Anti-D-dimer antibody and application thereof

Abstract

The invention discloses an anti-D-dimer antibody and application thereof, and relates to the field of antibodies. The anti-D-dimer antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for D-dimer detection, 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 antibody against D-dimer and its application. Background Art

[0002] D-dimer (DD) is a specific and simplest degradation product produced by the activation and hydrolysis of fibrin in the blood, and is a marker of the specific fibrinolysis process. The fibrinolysis system in the human body mainly maintains the normal permeability of blood vessels, the flow state of blood and tissue repair. Under pathological conditions, when the body undergoes coagulation, thrombin acts on fibrin to transform it into cross-linked fibrin. During the dissolution process of cross-linked fibrin, fragments such as X’, Y’, D’, E’ are released, and complexes such as DD, DD / E, YD / YD, YY / DD are formed. These fragments are further degraded into the smallest fragments DD and DD / E complexes. The molecular weight of DD is about 62000D, with a half-life in vivo > 3h, and it is mainly excreted through the kidneys.

[0003] An increase in the level of D-dimer reflects an important molecular marker of enhanced coagulation and fibrinolysis activity in the body, indicating the formation of blood clots in the vascular circulatory system, and is a sensitive marker for acute thrombosis. Myocardial infarction, cerebral infarction, pulmonary embolism, venous thrombosis, surgery, tumors, disseminated intravascular coagulation, infections, and tissue necrosis can all lead to an increase in D-dimer.

[0004] D-dimer is used as a specific monitoring indicator for thrombolytic therapy of thrombotic diseases in clinical practice. Because among the fibrin degradation products, only the cross-linked fragment of D-dimer can reflect the thrombolytic activity after thrombus formation. Therefore, the quantitative detection of D-dimer can quantitatively reflect the thrombolytic effect of drugs and can be used for the diagnosis and screening of newly formed thrombi.

[0005] Tumors can also cause an increase in the D-dimer concentration in patients and can be used as a criterion for staging, prognosis, etc. Masatoshi Oya et al. found in a study that the D-dimer in patients with colorectal cancer was significantly higher than that in patients with benign diseases, and the preoperative D-dimer was positively correlated with the pathological results and staging of the tumor. The postoperative survival period of patients with a high preoperative D-dimer level was significantly shorter than that of patients with a low level.

[0006] In liver diseases, the content of plasma D-dimer is significantly increased and is positively correlated with the severity of liver diseases. The D-dimer levels in patients with various types of hepatitis are significantly higher than those in the control group and show significant differences (P<0.01). The levels of D-dimer in patients are in the order of severe hepatitis group > liver cirrhosis group > severe group in chronic hepatitis group > acute hepatitis group > chronic hepatitis group, which may be related to the impairment of the anticoagulation system. In addition, the literature has also reported the application of D-dimer in the clinical detection of various diseases such as cerebral infarction, myocardial infarction, kidney diseases, neonatal asphyxia, etc.

[0007] The detection methods of D-dimer include the three P test, latex agglutination method (LATEX), ELISA method, immunofiltration colloidal gold color reaction method, etc. The most commonly used in clinics 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 all require antibodies against D-dimer.

[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 anti-D-dimer antibody, which provides an important raw material source for the detection of D-dimer and has good activity or affinity.

[0010] To achieve the above object, according to one aspect of the present invention, there is provided an anti-D-dimer antibody, which comprises three complementary determining regions of any one of the heavy chain variable regions having the amino acid sequences SEQ ID NO:21, 22, 23, 24 and three complementary determining regions of any one of the light chain variable regions having the amino acid sequences SEQ ID NO:29, 30, 31.

[0011] To achieve the above object, according to the second aspect of the present invention, there is provided an anti-D-dimer antibody, which comprises the following complementary determining regions:

[0012] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1 or consists of the same;

[0013] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2 or consists of the same;

[0014] HCDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO:3, 17 or consists of the same;

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

[0016] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and

[0017] LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6 or 19.

[0018] To achieve the above object, according to the third aspect of the present invention, there is provided an anti-D dimer antibody, 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 any one of SEQ ID NO:21, 22, 23, 24; and the amino acid sequence of the light chain variable region is any one of SEQ ID NO:29, 30, 31.

[0019] To achieve the above object, according to the fourth aspect of the present invention, there is provided an anti-D dimer antibody, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is any one of SEQ ID NO:25, 26, 27, 28; and the amino acid sequence of the light chain is any one of SEQ ID NO:32, 33, 34.

[0020] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody conjugate, which comprises the above-mentioned antibody.

[0021] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or a kit, which comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0022] To achieve the above object, according to the eighth aspect of the present invention, there is provided the use of the above-mentioned antibody and antibody conjugate in the preparation of a product for detecting D dimer.

[0023] To achieve the above object, the present invention also provides a nucleic acid, a vector, a cell and a method for preparing the above-mentioned antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 Results of reducing SDS-PAGE for Anti-DD 6B12 Rmb1 to Anti-DD 6B12 Rmb6. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] In a first aspect, embodiments of the present invention provide an antibody against D-dimer, which antibody comprises three complementarity determining regions having any one of the heavy chain variable regions of amino acid sequences SEQ ID NO: 21, 22, 23, 24 and three complementarity determining regions having any one of the light chain variable regions of amino acid sequences SEQ ID NO: 29, 30, 31.

[0027] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the same heavy chain variable region defined in the antibody described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the same light chain variable region defined in the antibody described in the first aspect.

[0028] For example, the HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 21; the LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 29.

[0029] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies or antigen-binding fragments, provided that they exhibit the desired biological activity.

[0030] The above antigen-binding fragments generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art can easily understand from the content described in the present invention that the above antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by methods of chemically reducing and cleaving disulfide bonds. Based on the disclosure of the structure of the complete antibody in the present invention, those skilled in the art can easily obtain the above antigen-binding fragments.

[0031] The above antigen-binding fragments 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 an automated peptide synthesizer sold by Applied BioSystems and the like.

[0032] In the present invention, the terms "complementarity determining region", "CDR" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0033] In the present invention, the heavy chain complementarity determining regions are denoted as HCDR and include HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted as LCDR and include LCDR1, LCDR2 and LCDR3.

[0034] The methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" can be found in Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR regions defined by Kabat, although they may be shortened or lengthened based on the prediction or experimental results of specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literatures are slightly different. 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 specific CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also fall within the scope of protection of the present disclosure.

[0035] Table 1: CDR Definitions 1

[0036]

[0037]

[0038] 1 The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". Those 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 used 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).

[0039] 2 "AbM" as used in Table 1 with a lower-case "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0040] 3 When neither H35A nor H35B is present, CDR-H1 ends at position 35; when only H35A is present, CDR-H1 ends at position 35A; when both H35A and H35B are present, CDR-H1 ends at position 35B.

[0041] 4 When neither H35A nor H35B is present, CDR-H1 ends at position 32; when only H35A is present, CDR-H1 ends at position 33; when both H35A and H35B are present, CDR-H1 ends at position 34.

[0042] 5 When neither H35A nor H35B is present, CDR-H1 ends at position 33; when only H35A is present, CDR-H1 ends at position 34; when both H35A and H35B are present, CDR-H1 ends at position 35.

[0043] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

[0044] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

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

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

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

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

[0049] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0050] In a second aspect, embodiments of the present invention provide an anti-D dimer antibody, the antibody comprising the following complementarity determining regions:

[0051] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, or consists of the same;

[0052] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2, or consists of the same;

[0053] HCDR3, which comprises the amino acid sequence shown in any one of SEQ ID NO:3 and 17, or consists of the same;

[0054] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4, or consists of the same;

[0055] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5, or consists of the same; and

[0056] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6 or 19, or consists of the same.

[0057] According to the embodiments of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0058] 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 regions other than the CDRs in the heavy chain variable region and the light chain variable region of the antibody; wherein, the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0059] In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0060] In an alternative embodiment, the antibody according to the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0061] In an alternative embodiment, the HFR1 comprises / is as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% identity therewith;

[0062] The HFR2 comprises / is as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity therewith;

[0063] The HFR3 comprises / is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity therewith;

[0064] The HFR4 comprises / is as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity therewith;

[0065] The LFR1 comprises / is as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity therewith;

[0066] The LFR2 comprises / is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity therewith;

[0067] The LFR3 comprises / is as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity therewith; and

[0068] The LFR4 comprises / is as shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity therewith.

[0069] It should be noted that in other embodiments, the amino acid sequences of the respective framework regions of the anti-D dimer antibody provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the corresponding framework regions (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) above.

[0070] In an alternative embodiment, the HFR3 comprises the amino acid sequence as shown in SEQ ID NO: 18.

[0071] In an alternative embodiment, the LFR1 comprises the amino acid sequence as shown in SEQ ID NO: 20.

[0072] In an alternative embodiment, the antibody binds to D-dimer with an affinity of KD < 8.70×10 -8 M.

[0073] In an alternative embodiment, the antibody binds to D-dimer with an affinity of KD ≤ 10 -8 M, KD ≤ 10 -9 M, KD ≤ 10 -10 M, KD ≤ 10 -11 M, or KD ≤ 10 -12 M.

[0074] In an alternative embodiment, the antibody binds to D-dimer with an affinity of KD ≤ 3.49×10 -9 M.

[0075] There are many methods for measuring antibody affinity (KD), which can be classified into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods according to the detection principle. Among them, common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biolayer interferometry (BLI); common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA), etc.

[0076] In an alternative embodiment, the KD is measured by a kinetic detection method; optionally, surface plasmon resonance, for example, by using a biosensor system such as the system.

[0077] In a third aspect, an embodiment of the present invention provides an antibody against D-dimer, 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 as shown in any one of SEQ ID NO: 21, 22, 23, and 24, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 29, 30, and 31.

[0078] In an alternative embodiment, the heavy chain variable region and the light chain variable region described in the first aspect or the third aspect above are selected from any one of the following combinations:

[0079] Combination VH VL 1 SEQ ID NO:21 SEQ ID NO:29 2 SEQ ID NO:23 SEQ ID NO:29 3 SEQ ID NO:22 SEQ ID NO:29 4 SEQ ID NO:24 SEQ ID NO:29 5 SEQ ID NO:21 SEQ ID NO:30 6 SEQ ID NO:21 SEQ ID NO:31

[0080] .

[0081] In an alternative embodiment, the antibody described in the first, second, and third aspects above further comprises a constant region.

[0082] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0083] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0084] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0085] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0086] In an alternative embodiment, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0087] In an alternative embodiment, the species origin of the constant region is bovine, equine, dairy cow, porcine, ovine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting chicken or human.

[0088] In an alternative embodiment, the species origin of the constant region is mouse.

[0089] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:16.

[0090] It should be noted that in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above constant region (SEQ ID NO:15 or 16).

[0091] In an alternative embodiment, the antibody includes any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv and scFv.

[0092] Fourth aspect, the present invention provides an antibody against D-dimer, comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO:25, 26, 27, 28, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:32, 33, 34.

[0093] In an alternative embodiment, the antibody described in the first, second, third, or fourth aspect above comprises a heavy chain and a light chain of any of the following combinations:

[0094] Combination VH VL 1 SEQ ID NO:25 SEQ ID NO:32 2 SEQ ID NO:27 SEQ ID NO:32 3 SEQ ID NO:26 SEQ ID NO:32 4 SEQ ID NO:28 SEQ ID NO:32 5 SEQ ID NO:25 SEQ ID NO:33 6 SEQ ID NO:25 SEQ ID NO:34

[0095] 。

[0096] In a fifth aspect, the present invention provides an antibody conjugate, which comprises the antibody described above.

[0097] In an alternative embodiment, the above antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0098] In an alternative embodiment, the antibody conjugate further comprises a label conjugated to the antibody.

[0099] In an alternative embodiment, the above label refers to a class of substances having characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or probed by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through these characteristics.

[0100] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0101] In actual use, those skilled in the art can select a suitable label according to the detection conditions or actual needs. No matter which label is used, it falls within the protection scope of the present invention.

[0102] In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (e.g., including but not limited to rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (e.g., including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (e.g., including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0103] In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0104] In alternative embodiments, the radioisotopes 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.

[0105] In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rosamide and its derivatives, and peroxyoxalate and its derivatives.

[0106] In alternative embodiments, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0107] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0108] In alternative embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0109] In alternative embodiments, the colloidal metal is colloidal gold.

[0110] In alternative embodiments, the above antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0111] In alternative embodiments, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0112] In alternative embodiments, the solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0113] In a sixth aspect, the present invention provides a reagent or kit, which includes the above antibody or the above antibody conjugate.

[0114] As described above, the antibodies in some embodiments or examples of the present invention can effectively bind to D-dimer. Therefore, the reagent or kit containing the D-dimer antibody can effectively perform qualitative or quantitative detection of D-dimer. Applying the reagent or kit provided by the present invention, for example, it can be used in detections such as immunoblotting and immunoprecipitation that involve the specific binding property of D-dimer and its antibody. As described above, the antibodies in some embodiments or examples of the present invention have higher binding activity or affinity with D-dimer. Therefore, the reagent or kit containing the antibody has higher detection sensitivity or specificity.

[0115] In a seventh aspect, the present invention provides a method for detecting D-dimer, including: a) contacting the above antibody, antibody conjugate, reagent, or kit with D-dimer in a test sample under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample;

[0116] In alternative embodiments, the immune complex further includes a second antibody that binds to the antibody.

[0117] In alternative embodiments, the immune complex further includes a second antibody that binds to D-dimer.

[0118] In an eighth aspect, the present invention provides the use of the above anti-D-dimer antibody and antibody conjugate in the preparation of products for detecting D-dimer.

[0119] It should be noted that the products in the present invention include, but are not limited to, reagents, reagent kits or detection plates.

[0120] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0121] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0122] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0123] In a twelfth aspect, the present invention provides a method for preparing an anti-D dimer antibody, which includes: culturing the cells as described above.

[0124] Based on the disclosure of the amino acid sequence of the anti-D dimer antibody in the present invention, those skilled in the art can easily think of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the anti-D dimer antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells 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, no matter what technique is used to prepare the anti-D dimer antibody of the present invention, it falls within the protection scope of the present invention.

[0125] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0126] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those 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. Unless otherwise stated, the techniques employed or contemplated herein are standard methods. The materials, methods and examples are illustrative only and not restrictive.

[0127] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second 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., 1991), each of which is hereby expressly incorporated by reference.

[0128] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0129] Example 1 Preparation of Anti-DD 6B12 Monoclonal Antibody

[0130] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the Anti-DD 6B12 monoclonal antibody was the hybridoma cell line prepared in this laboratory and was revived for standby.

[0131] (1) Preparation of antibody genes

[0132] mRNA was extracted from the hybridoma cell line secreting the Anti-DD 6B12 monoclonal antibody, and DNA products were obtained by RT-PCR. After the addition of A reaction to the products with rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0133] (2) Sequence analysis of the variable region genes of Anti-DD 6B12 antibody

[0134] The gene sequences obtained from the above sequencing were analyzed in the kabat antibody database, and the 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 the gene fragments amplified by the Light Chain, the VL gene sequence was 333 bp, and there was a 57 bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 363 bp, belonging to the VH1 gene family, and there was a 57 bp leader peptide sequence in front of it.

[0135] (3) Construction of recombinant antibody expression plasmid

[0136] pcDNA TM 3.4 The pcDNA 3.4 vector was the recombinant antibody eukaryotic expression vector constructed. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI had been introduced into this expression vector, and it was named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protection bases at both ends, and a 0.71 kb Light Chain gene fragment and a 1.39 kb Heavy Chain gene fragment were amplified by PCR amplification.

[0137] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain.

[0138] 2. Recombinant antibody production

[0139] Resuscitate HEK293 cells in advance, passage and culture them to a 200 ml system to make the cell density reach 3 - 5×10 6 cells / ml. Select the antibody concentration and cells with a cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the medium, and at the same time, use it 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 them 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. Affinity purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. After reducing SDS-PAGE, two bands are shown, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0140] The obtained antibody was named Anti-DD 6B12Rmb1, and the Anti-DD 6B12Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0141] Table 2 Antibody sequences

[0142] Antibody Name Heavy Chain Light Chain Anti-DD 6B12Rmb1 SEQ ID NO:25 SEQ ID NO:32 Anti-DD 6B12Rmb2 SEQ ID NO:27 SEQ ID NO:32 Anti-DD 6B12Rmb3 SEQ ID NO:26 SEQ ID NO:32 Anti-DD 6B12Rmb4 SEQ ID NO:28 SEQ ID NO:32 Anti-DD 6B12Rmb5 SEQ ID NO:25 SEQ ID NO:33 Anti-DD 6B12Rmb6 SEQ ID NO:25 SEQ ID NO:34

[0143] Example 2 Performance detection of the antibody

[0144] 1. Affinity analysis

[0145] Dilute the purified antibody in advance and perform gradient dilution on D-dimer antigen (from Fapon Biotech); use the CM5 chip pre-coupled with goat anti-mouse IgG to test the binding and dissociation curves of the antigen and antibody on the Biacore 8K+ device, and the instrument automatically fits to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0146] Table 3 Affinity Data

[0147] Sample Name KD ka kd Control 8.70E-08 2.32E+03 2.01E-04 Anti-DD 6B12Rmb1 2.16E-10 3.94E+05 8.52E-05 Anti-DD 6B12Rmb2 2.39E-10 3.52E+05 8.41E-05 Anti-DD 6B12Rmb3 4.10E-10 7.56E+05 3.10E-04 Anti-DD 6B12Rmb4 6.29E-10 8.13E+05 5.11E-04 Anti-DD 6B12Rmb5 3.49E-09 1.02E+04 3.56E-05 Anti-DD 6B12Rmb6 2.70E-10 4.49E+05 1.21E-04

[0148] 2. Activity Identification

[0149] Dilute D-dimer antigen (from Fapon Biotech) to 3 μg / ml with coating buffer (main component: NaHCO3), 100 μL per well, and incubate overnight at 4°C; the next day, wash 2 times with washing buffer (main components: Na2HPO4 + NaCl), and pat dry; add blocking buffer (20% BSA + 80% PBS), 120 μL per well, incubate at 37°C for 1 h, and pat dry; add the diluted purified antibody and control antibody, 100 μL per well, incubate at 37°C for 30 min; wash 5 times with washing buffer, and pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, incubate at 37°C for 30 min; wash 5 times with washing buffer, and pat dry; add chromogenic solution A (50 μL per well), add chromogenic solution B (50 μL per well), for 10 min; add stop solution, 50 μL per well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0150] Note: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0151] Table 4 Activity Data

[0152] Concentration (ng / ml) 90 45 22.5 11.25 5.625 0 Control 0.901 0.529 0.317 0.186 0.062 0.012 Anti-DD 6B12Rmb1 1.531 1.117 0.785 0.524 0.371 0.013 Anti-DD 6B12Rmb2 1.551 1.057 0.626 0.333 0.232 0.051 Anti-DD 6B12Rmb3 1.254 0.899 0.444 0.236 0.147 0.044 Anti-DD 6B12Rmb4 1.449 1.126 0.623 0.346 0.181 0.062 Anti-DD 6B12Rmb5 1.378 0.836 0.495 0.308 0.156 0.065 Anti-DD 6B12Rmb6 1.194 0.71 0.447 0.299 0.174 0.052

[0153] 3. Stability Assessment

[0154] Place the above antibody at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Take samples on the 7th, 14th, and 21st days for status observation, and perform activity detection on the 21-day samples. The results show that no obvious protein state changes are observed after the antibody is placed for 21 days under the three assessment conditions, and the activity does not show a downward trend with the increase of the assessment temperature, indicating that the above antibody is stable. The following Table 5 shows the OD results of the enzyme immunoassay activity detection of antibody Anti-DD 6B12Rmb2 after 21 days of assessment.

[0155] Table 5 Stability Data

[0156] Sample Concentration (ng / ml) 90 45 0 4°C, 21-day sample 1.666 1.031 0.016 -80°C, 21-day sample 1.613 1.201 0.017 37°C, 21-day sample 1.645 1.071 0.011

[0157] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0158] Some of the amino acid sequences involved in this application are shown in Table 6 as follows:

[0159]

[0160]

[0161]

[0162]

Claims

1. An anti-D dimer antibody, the antibody comprising three complementarity determining regions of any one of the heavy chain variable regions having the amino acid sequences SEQ ID NO: 21, 22, 23, 24 and three complementarity determining regions of any one of the light chain variable regions having the amino acid sequences SEQ ID NO: 29, 30, 31.

2. The antibody according to claim 1, wherein, the complementarity determining regions of the variable regions are defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

3. An anti-D dimer antibody, wherein, the antibody comprises the following complementarity determining regions: The amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 2; The amino acid sequence of HCDR3 is as shown in any one of SEQ ID NO: 3, 17; The amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4; The amino acid sequence of LCDR2 is as shown in SEQ ID NO: 5; and The amino acid sequence of LCDR3 is as shown in any one of SEQ ID NO: 6 or 19; Optionally, 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; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto; Optionally, the antibody binds to D dimer with an affinity of KD < 8.70×10 -8 M.

4. An anti-D dimer antibody 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 as shown in any one of SEQ ID NO: 21, 22, 23, 24; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 29, 30, 31; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations: ； Optionally, the antibody further comprises a constant region; Optionally, the constant region comprises a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments; Optionally, the heavy chain constant region comprises CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, canine, camel, feline, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto; Optionally, the antibody comprises any one of F(ab’)2, Fab’, Fab, Fv and scFv.

5. An antibody against D-dimer, comprising a heavy chain and / or a light chain, characterized in that the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 25, 26, 27, 28; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 32, 33, 34.

6. An antibody conjugate, characterized in that the antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a label conjugated to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle-based labels; Optionally, the antibody conjugate further comprises a solid-phase carrier conjugated to the antibody.

7. A reagent or kit, characterized in that the reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1-5 or the antibody conjugate according to claim 6 in the preparation of a product for detecting D-dimer; Optionally, the use comprises: a) contacting the antibody according to any one of claims 1-5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with D-dimer in a test sample under conditions sufficient to effect an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody that binds to the antibody; Optionally, the immune complex further comprises a second antibody that binds to D-dimer.

9. A nucleic acid, vector, cell or method for preparing the antibody according to any one of claims 1-5, wherein the nucleic acid encodes the antibody according to any one of claims 1 to 5; the vector contains the nucleic acid encoding the antibody according to any one of claims 1 to 5; the cell contains the above nucleic acid or vector; the method comprises the above cell.

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