic acid molecule includes DNA or RNA.

[0159] It should be noted that for the nucleic acid molecules mentioned herein, those skilled in the art should understand that it actually includes either any one of the complementary double strands or both. For convenience, in this text, although only one strand is given in most cases, in fact, the other complementary strand is also disclosed. In addition, the molecular sequences in the present invention include DNA form or RNA form. Disclosing one of them means that the other is also disclosed.

[0160] In the fifth aspect of the present invention, the present invention provides a vector. According to an embodiment of the present invention, the vector includes the nucleic acid molecule described in the fourth aspect. When ligating the above nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule, etc. Of course, these control elements can directly come from the vector itself or be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control elements are operably connected. As used herein, "operably connected" means connecting an exogenous gene to a vector such that the control elements in the vector, such as transcriptional control sequences and translational control sequences, etc., can exert their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After the vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the aforementioned antibody can be effectively achieved, and thus a large amount of the antibody can be obtained in vitro.

[0161] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a phage.

[0162] In an alternative embodiment of the present invention, the expression vector is a plasmid expression vector.

[0163] In a sixth aspect of the present invention, the present invention provides a cell or host. According to an embodiment of the present invention, the cell or host comprises: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expresses the antibody described in the first aspect, the second aspect or the third aspect. Using this cell under suitable conditions, the aforementioned antibody can be effectively expressed intracellularly.

[0164] According to an embodiment of the present invention, the cell is obtained by introducing the vector described in the fifth aspect into the cell.

[0165] It should be noted that the cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or phages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, etc., insect cells such as Spodoptera frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0166] In an alternative embodiment of the present invention, the cell is a mammalian cell, including BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0167] It should be noted that the "suitable conditions" described in the present invention refer to the conditions suitable for the expression of the antibody of the present invention. It is easily understood by those skilled in the art that the conditions suitable for the expression of the antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell densities, suitable cell culture environments, and suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the antibody according to the specific environment of the laboratory.

[0168] In a seventh aspect of the present invention, the present invention provides a method for preparing the antibody described in the first aspect, the second aspect or the third aspect. According to an embodiment of the present invention, the method comprises culturing the cell or host described in the sixth aspect. According to the methods of some specific embodiments of the present invention, the antibody can be effectively obtained in large quantities.

[0169] Based on the amino acid sequence of the antibody of the present disclosure, those skilled in the art can easily conceive of preparing the antibody using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression). For example, the antibody can be isolated and purified from the culture product of a recombinant cell capable of recombinantly expressing the antibody described in any one of the above. This is easily achievable for those skilled in the art. Based on this, regardless of the technique used to prepare the antibody of the present disclosure, it falls within the protection scope of the present disclosure.

[0170] Those skilled in the art can understand that the features and advantages described for the antibodies in the first, second, and third aspects also apply to the nucleic acid molecule, vector, cell or host, and method for preparing the antibody, which will not be elaborated here.

[0171] Conjugate, reagent or kit and their uses

[0172] The antibody or its functional fragment can be used in combination with any detection reagent or therapeutic agent. For example, it can be used in combination with diagnostic radionuclides, nanomaterials, etc. The radioactive target site can be detected by the radioactivity of the radionuclide, and then information about the target site can be obtained; it can also be used in combination with therapeutic radionuclides to specifically kill target cells, tissues, etc. using the radioactivity of the radionuclide.

[0173] In the eighth aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate includes: the antibody described in the first, second, or third aspect and a conjugate moiety conjugated thereto. The conjugate according to the embodiment of the present invention can specifically target and bind to D-dimer, and can be used for qualitative or quantitative detection of D-dimer, or for diagnosing diseases related to abnormal D-dimer.

[0174] According to an embodiment of the present invention, the above conjugate may further include at least one of the following additional technical features:

[0175] According to an embodiment of the present invention, the conjugate moiety is selected from carriers. Exemplarily, the carrier includes a purification tag or a label.

[0176] In this text, the carrier can be a substance capable of being suspended or dispersed in a liquid phase (e.g., solid-phase carriers such as particles, magnetic beads, etc.), or a solid phase capable of accommodating or carrying a liquid phase (e.g., supports such as plates, membranes, test tubes, etc., and containers such as microtiter plates, microfluidic channels, glass capillaries, nano-columns, monolithic columns, etc.); it can also be a labeling carrier for labeling antibodies, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), affinity substances (e.g., one of streptavidin and biotin, one of nucleic acids of sense and antisense strands that are complementary to each other), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioisotopes (e.g., 3 H, 14 C, 32 P, 35 S, 125 I), and gold colloids, etc.

[0177] In some specific embodiments of the present invention, the purification tag or label includes at least one selected from colloidal gold, radioactive label, luminescent substance, colored substance, enzyme, biotin / avidin, and spin label.

[0178] In some specific embodiments of the present invention, the purification tag or label includes at least one selected from fluorescent label, chromophore label, and electron-dense label.

[0179] In some specific embodiments of the present invention, the purification tag or label includes one or more selected from radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, fluorescein, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase.

[0180] In some specific embodiments of the present invention, the coupling part includes at least one selected from magnetic microspheres, plastic microspheres, plastic microparticles, microtiter plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0181] In the ninth aspect of the present invention, the present invention provides a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit includes: the antibody according to the first, second or third aspect or the conjugate according to the eighth aspect. As described above, the antibodies in some specific embodiments or examples of the present invention can effectively bind to D-dimer. Therefore, the reagent or the kit containing the antibody can effectively qualitatively or quantitatively detect D-dimer. By applying the reagent or the kit provided by the present invention, for example, it can be used for detections such as immunoblotting and immunoprecipitation that involve the specific binding performance of D-dimer and its antibody. As described above, the antibodies in some specific embodiments or examples of the present invention have a higher binding activity with D-dimer. Therefore, the reagent or the kit containing the antibody has a higher detection sensitivity.

[0182] These kits may include any one or more of the following: treatment solution, anti-D-dimer antibody, D-dimer control product, anti-IgG antibody, instructions or literature, etc. The anti-D-dimer antibody can be used in different types of diagnostic tests. For example, it can detect the presence of various diseases, drugs, or other proteins in vitro or in vivo. For example, it can be used to test related diseases by detecting the serum or blood of a subject.

[0183] In the tenth aspect of the present invention, the present invention provides the use of the antibody according to the first, second or third aspect, the conjugate according to the eighth aspect, or the reagent or the kit according to the ninth aspect in the preparation of a product for detecting D-dimer for diagnosing D-dimer related diseases.

[0184] In this document, the D-dimer related diseases include but are not limited to at least one of thrombotic diseases, disseminated intravascular coagulation, systemic lupus erythematosus, myocardial infarction, cirrhosis or hepatitis, cancer or tumor, mycoplasma pneumonia, diabetes, and allergic purpura.

[0185] In this document, the term "thrombotic disease" refers to a disease caused by a thrombus, including but not limited to arterial and venous thrombi (e.g., deep vein thrombosis (DVT)), pulmonary embolism, etc.

[0186] In the eleventh aspect of the present invention, the present invention provides a method for detecting D-dimer. According to an embodiment of the present invention, the method includes: contacting a sample to be detected with the antibody according to the first, second or third aspect, the conjugate according to the eighth aspect, or the reagent or the kit according to the ninth aspect to form an immune complex.

[0187] According to an embodiment of the present invention, based on the signal of the immune complex, it is determined whether the sample to be detected contains D-dimer or the content of D-dimer.

[0188] According to an embodiment of the present invention, the immune complex further comprises a second antibody, and the second antibody binds to the antibody.

[0189] According to an embodiment of the present invention, the immune complex further comprises a second antibody, and the second antibody binds to D-dimer.

[0190] According to an embodiment of the present invention, the signal includes a fluorescence signal.

[0191] In a twelfth aspect of the present invention, the present invention provides a method for screening D-dimer antibodies. According to an embodiment of the present invention, the method comprises: a) designing primers for amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8, or 9 sites among X1, X2, X3, X4, X5, X6, X7, X8, and X9 defined in the antibody described in the second aspect; b) constructing a mutant library with the primers described in a) using the nucleic acid molecule described in the fourth aspect, the vector described in the fifth aspect, or the cell described in the sixth aspect as a template; c) screening D-dimer antibodies from the mutant library.

[0192] According to an embodiment of the present invention, the mutant library is a single-site saturation mutant library.

[0193] According to an embodiment of the present invention, the D-dimer antibody comprises or is the antibody described in the first aspect, the second aspect, or the third aspect.

[0194] In a thirteenth aspect of the present invention, the present invention provides a mutant library, which comprises variants of SEQ ID NO:1 and variants of SEQ ID NO:2 defined in the antibody described in the first aspect, the second aspect, or the third aspect.

[0195] According to an embodiment of the present invention, the mutant library is constructed with the primers in the method described in the twelfth aspect.

[0196] In a fourteenth aspect of the present invention, the present invention provides an antibody described in the first aspect, the second aspect, or the third aspect, a conjugate described in the eighth aspect, or a reagent or kit described in the ninth aspect, for detecting D-dimer or for diagnosing D-dimer-related diseases.

[0197] In a fifteenth aspect of the present invention, the present invention provides the use of an antibody described in the first aspect, the second aspect, or the third aspect, a conjugate described in the eighth aspect, or a reagent or kit described in the ninth aspect in the preparation of a product for detecting D-dimer or for diagnosing D-dimer-related diseases.

[0198] Those skilled in the art can understand that the antibodies, the described features and advantages mentioned for the first, second and third aspects above are equally applicable to the conjugate, reagent or kit and their uses and methods, which will not be elaborated here.

[0199] The amino acid sequences or nucleotide sequences involved in this article are shown in Table 2. Among them, the mutation sites of the heavy chain variable region or HCDRs are referenced to the amino acid sequence shown in SEQ ID NO:1 or the HCDRs in SEQ ID NO:1, and the mutation sites of the light chain variable region or LCDRs are referenced to the amino acid sequence shown in SEQ ID NO:2 or the LCDRs in SEQ ID NO:2. WT indicates no mutation:

[0200] Table 2: Amino Acid Sequences

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213] The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0214] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. The techniques employed or contemplated herein are standard methods unless otherwise indicated. The materials, methods, and examples are illustrative only and not limiting.

[0215] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Coligan et al., eds., 2011), each of which is hereby expressly incorporated by reference.

[0216] In this example, the restriction endonuclease, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs, the Taq DNA polymerase was purchased from TaKaRa, the V01 expression vector was constructed by our laboratory, the gel extraction kit and plasmid extraction kit were commercially available, and primer synthesis and gene sequencing were completed by an outsourcing company. The sequence of the D-dimer monoclonal antibody (hereinafter referred to as the WT antibody) was derived from the sequencing of mouse hybridoma cells.

[0217] Example 1: Construction and screening of the mutant library

[0218] 1. Construction of the wild-type (WT) D-dimer antibody (referred to as the WT antibody) template plasmid

[0219] (1) Synthesis of the WT antibody gene:

[0220] The VH (amino acid sequence shown in SEQ ID NO: 1) and VL (amino acid sequence shown in SEQ ID NO: 2) of the WT antibody sequence were optimized for E. coli codons, and then the antibody gene sequence was handed over to an outsourcing company for gene synthesis.

[0221] (2) Amplification of the WT antibody gene fragment:

[0222] The nucleotide sequences of the antibody VH and VL synthesized in step (1) were amplified by PCR using DNA polymerase, and then the antibody bands were separated by agarose gel electrophoresis. Then, the antibody gene fragment was purified using a gel extraction kit.

[0223] (3) Enzyme digestion and ligation of the WT antibody gene fragment:

[0224] The antibody gene fragment obtained in step (2) and the V01 vector plasmid were digested with a restriction endonuclease at the same time, and then the antibody gene fragment and the V01 vector with sticky ends were purified using a gel extraction kit. Then, the antibody gene fragment and the V01 vector were ligated with T4 DNA ligase at 22 °C for 4 hours. After that, the ligation reaction product was recovered and purified, and the DNA concentration was measured. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 E. coli competent cells to obtain a bacterial solution. Then, the entire bacterial solution was spread on a plate containing ampicillin resistance and cultured overnight at 37 °C.

[0225] (4) Extraction and sequencing verification of the WT template plasmid

[0226] Ten monoclonal colonies cultured overnight in step (3) were selected, and colony PCR and gel electrophoresis were performed using Taq DNA polymerase. The bacteria with the correct insertion of the antibody gene sequence were selected for culture and amplification. The WT template plasmid was obtained using a plasmid extraction kit and sent to a sequencing company for gene sequencing verification.

[0227] 2. Construction of single-point mutation library

[0228] In this part of the experiment, single-point saturation mutagenesis was performed on the VH and VL full CDR regions of the WT antibody obtained in step 1 to construct a single-point mutation library.

[0229] (1) Primer design and synthesis

[0230] Using degenerate base codons, 71 pairs of upstream and downstream primers for single-point saturation mutagenesis of the VH and VL full CDR regions (71 amino acid sites) were designed and handed over to an outsourcing company for primer synthesis.

[0231] (2) PCR amplification of single-point saturation mutagenesis plasmid

[0232] Using the primers obtained in step (1), PCR amplification of the single-point saturation mutagenesis plasmid was performed by the PCR method. The reaction system was configured according to Table 3, and then the PCR reaction conditions in Table 4 were used for the amplification and preparation of the single-point saturation mutation library plasmid. Finally, the WT template plasmid obtained in step 1 was digested with a restriction enzyme at 37°C for 1 hour to obtain 71 plasmids of the mutant library.

[0233] Table 3: Reaction system for PCR amplification

[0234] WT template plasmid 50 ng DNA polymerase 1 μl DNA polymerase buffer 10 μl dNTP (2.5 mM) 4 μl Forward primer (10 μM) 1 μl Reverse primer (10 μM) 1 μl <![CDATA[ddH2O]]> Volume made up to 50 μl

[0235] Table 4: PCR reaction conditions

[0236] Step 1 Step 2 Step 3 Step4 Step5 Step6 Temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ Time 5min 30s 30s 2min 5min ∞

[0237] Note: 22 cycles were performed for Step2 - Step4.

[0238] (3) Transformation of single-point saturation mutagenesis plasmid

[0239] Take 10 μl of the reaction products of the 71 plasmids of the mutant library obtained in step (2) and transform them into 100 μl of TG1 Escherichia coli competent cells to obtain a bacterial solution, and then spread all the bacterial solution on a plate containing ampicillin resistance and culture overnight at 37°C.

[0240] 3. Screening of single-point mutation library

[0241] (1) Expression of mutant library antibodies

[0242] Pre-add 500 μl of culture medium into a 96-well culture plate. For each single-point mutation library, pick 92 monoclonal colonies transformed with the single-point saturated mutation plasmid cultured overnight in step 2-(3), and set up WT, negative control (i.e., colonies without inserting the VH\VL gene-containing colonies), and blank control (i.e., only culture medium without colonies). After culturing at 37 °C for 5-6 hours, transfer the bacterial liquid to a new 96-well culture plate. Then, after culturing at 37 °C for 1-2 hours, finally add the induction medium and culture overnight at 37 °C to express antibodies, obtaining the antibody expression supernatants of 71 mutation libraries.

[0243] (2) Screening and sequencing of mutation libraries

[0244] Add commercially available D-dimer protein at a quantity of 0.2 μg / ml and 100 μl / well to 71 ELISA plates, and react overnight at 4 °C for coating. The next day, block with 1-2% skim milk powder. Add the antibody expression supernatants of the 71 mutation libraries obtained in step (1) to the ELISA plate wells at a quantity of 100 μl / well, and set up WT, negative control (i.e., colonies without inserting the VH\VL gene-containing colonies), and blank control (i.e., only culture medium without colonies). Incubate at room temperature for 2 hours, and perform subsequent plate washing, color development, and reading using the detection method of conventional ELISA; finally, organize and analyze the data results, send the clones with improved performance for sequencing, and finally analyze the sequencing results to select the mutation sites of 16 unique mutation candidate clones (see Table 5) for constructing a combinatorial mutation library. (The Ratio value in Table 5 represents the degree of affinity improvement. When the Ratio value is equal to 1, it indicates that the affinity of the mutant clone is the same as that of WT; when the Ratio value is greater than 1, it indicates that the affinity has been improved, that is, the larger the Ratio value, the greater the degree of affinity improvement).

[0245] Table 5: Screening results and mutation sites of candidate clones

[0246]

[0247] 4. Construction of combinatorial mutation library

[0248] (1) Design and synthesis of library primers:

[0249] According to the mutation sites on VH and VL of the D-dimer antibody obtained in step 3-(2), design amplification primers for the combinatorial mutation library and hand them over to an outsourcing company for primer synthesis.

[0250] (2) Fragment amplification and ligation

[0251] According to the PCR system in Table 3 and the PCR reaction conditions in Table 4, the antibody mutant fragments were amplified, and then the antibody mutant fragments were recovered by gel extraction. The antibody mutant fragments were spliced into complete antibody fragments (heavy chain variable region or light chain variable region) by the method of Overlap PCR.

[0252] Finally, the antibody fragment was inserted into the V01 vector by enzymatic digestion and ligation to form a complete antibody expression plasmid (for the specific steps, see the steps of "Enzymatic digestion and ligation of WT antibody gene fragment" in step 1-(3)). 100 ng of the plasmid was transformed into 100 μl of TG1 Escherichia coli competent cells, and all the bacterial solution was spread on a plate containing ampicillin resistance and cultured overnight at 37°C.

[0253] 5. Screening of the combinatorial mutation library

[0254] 62 combinatorial mutant antibodies were randomly selected for supernatant expression, ELISA screening and detection, and positive clone sequencing analysis. The specific screening results, mutation site information and Ratio value determination are shown in Table 6.

[0255] Table 6: Information of combinatorial mutation candidate clones

[0256]

[0257]

[0258] Example 2: Expression of mutant D-dimer antibody

[0259] In this example, the mutant D-dimer antibody obtained by screening in Example 1 was expressed. The specific experimental operations are as follows:

[0260] 1. Construction of eukaryotic recombinant expression plasmid

[0261] pcDNA TM 3.4 The vector is the eukaryotic expression vector of the recombinant antibody constructed. Multiple cloning sites such as HindIII, BamHI, and EcoRI have been introduced into this expression vector, which is named the pcDNA3.4A expression vector, hereinafter simply referred to as the 3.4A expression vector. According to the variable region gene sequences in the 62 candidate clones screened from the above-mentioned combinatorial mutant library (see Table 6, and the specific amino acid sequences are shown in Table 2), VL and VH gene-specific amplification primers for the corresponding antibody sequences and overlap primers for the constant regions (the amino acid sequences of the heavy chain constant region and the light chain constant region are shown in Table 2. Among them, the N-terminus of the heavy chain constant region is connected to the C-terminus of VH to form the heavy chain, and the N-terminus of the light chain constant region is connected to the C-terminus of VL to form the light chain) are designed. The primers at both ends carry HindIII and EcoRI restriction enzyme sites and protective bases. The light chain gene fragment and the heavy chain gene fragment are amplified by the PCR amplification method.

[0262] The heavy chain and light chain gene fragments are respectively digested with HindIII / EcoRI double enzymes, and the 3.4A vector is digested with HindIII / EcoRI double enzymes. The digested antibody light and heavy chain gene fragments and the vector are purified and recovered. Then, the gene fragments encoding the antibody light and heavy chains are respectively ligated into the 3.4A expression vector and transformed into DH5α Escherichia coli competent cells. After colonies grow, single colonies are respectively picked for PCR identification of positive clones. The positive clones are picked for sequencing to determine the correctness of the sequences. The clones with correct sequencing are selected for plasmid extraction for standby.

[0263] 2. Preparation of recombinant antibody samples

[0264] Resuscitate HEK293 cells in advance and passage them to a 200 ml system to make the cell density reach (3 - 5)×10 6 cells / ml, and the cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 3.7×10 6 cells / ml as the cell dilution solution. Prepare the plasmid DNA and transfection reagent dilution solutions with the medium respectively. Add the transfection reagent dilution solution to the plasmid DNA dilution solution, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell dilution solution within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place it in a 35 °C constant temperature incubator for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, centrifuge to collect the samples. Use a protein A affinity chromatography column for affinity purification to obtain 62 antibodies. Among them, the amino acid sequences of the heavy chain variable region, light chain variable region, heavy chain, and light chain of the 62 antibodies are shown in Table 7.

[0265] Table 7: Amino acid sequences of the heavy chain variable region, light chain variable region, heavy chain, and light chain of 37 antibodies

[0266]

[0267]

[0268] 3. Affinity Analysis

[0269] The wild-type antibody (the amino acid sequence of the heavy chain is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain is shown in SEQ ID NO: 26), 16 candidate cloned antibodies with single-site mutations obtained in step 3 of Example 1, and 62 combinatorial mutant antibodies obtained in step 2 of Example 2 were subjected to affinity detection and analysis. The specific steps were as follows: The binding and dissociation curves of D-dimer and mutant D-dimer antibodies were tested on a Biacore8K+ device, and the instrument automatically fitted to obtain the affinity constant, binding rate, and dissociation rate. In the affinity detection results, the affinity of the above 62 antibodies for D-dimer was 10×10 -12 M < KD < 10×10 - 10 M, and the affinity of the mutant antibody for D-dimer was better than that of the wild-type antibody for D-dimer. (KD represents the equilibrium dissociation constant, i.e., the affinity constant. The smaller the KD value, the higher the affinity; ka represents the binding rate; kd represents the dissociation rate)

[0270] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0271] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-D-dimer antibody, characterized in that include: heavy chain variable region and / or light chain variable region; Wherein, the heavy chain variable region includes or is the amino acid sequence shown in SEQ ID NO: 1 or a variant of SEQ ID NO: 1, and the variant of SEQ ID NO: 1 includes mutations at the following sites compared with the amino acid sequence shown in SEQ ID NO: 1: at least one of the 52nd, 59th, 102nd, 107th and 110th; The light chain variable region includes or is an amino acid sequence as shown in SEQ ID NO: 2 or a variant of SEQ ID NO: 2, wherein the variant of SEQ ID NO: 2 includes mutations at the following sites compared with the amino acid sequence shown in SEQ ID NO: 2: at least one of the 30th, 60th, 61st and 98th; The heavy chain variable region includes or is the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region includes or is the amino acid sequence shown in SEQ ID NO: 2, but they do not exist at the same time.

2. The antibody according to claim 1, characterized in that The variant of SEQ ID NO: 1 comprises at least one of the following mutations compared to the amino acid sequence shown in SEQ ID NO: 1: S52D, D59L / M / W, Y102F, F107W, and H110V; Optionally, the mutation at position 52 is S52D; Optionally, the mutation at position 59 is D59L; Optionally, the mutation at position 59 is D59M; Optionally, the mutation at position 59 is D59W; Optionally, the mutation at position 102 is Y102F; Optionally, the mutation at position 107 is F107W; Optionally, the mutation at position 110 is H110V; Optionally, compared with the amino acid sequence shown in SEQ ID NO: 1, the variant of SEQ ID NO: 1 includes mutations at the following sites: Optionally, the variant of SEQ ID NO: 2 comprises at least one of the following mutations compared to the amino acid sequence shown in SEQ ID NO: 2: V30K, F60W, S61W / D / E, and H98G / L / A / M; Optionally, the mutation at position 30 is V30K; Optionally, the mutation at position 60 is F60W; Optionally, the mutation at position 61 is S61W; Optionally, the mutation at position 61 is S61D; Optionally, the mutation at position 61 is S61E; Optionally, the mutation at position 98 is H98G; Optionally, the mutation at position 98 is H98L; Optionally, the mutation at position 98 is H98A; Optionally, the mutation at position 98 is H98M; Optionally, compared with the amino acid sequence shown in SEQ ID NO: 2, the variant of SEQ ID NO: 2 includes mutations at the following sites: ； Optionally, the antibody comprises the heavy chain variable region and light chain variable region as shown in the following table: Among them, WT means there is no mutation site.

3. An anti-D-dimer antibody comprising HCDRs and LCDRs, characterized in that: The HCDRs include or are HCDRs that are consistent with the HCDRs of the heavy chain variable region defined by the antibody according to any one of claims 1 to 3, and the LCDRs include or are LCDRs that are consistent with the LCDRs of the light chain variable region defined by the antibody according to any one of claims 1 to 3; Optionally, the HCDRs and / or LCDRs are defined by the Kabat, Chothia, AbM, Contact or IMGT systems; Optionally, the HCDRs and LCDRs include or are the amino acid sequences shown below: HCDR 1:GYRFTDYSIH; HCDR 2:VIX1TYSGNPX2YNQKFKG, wherein X1 is S or D, and X2 is D, L, M or W; HCDR 3:MNDX3YGDYX4FDX5, wherein X3 is Y or F, X4 is F or W, and X5 is H or V; LCDR1:RSSQSLX6HTNGNTYLH, where X6 is V or K; LCDR 2: KVSNRX7X8, where X7 is F or W, and X8 is S, W, D or E; LCDR 3: SQSRX9VPLT, where X9 is H, G, L, A, or M; Among them, X1 is S, X2 is D, X3 is Y, X4 is F, X5 is H, X6 is V, X7 is F, X8 is S and X9 is H do not exist at the same time; Optionally, X1 is S; Optionally, X1 is D; Optionally, X2 is D; Optionally, X2 is L; Optionally, X2 is M; Optionally, X2 is W; Optionally, X3 is Y; Optionally, X3 is F; Optionally, X4 is F; Optionally, X4 is W; Optionally, X5 is H; Optionally, X5 is V; Optionally, X6 is V; Optionally, X6 is K; Optionally, X7 is F; Optionally, X7 is W; Optionally, X8 is S; Optionally, X8 is W; Optionally, X8 is D; Optionally, X8 is E; Optionally, X9 is H; Optionally, X9 is G; Optionally, X9 is L; Optionally, X9 is A; Optionally, X9 is M; Optionally, the antibody comprises or is HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 as shown in the following table, Optionally, the antibody further comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4; Wherein, at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 is derived from at least one of mouse antibody, human antibody, primate antibody, bovine antibody, horse antibody, dairy cow antibody, porcine antibody, sheep antibody, goat antibody, dog antibody, cat antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting cock antibody or mutants thereof; Optionally, the HFR1 comprises the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80% homology thereto; The HFR2 comprises an amino acid sequence as shown in SEQ ID NO:41 or an amino acid sequence having at least 80% homology thereto; The HFR3 comprises the amino acid sequence shown in SEQ ID NO:42 or an amino acid sequence having at least 80% homology thereto; The HFR4 comprises an amino acid sequence as shown in SEQ ID NO:43 or an amino acid sequence having at least 80% homology thereto; The LFR1 comprises an amino acid sequence as shown in SEQ ID NO: 44 or an amino acid sequence having at least 80% homology thereto; The LFR2 comprises an amino acid sequence as shown in SEQ ID NO: 45 or an amino acid sequence having at least 80% homology thereto; The LFR3 comprises the amino acid sequence shown in SEQ ID NO:46 or an amino acid sequence having at least 80% homology thereto; The LFR4 comprises an amino acid sequence as shown in SEQ ID NO: 47 or an amino acid sequence having at least 80% homology thereto; Optionally, the antibody comprises the heavy chain variable region and / or light chain variable region according to any one of claims 1 to 3.

4. The antibody according to any one of claims 1 to 3, characterized in that The antibody further comprises a constant region; Wherein, the constant region includes at least one of a heavy chain constant region and a light chain constant region; Optionally, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof; Optionally, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or The light chain constant region comprises a light chain constant region selected from a κ type or a λ type; Optionally, the light chain constant region and the heavy chain constant region are both from a murine antibody or a mutant thereof; Optionally, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the N-terminus of the light chain variable region; Optionally, the heavy chain constant region comprises or is the heavy chain constant region shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% identity thereto; or The light chain constant region comprises or is the light chain constant region shown in SEQ ID NO:4 or an amino acid sequence having at least 80% identity thereto; Optionally, the antibody comprises the heavy chain and light chain as shown in the following table: Optionally, the antibody comprises at least one selected from polyclonal antibody, full-length monoclonal antibody, Fab antibody, Fab' antibody, F(ab')2 antibody, Fv antibody, single-chain antibody, single-domain antibody and minimum recognition unit.

5. A nucleic acid molecule, vector, cell or host or a method for preparing the antibody according to any one of claims 1 to 4, characterized in that: The nucleic acid molecule encodes the antibody according to any one of claims 1 to 4; the vector comprises the nucleic acid molecule; the cell or host comprises the nucleic acid molecule or the vector or expresses the antibody according to any one of claims 1 to 4; the method comprises culturing the cell or host.

6. A conjugate, characterized in that: include: The antibody according to any one of claims 1 to 4 and the conjugated part thereof; Optionally, the coupling moiety comprises a purification tag or label selected from; Optionally, the purification tag or label comprises at least one selected from colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, biotin / avidin and a spin label; Optionally, the purification tag or label comprises at least one selected from a fluorescent label, a chromophore label and an electron-dense label; Optionally, the purification tag or marker comprises one or more selected from the group consisting of radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; Optionally, the coupling part includes at least one selected from magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

7. A reagent or a kit, characterized in that: include: The antibody according to any one of claims 1 to 4 or the conjugate according to claim 16.

8. Use of the antibody according to any one of claims 1 to 4, the conjugate according to claim 6, or the reagent or kit according to claim 7 in detecting D-dimer or preparing a product for diagnosing D-dimer-related diseases; Optionally, the D-dimer related diseases include: At least one of thrombotic disease, disseminated intravascular coagulation, systemic lupus erythematosus, myocardial infarction, cirrhosis or hepatitis, cancer or tumor, mycoplasma pneumonia, diabetes mellitus and Henoch-Schonlein purpura.

9. A method for detecting D-dimer, characterized in that: include: The antibody according to any one of claims 1 to 4, the conjugate according to claim 6, or the reagent or kit according to claim 7 is contacted with a sample to be detected to form an immune complex; Optionally, based on the signal of the immune complex, determining whether the sample to be tested contains D-dimer or the content of the D-dimer; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to D-dimer; Optionally, the signal comprises a fluorescent signal.

10. A method for screening D-dimer antibodies, characterized in that: include: a) designing primers for amino acid substitution at 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the positions X1, X2, X3, X4, X5, X6, X7, X8 and X9 defined in the antibody of claim 3; b) using the nucleic acid molecule, vector or cell of claim 5 as a template and constructing a mutation library using the primers described in a); c) screening D-dimer antibodies from the mutant library; Optionally, the mutation library is a single-site saturation mutation library; Optionally, the D-dimer antibody includes or is the antibody according to any one of claims 1 to 4.

11. A mutation library, characterized in that The mutant library includes variants of SEQ ID NO: 1 and variants of SEQ ID NO: 2 defined in the antibody according to any one of claims 1 to 4; Optionally, the mutant library is constructed using the primers in the method of claim 10.

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