Anti-D-dimer antibodies, reagents and kits for detecting D-dimer

Abstract

The present invention discloses an anti-D-dimer antibody or a reagent and kit for detecting D-dimer, relating to the field of antibodies. The anti-D-dimer antibody disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region. The antibody provides an important source of raw materials for the detection of D-dimer and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an anti-D-dimer antibody, a reagent and a kit for detecting D-dimer. Background Art

[0002] D-dimer (DD) is a specific, simplest degradation product of fibrin in the blood, produced through activation and hydrolysis. It serves as a specific marker of the fibrinolytic process. The human fibrinolytic system primarily maintains normal vascular permeability, blood flow, and tissue repair. Under pathological conditions, when coagulation occurs, thrombin acts on fibrin to convert it into cross-linked fibrin. During the dissolution of cross-linked fibrin, fragments such as X', Y', D', and E' are released, forming complexes such as DD, DD / E, YD / YD, and YY / DD. These fragments are further degraded into the smallest fragment, DD, and the DD / E complex. DD has a molecular weight of approximately 62,000 D, a half-life of >3 hours in vivo, and is primarily excreted through the kidneys.

[0003] Elevated D-dimer levels reflect the presence of enhanced coagulation and fibrinolytic activity in the body, representing clot formation in the vascular system and a sensitive marker for acute thrombosis. Elevated D-dimer levels can be caused by conditions such as myocardial infarction, cerebral infarction, pulmonary embolism, venous thrombosis, surgery, tumors, disseminated intravascular coagulation, infection, and tissue necrosis.

[0004] D-dimer is clinically used as a specific monitoring indicator for thrombolytic therapy in thrombotic diseases. Among fibrinolytic protein degradation products, only D-dimer cross-linked fragments can reflect thrombolytic activity after thrombus formation. Therefore, quantitative D-dimer measurement can quantitatively reflect the thrombolytic effect of drugs and can be used to diagnose and screen newly formed thrombi.

[0005] Tumors can also cause elevated D-dimer levels in patients, and this can be used as a criterion for staging and prognosis. A study by Masatoshi Oya et al. found that D-dimer levels in patients with colorectal cancer were significantly higher than those in patients with benign diseases. Preoperative D-dimer levels were positively correlated with tumor pathology and stage. Patients with high preoperative D-dimer levels had significantly shorter postoperative survival than those with low levels.

[0006] In liver diseases, plasma D-dimer levels are significantly elevated and positively correlated with the severity of the disease. D-dimer levels were significantly higher in patients with various hepatitis types compared to controls (P < 0.01). The order of D-dimer levels was severe hepatitis > cirrhosis > chronic hepatitis, and severe > acute hepatitis > chronic hepatitis, potentially related to an impaired anticoagulant system. Furthermore, the literature has also reported the clinical application of D-dimer in the detection of various conditions, including cerebral infarction, myocardial infarction, kidney disease, and neonatal asphyxia.

[0007] The detection methods for D-dimers include the triple P test, latex agglutination method (LATEX), ELISA method, immunofiltration colloidal gold colorimetric reaction method, etc. The most commonly used methods in clinical practice are: ELISA, LATEX and immunofiltration colloidal gold staining method. The detection principles of these methods are all based on the binding reaction of antigen and antibody. Different methods have their own advantages and disadvantages, but they all require monoclonal antibodies against D-dimers.

[0008] Therefore, those skilled in the art have a strong demand for anti-D-dimer antibodies with good performance. Summary of the Invention

[0009] The present application provides an antibody or an antigen-binding fragment thereof, which provides an important source of raw materials for the detection of D-dimer and has good activity or affinity.

[0010] To achieve the above-mentioned object, according to one aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof comprises three complementarity determining regions having a heavy chain variable region having the amino acid sequence of any one of SEQ ID NOs: 21, 22, 23, and 24 and three complementarity determining regions having a light chain variable region having the amino acid sequence of any one of SEQ ID NOs: 29 and 30.

[0011] To achieve the above object, according to a second aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the following complementarity determining regions:

[0012] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0013] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0014] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, 17 or 18;

[0015] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0016] LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5; and

[0017] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0018] To achieve the above-mentioned object, according to the third aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 21, 22, 23, and 24; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 29 and 30.

[0019] In order to achieve the above-mentioned object, according to the fourth aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 25, 26, 27, and 28; and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 31 and 32.

[0020] In order to achieve the above object, according to a fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or antigen-binding fragment thereof.

[0021] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or kit is provided, wherein the reagent or kit comprises the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0022] To achieve the above-mentioned object, according to the seventh aspect of the present invention, a method for detecting D-dimer is provided, comprising: a) contacting the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, or reagent or kit with D-dimer in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0023] In order to achieve the above object, according to the eighth aspect of the present invention, a nucleic acid is provided, which encodes the above-mentioned antibody or antigen-binding fragment thereof.

[0024] In order to achieve the above object, according to the ninth aspect of the present invention, a vector is provided, wherein the vector comprises the above nucleic acid.

[0025] To achieve the above object, according to the tenth aspect of the present invention, a cell is provided, wherein the cell comprises the above nucleic acid, vector or expresses the above antibody or antigen-binding fragment thereof.

[0026] To achieve the above object, according to the eleventh aspect of the present invention, there is provided a method for preparing the above antibody or antigen-binding fragment thereof, the method comprising culturing the above cell.

[0027] In order to achieve the above-mentioned object, according to the twelfth aspect of the present invention, there is provided a use of the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit in the preparation of a product for detecting D-dimer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 The results of reducing SDS-PAGE of Anti-DD 13F8 Rmb1 to Anti-DD 13F8 Rmb5 are shown. DETAILED DESCRIPTION

[0030] In a first aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, comprising three complementarity determining regions having a heavy chain variable region of any one of the amino acid sequences of SEQ ID NOs: 21, 22, 23, and 24 and three complementarity determining regions having a light chain variable region of any one of the amino acid sequences of SEQ ID NOs: 29 and 30.

[0031] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or antigen-binding fragment thereof described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or antigen-binding fragment thereof described in the first aspect.

[0032] For example, the HCDR1, HCDR2, and HCDR3 have amino acid sequences consistent with those of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in SEQ ID NO: 21; and the LCDR1, LCDR2, and LCDR3 have amino acid sequences consistent with those of LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in SEQ ID NO: 29.

[0033] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0034] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0035] In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0036] Methods for defining CDRs are well known in the art, including the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200200201154, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196: 901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes, but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literature vary slightly. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0037] Table 1: CDR Definition 1

[0038] CDR Kabat AbM2 IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0039] 1The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+number" and amino acid numbers on the light chain represented by "L+number." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0040] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0041] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0042] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0043] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0044] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

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

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

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

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

[0049] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0050] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0051] According to an embodiment of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, Chothia, AbM or IMGT systems are as follows:

[0052]

[0053] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0054] In a second aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the following complementarity determining regions:

[0055] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0056] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0057] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3, 17 or 18;

[0058] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0059] LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5; and

[0060] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0061] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0062] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0063] In an optional embodiment, the antibody or antigen-binding fragment thereof of the first aspect or the second aspect further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

[0064] The HFR1 comprises / is as SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0065] The HFR2 comprises / is as SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0066] The HFR3 comprises / is SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0067] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0068] The LFR1 comprises / is SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0069] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0070] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto;

[0071] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0072] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or antigen-binding fragments thereof provided by the present invention may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0073] In an optional embodiment, the HFR1 comprises / is represented by the amino acid sequence of SEQ ID NO: 7 or 19.

[0074] In an optional embodiment, the LFR1 includes / is represented by the amino acid sequence of SEQ ID NO: 11 or 20.

[0075] In an alternative embodiment, the antibody or antigen-binding fragment thereof has a KD < 4.09×10 -10 M binds D-dimer with high affinity.

[0076] In an alternative embodiment, the antibody or antigen-binding fragment thereof has a KD ≤ 10 -9 M, KD≤10 -10 M, KD≤10 -11 M, KD≤10 -12 M or KD≤10 -13 M binds D-dimer with high affinity.

[0077] In an alternative embodiment, the antibody or antigen-binding fragment thereof has a KD ≤ 4.88×10 -10 M binds D-dimer with high affinity.

[0078] There are many methods for determining antibody affinity (KD). Based on the detection principle, they can be divided into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods. Among them, thermodynamic detection methods such as isothermal titration calorimetry (ITC) are common; kinetic detection methods such as surface plasmon resonance (SPR) and biofilm interferometry (BLI) are common; and dynamic equilibrium detection methods such as enzyme-linked immunosorbent assay (ELISA) are common.

[0079] In alternative embodiments, KD is determined using a kinetic assay; optionally, surface plasmon resonance, for example, by using a kinetic assay such as System of biosensor systems.

[0080] In a third aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 21, 22, 23, and 24, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 29 and 30.

[0081] In an optional embodiment, the heavy chain variable region and light chain variable region of the first aspect and the third aspect are selected from any one of the following combinations:

[0082] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:21 SEQ ID NO:29 2 SEQ ID NO:24 SEQ ID NO:29 3 SEQ ID NO:22 SEQ ID NO:29 4 SEQ ID NO:23 SEQ ID NO:29 5 SEQ ID NO:21 SEQ ID NO:30 .

[0083] In an alternative embodiment, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0084] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0085] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0086] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

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

[0088] In an alternative embodiment, the light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0089] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0090] In an alternative embodiment, the species origin of the constant region is mouse.

[0091] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO:15, and the light chain constant region (CL) sequence is shown as SEQ ID NO:16.

[0092] It should be noted that, in other embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0093] In an optional embodiment, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0094] The antigen-binding fragments of the aforementioned antibodies generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that the antigen-binding fragments of the aforementioned antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0095] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0096] In a fourth aspect, the present invention provides an antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 25, 26, 27, and 28, and the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 31 and 32.

[0097] In an optional embodiment, the antibody described in the first, second, third, and fourth aspects above comprises any combination of the following heavy chains and light chains:

[0098] combination heavy chain light chain 1 SEQ ID NO:25 SEQ ID NO:31 2 SEQ ID NO:28 SEQ ID NO:31 3 SEQ ID NO:26 SEQ ID NO:31 4 SEQ ID NO:27 SEQ ID NO:31 5 SEQ ID NO:25 SEQ ID NO:32

[0099] In a fifth aspect, the present invention provides an antibody conjugate comprising the above-mentioned antibody or antigen-binding fragment thereof.

[0100] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.

[0101] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof.

[0102] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0103] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0104] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0105] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0106] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0107] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0108] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0109] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0110] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0111] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0112] In an optional embodiment, the colloidal metal is colloidal gold.

[0113] In an optional embodiment, the above-mentioned antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

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

[0115] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0116] In a sixth aspect, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0117] As previously mentioned, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention can effectively bind to D-dimer, and therefore, the reagents or kits comprising the D-dimer antibodies or antigen-binding fragments thereof can effectively detect D-dimer qualitatively or quantitatively. The reagents or kits provided by the present invention can be used, for example, for detection involving the use of D-dimer and its antibody specific binding properties by immunoblotting, immunoprecipitation, etc. As previously mentioned, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity with D-dimer, and therefore the reagents or kits comprising the antibodies or antigen-binding fragments thereof have higher detection sensitivity or specificity.

[0118] In a seventh aspect, the present invention provides a method for detecting D-dimer, comprising: a) contacting the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit with D-dimer in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0119] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or antigen-binding fragment thereof.

[0120] In an alternative embodiment, the immune complex further comprises a second antibody, which binds to D-dimer.

[0121] In an eighth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0122] In a ninth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0123] In a tenth aspect, the present invention provides a cell containing the above-mentioned vector.

[0124] In an eleventh aspect, the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof, comprising: culturing the cell as described above.

[0125] In a twelfth aspect, the present invention provides use of the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, or the above-mentioned reagent or kit in the preparation of a product for detecting D-dimer.

[0126] Based on the amino acid sequence of the antibody or antigen-binding fragment thereof disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or antigen-binding fragment thereof. For example, the antibody or antigen-binding fragment thereof can be isolated and purified from the culture product of recombinant cells that can recombinantly express the antibody or antigen-binding fragment thereof as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, regardless of the technology used to prepare the antibody or antigen-binding fragment of the present invention, it falls within the scope of protection of the present invention.

[0127] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0128] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled 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 preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0129] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0130] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0131] Example 1 Preparation of Anti-DD 13F8 Monoclonal Antibody

[0132] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM The RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting the Anti-DD 13F8 monoclonal antibody was generated in our laboratory and revived for future use.

[0133] (1) Antibody gene preparation

[0134] mRNA was extracted from a hybridoma cell line secreting the Anti-DD 13F8 monoclonal antibody. DNA products were obtained by RT-PCR. This product was PCR-amplified with rTaq DNA polymerase and inserted into the pMD-18T vector. The cells were then transformed into DH5α competent cells. After colonies grew, the Heavy Chain and Light Chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing.

[0135] (2) Sequence analysis of the variable region gene of Anti-DD 13F8 antibody

[0136] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among them, in the gene fragment amplified by the Light Chain, the VL gene sequence was 336 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 366 bp, belonging to the VH1 gene family, and preceded by a 57 bp leader peptide sequence.

[0137] (3) Construction of recombinant antibody expression plasmid

[0138] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed by using the pMD-18T vector. Multiple cloning restriction sites such as HindIII, BamHI, and EcoRI have been introduced into the vector, and the vector is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region genes in the pMD-18T vector, specific primers for the VL and VH genes of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. PCR amplification was used to amplify a 0.71kb Light Chain gene fragment and a 1.40kb Heavy Chain gene fragment.

[0139] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0140] 2. Recombinant Antibody Production

[0141] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6 cells / ml cell density reaches the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 Cells were washed with 100 μg of culture medium and re-dissolved. This was also used as a cell diluent. Plasmid DNA and transfection reagent diluents were prepared separately using culture medium. The transfection reagent diluent was added to the plasmid DNA diluent, mixed, and allowed to stand at room temperature for 15 minutes. The mixture was slowly added to the cell diluent over 1 minute, mixed, and sampled. The viability of the cells after transfection was recorded and observed. The cells were cultured in a 35°C constant temperature incubator at 120 rpm and 8% CO2. After 13 days, the samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE. The electrophoresis pattern is shown in the figure. Two bands were shown after reducing SDS-PAGE, one with an Mr of 50 KD (heavy chain) and the other with an Mr of 28 KD (light chain).

[0142] The resulting antibody was named Anti-DD 13F8Rmb1. Anti-DD 13F8Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table:

[0143] Table 2 Antibody sequences

[0144] Antibody name Heavy chain light chain Anti-DD 13F8Rmb1 SEQ ID NO:25 SEQ ID NO:31 Anti-DD 13F8Rmb2 SEQ ID NO:28 SEQ ID NO:31 Anti-DD 13F8Rmb3 SEQ ID NO:26 SEQ ID NO:31 Anti-DD 13F8Rmb4 SEQ ID NO:27 SEQ ID NO:31 Anti-DD 13F8Rmb5 SEQ ID NO:25 SEQ ID NO:32

[0145] Example 2 Performance testing of antibodies

[0146] 1. Affinity Analysis

[0147] Purified antibodies were diluted in advance, and D-dimer antigen (from Feipeng Bio) was serially diluted. Binding and dissociation curves of the antigen and antibody were measured on a Biacore 8K+ instrument using a CM5 chip pre-coupled with goat anti-mouse IgG. The instrument automatically fitted the affinity constant, association rate, and dissociation rate. (KD represents the equilibrium dissociation constant, also known as the affinity constant; ka represents the association rate; and kd represents the dissociation rate.)

[0148] Table 3 Affinity data

[0149] Sample name KD ka kd comparison 4.09E-10 3.27E+03 1.34E-06 Anti-DD 13F8Rmb1 2.38E-11 1.86E+04 4.42E-07 Anti-DD 13F8Rmb2 2.43E-11 1.84E+04 4.47E-07 Anti-DD 13F8Rmb3 2.56E-11 1.72E+04 4.40E-07 Anti-DD 13F8Rmb4 2.49E-11 1.79E+04 4.46E-07 Anti-DD 13F8Rmb5 4.88E-11 3.64E+04 1.78E-06

[0150] 2. Activity Identification

[0151] D-dimer antigen (from Feipeng Bio) was diluted to 3 μg / ml in coating buffer (main component: NaHCO₃), and 100 μL was added to each well at 4°C overnight. The next day, the wells were washed twice with wash buffer (main component: Na₂HPO₄ + NaCl) and patted dry. Blocking buffer (20% BSA + 80% PBS) was added at 120 μL per well and incubated at 37°C for 1 hour, then patted dry. Diluted purified antibody and control antibody were added at 100 μL / well at 37°C for 30 minutes. The wells were washed five times with wash buffer and patted dry. Goat anti-mouse IgG-HRP was added at 100 μL per well at 37°C for 30 minutes. The wells were washed five times with wash buffer and patted dry. Chromogenic Buffer A (50 μL / well) and Chromogenic Buffer B (50 μL / well) were added for 10 minutes. Stop buffer (50 μL / well) was added. The OD values were read on a microplate reader at 450 nm (reference: 630 nm).

[0152] Note: Solution A (main ingredients: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main ingredients: citric acid + EDTA·2Na + TMB + concentrated HCL); Stop solution (EDTA·2Na + concentrated H2SO4)

[0153] Table 4 Activity data

[0154] Concentration (ng / ml) 9.766 4.883 2.441 1.221 0.61 0 comparison 1.022 0.552 0.311 0.118 0.019 0.013 Anti-DD 13F8Rmb1 1.559 1.19 0.792 0.511 0.243 0.03 Anti-DD 13F8Rmb2 1.548 1.134 0.791 0.521 0.246 0.037 Anti-DD 13F8Rmb3 1.557 1.123 0.788 0.532 0.227 0.042 Anti-DD 13F8Rmb4 1.539 1.131 0.784 0.529 0.231 0.036 Anti-DD 13F8Rmb5 1.446 0.945 0.391 0.135 0.088 0.036

[0155] 3. Stability assessment

[0156] The above-mentioned antibody was placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected at 7, 14, and 21 days for observation, and the 21-day sample was tested for activity. The results showed that no significant changes in protein status were observed under all three test conditions for 21 days, and the activity did not decrease with increasing test temperature, indicating that the above-mentioned antibody is stable. Table 5 below shows the OD results of the enzyme immunoassay activity assay of the antibody Anti-DD 13F8Rmb1 after 21 days of testing.

[0157] Table 5 Stability data

[0158]

[0159]

[0160] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0161] Some of the amino acid sequences involved in this application are shown in Table 6:

[0162]

[0163]

Claims

1. An anti-D-dimer antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof has three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 21, 22, 23, and 24, and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 29 and 30, and the complementarity determining regions of the heavy chain variable region and the light chain variable region are defined by the same system, and the system is selected from any one of Kabat, Chothia, IMGT, AbM or Contact.

2. An anti-D-dimer antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1; The amino acid sequence of HCDR2 is shown in SEQ ID NO: 2; The amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, 17 or 18; The amino acid sequence of LCDR1 is shown in SEQ ID NO:4; The amino acid sequence of LCDR2 is shown in SEQ ID NO: 5; and The amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The antibody or antigen-binding fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

4. The antibody or antigen-binding fragment thereof according to claim 3, characterized in that The HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and 4, characterized in that: The antibody or antigen-binding fragment thereof has a KD of less than 4.09×10 -10 M binds to D-dimer with high affinity.

6. An anti-D-dimer antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region and light chain variable region are selected from any one of the following combinations:

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4, and 6, wherein: The antibody or antigen-binding fragment thereof further comprises a constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that The constant region includes a heavy chain constant region and a light chain constant region.

9. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that The heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

10. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that The heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

11. The antibody or antigen-binding fragment thereof according to claim 7, wherein The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

12. The antibody or antigen-binding fragment thereof according to claim 7, wherein The species origin of the constant region is mouse.

13. The antibody or antigen-binding fragment thereof according to claim 8, wherein The heavy chain constant region sequence is shown in SEQ ID NO: 15 or has at least 80% identity thereto; the light chain constant region sequence is shown in SEQ ID NO: 16 or has at least 80% identity thereto.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4, and 6, wherein: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

15. An anti-D-dimer antibody comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain and the amino acid sequence of the light chain are selected from any one of the following groups: 。 16. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the antibody according to claim 15 and biotin or a label conjugated to the antibody or antigen-binding fragment thereof.

17. The antibody conjugate according to claim 16, characterized in that The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

18. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the antibody according to claim 15 and a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

19. The antibody conjugate according to claim 18, characterized in that The solid support is selected from microspheres, plates and membranes.

20. A reagent or kit, characterized in that The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the antibody according to claim 15, or the antibody conjugate according to any one of claims 16 to 19.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the antibody according to claim 15, the antibody conjugate according to any one of claims 16 to 19, or the reagent or kit according to claim 20 in the preparation of a product for detecting D-dimer, characterized in that: include: a) contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the antibody according to claim 15, the antibody conjugate according to any one of claims 16 to 19, or the reagent or kit according to claim 20 with D-dimer in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

22. The use according to claim 21, characterized in that The immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment thereof.

23. The use according to claim 21, characterized in that The immune complex further includes a second antibody that binds to the D-dimer.

24. A nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the antibody according to claim 15.

25. A carrier, characterized in that It contains the nucleic acid according to claim 24.

26. A cell, characterized in that It contains the nucleic acid according to claim 24 or the vector according to claim 25.

27. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the antibody according to claim 15, characterized in that: It includes: Cultivate the cell of claim 26.

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