WO2023088445A1 - 一种抗d-二聚体的抗体及其制备方法和用途 - Google Patents

一种抗d-二聚体的抗体及其制备方法和用途 Download PDF

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WO2023088445A1
WO2023088445A1 PCT/CN2022/132982 CN2022132982W WO2023088445A1 WO 2023088445 A1 WO2023088445 A1 WO 2023088445A1 CN 2022132982 W CN2022132982 W CN 2022132982W WO 2023088445 A1 WO2023088445 A1 WO 2023088445A1
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amino acid
acid sequence
seq
antibody
heavy chain
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French (fr)
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孟媛
钟冬梅
覃文新
熊俊文
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东莞市朋志生物科技有限公司
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Definitions

  • the present disclosure belongs to the technical field of antibodies. More specifically, it relates to an anti-D-dimer antibody and its preparation method and use.
  • D-dimer (D-Dimer, DD) is a specific and simplest degradation product produced by fibrin in blood after activation and hydrolysis, and is a specific marker of fibrinolysis process.
  • fragments such as X’, Y’, D’, E’ are released, and complexes such as DD, DD/E, YD/YD, and YY/DD are formed. These fragments are further degraded into the smallest fragments DD and DD/E complex.
  • DD has a molecular weight of about 62000D and a half-life of >3h in the body, mainly excreted by the kidneys.
  • D-dimer level reflects the enhanced coagulation and fibrinolytic activity in the body, and it is a sensitive marker of acute thrombosis.
  • Myocardial infarction, cerebral infarction, pulmonary embolism, venous thrombosis, surgery, tumor, disseminated intravascular coagulation, infection and tissue necrosis can all lead to elevated D-dimer.
  • thrombus-directed thrombolytic agents have been introduced using monoclonal antibodies and protein linking technology.
  • the basic principle is to use the affinity between antigen [D-dimer] and antibody [anti-D-dimer monoclonal antibody]
  • the monoclonal antibody of the thrombus-specific component is linked with the thrombolytic drug to form an antibody-thrombolytic complex.
  • the monoclonal antibody is like a missile, which can carry a thrombolytic agent to specifically bind to the thrombus, so that the thrombolytic agent at the thrombus site is highly aggregated, thereby enhancing the dissolution of the thrombus.
  • the anti-D-dimer monoclonal antibody labeled radionuclide can carry the radionuclide to the thrombus during the specific binding process between the antibody and the antigen, and then monitor the radionuclide concentration in the body with a radionuclide detector. distribution, so as to achieve the purpose of using guided tracers to locate and diagnose thrombus.
  • the detection methods of D-dimer include triple P test, latex agglutination method (LATEX), ELISA method, immunodiafiltration colloidal gold chromogenic reaction method and so on. These currently used detection methods are all based on specific monoclonal antibodies, which means that the preparation of monoclonal antibodies against D-dimer becomes the key to improving detection sensitivity and specificity.
  • the present disclosure provides an anti-D-dimer antibody or a functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include or are the same as SEQ ID NO: 15, 30, The amino acid sequence consistent with HCDR1-HCDR3 of any one of the heavy chain variable regions shown in 31 and 32; the LCDR1-LCDR3 includes or is the same as any of the light chains shown in SEQ ID NO:16, 33, 34, 35 and 36 The consistent amino acid sequence of LCDR1-LCDR3 in the variable region.
  • the CDRs are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.
  • the present disclosure provides an anti-D-dimer antibody or a functional fragment thereof, the anti-D-dimer antibody or a functional fragment thereof contains the following CDRs:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of it;
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of it;
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of it;
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of it;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of it.
  • the antibody or its functional fragments also has the following framework regions: the amino acid sequence of HFR1 is as shown in SEQ ID NO: 7 or has at least 80% homology therewith; HFR2 The amino acid sequence is as shown in SEQ ID NO: 8 or has at least 80% homology therewith; the HFR3 amino acid sequence is as shown in any of SEQ ID NO: 9, 21, 22, 23, or has at least 80% homology therewith; The amino acid sequence of HFR4 is as shown in SEQ ID NO: 10 or has at least 80% homology therewith; the amino acid sequence of LFR1 is as shown in any of SEQ ID NO: 11, 24, 25, 26, or has at least 80% homology therewith The amino acid sequence of LFR2 is as shown in SEQ ID NO: 12 or has at least 80% homology with it; the amino acid sequence of LFR3 is as shown in any of SEQ ID NO: 13, 27, 28, 29, or it has at least 80% homology with it Sex; the LFR4 amino acid sequence is shown in SEQ ID NO:
  • the present disclosure provides an anti-D-dimer antibody or a functional fragment thereof, the anti-D-dimer antibody or a functional fragment thereof comprising a heavy chain variable region and/or a light chain variable region,
  • the heavy chain variable region includes the above-mentioned HCDR1-3 and the above-mentioned HFR1-4
  • the light chain variable region includes the above-mentioned LCDR1-3 and the above-mentioned LFR1-4.
  • the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 15, 30, 31 and 32, or consists of it;
  • the light chain variable region comprises SEQ ID NO: The amino acid sequence shown in any one of 16, 33, 34, 35 and 36, or consists of it.
  • the anti-D-dimer antibody or functional fragment thereof further includes a constant region.
  • the constant region comprises a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.
  • the light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.
  • the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose , Turkey, Fighting Cock Or Man.
  • the species source of the constant region is mouse.
  • the heavy chain constant region is SEQ ID NO: 17 or an amino acid sequence having more than 80% homology with SEQ ID NO: 17;
  • the light chain constant region is SEQ ID NO: 18 or an amino acid sequence with SEQ ID NO: 17 NO:18 is an amino acid sequence with more than 80% homology.
  • the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.
  • the present disclosure also provides an anti-D-dimer antibody or a functional fragment thereof, including a heavy chain and/or a light chain, and the heavy chain includes the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region;
  • the light chain includes the above-mentioned light chain variable region and the above-mentioned light chain constant region.
  • amino acid sequence of the heavy chain is as shown in any of SEQ ID NO: 19, 37, 38, 39; the amino acid sequence of the light chain is as shown in any of SEQ ID NO: 20, 40, 41, 42, 43 one shown.
  • the present disclosure also provides a nucleic acid encoding the antibody or a functional fragment thereof of any one of the above.
  • the present disclosure also provides a cell comprising the above-mentioned nucleic acid.
  • the present disclosure also provides a method for preparing any of the above-mentioned antibodies or functional fragments thereof, the method comprising culturing the above-mentioned cells.
  • the present disclosure also provides an antibody conjugate, which comprises any one of the above-mentioned antibodies or functional fragments thereof and a conjugation moiety conjugated thereto.
  • the coupling moiety is selected from a purification label or a detectable label, such as colloidal gold, radioactive label, luminescent substance, colored substance, enzyme, such as a fluorescent label, a chromophore label, an electron-dense label , such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, sugar One or more of oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin label.
  • a detectable label such as colloidal gold, radioactive label, luminescent substance, colored substance, enzyme, such as a fluorescent label, a chromophore label, an electron-dense label , such as radioiso
  • the coupling moiety is selected from magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
  • the present disclosure also provides a reagent or a kit, the reagent or kit comprising any of the above-mentioned antibodies or functional fragments thereof or the above-mentioned antibody conjugates.
  • the present disclosure also provides the use of any one of the above-mentioned antibodies or functional fragments thereof or the antibody conjugates in the preparation of reagents or kits.
  • the reagent or kit is used for detecting D-dimer or diagnosing D-dimer-related diseases.
  • the present disclosure also provides the antibody or functional fragment thereof or the antibody conjugate or the reagent or kit described in any one of the above for detecting D-dimer or diagnosing D-dimer Use in related diseases.
  • the D-dimer-related diseases include myocardial infarction, cerebral infarction, preeclampsia, deep vein thrombosis, main thoracic dissection, disseminated intravascular coagulation, pulmonary hypertension, liver injury, systemic Lupus, tumor, or tissue death.
  • the present disclosure also provides a method for diagnosing whether a subject suffers from a D-dimer-related disease, comprising:
  • the D-dimer-related diseases include myocardial infarction, cerebral infarction, preeclampsia, deep vein thrombosis, main thoracic dissection, disseminated intravascular coagulation, pulmonary hypertension, liver injury, systemic Lupus, tumor, or tissue death.
  • the present disclosure also provides a method of detecting D-dimer in a test sample, comprising:
  • the immune complex further includes a second antibody that binds to the anti-D-dimer antibody or a functional fragment thereof.
  • Fig. 1 is the reducing SDS-PAGE results of 14D2RMb1 to 14D2RMb11 antibodies (from left to right).
  • Some embodiments of the present disclosure provide an anti-D-dimer antibody or a functional fragment thereof, including HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 are included with SEQ ID NO: 15, 30, 31 and 32 any amino acid sequence consistent with HCDR1-HCDR3 of the heavy chain variable region shown in 32; said LCDR1-LCDR3 includes any light chain that can be shown in SEQ ID NO: 16, 33, 34, 35 and 36 The consistent amino acid sequence of LCDR1-LCDR3 in the variable region.
  • the HCDR1-HCDR3 are amino acid sequences consistent with the HCDR1-HCDR3 of any one of the heavy chain variable regions shown in SEQ ID NO: 15, 30, 31 and 32; the LCDR1-LCDR3 are An amino acid sequence consistent with LCDR1-LCDR3 of any one of the light chain variable regions shown in SEQ ID NO: 16, 33, 34, 35 and 36.
  • antibody is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.
  • CDRs complementarity determining regions
  • CDRs complementarity determining regions
  • CDRs refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain one or more, or even all, of the antibodies or The region of major amino acid residues responsible for the binding affinity of an antigen-binding fragment to the antigen or epitope it recognizes.
  • CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.
  • the heavy chain complementarity determining region is denoted by “HCDR”, which includes HCDR1, HCDR2 and HCDR3;
  • the light chain complementarity determining region is denoted by “LCDR”, which includes LCDR1, LCDR2 and LCDR3.
  • CDR labeling methods in the field include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions.
  • the system for defining the CDR is not particularly limited, and the CDR sequences defined by conventional systems in the art are within the protection scope of the present application.
  • the CDR definition method is referred to such as Kabat et al., U.S.Dept.of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) or Chothia et al., J Mol Biol 196:901-917 (1987).
  • Exemplary defined CDRs are listed in Table 1 below. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR.
  • Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody.
  • One of ordinary skill in the art can unambiguously map this Kabat numbering system to any variable region sequence, without reliance on any experimental data other than the sequence itself.
  • 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).
  • the polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system. However, those of ordinary skill in the art are fully able to convert the sequence numbers of the sequence listing into Kabat numbers.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.
  • the HCDR1, HCDR2, and HCDR3 sequentially comprise amino acid sequences at positions 31-35, 50-65, and 95-100 of the heavy chain variable region, or sequentially such as 31 amino acid sequences of the heavy chain variable region. ⁇ 35, 50 ⁇ 65, 95 ⁇ 100 amino acid sequences;
  • the LCDR1, LCDR2 and LCDR3 sequentially comprise amino acid sequences at positions 24-34, 50-56, and 89-95 of the light chain variable region, or sequentially such as 24-34 and 50-56 of the light chain variable region , 89-95 amino acid sequence.
  • the numbering of the amino acid positions is based on the Kabat numbering system.
  • the anti-D-dimer antibody or functional fragment thereof comprises the following CDRs:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of it;
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of it;
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of it;
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of it;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of it.
  • framework region includes heavy chain framework region and light chain framework region, and refers to the antibody heavy chain variable region and light chain variable region except CDR regions; where the heavy chain framework region can be further subdivided into contiguous regions separated by CDRs, including the HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into contiguous regions separated by CDRs Contiguous regions, including the framework regions of HFR1, HFR2, HFR3 and HFR4.
  • the heavy chain variable region is obtained by joining the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs The following combinations and permutations are obtained: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
  • the terms "subject” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
  • the antibody or its functional fragment also has the following framework regions:
  • HFR1 The amino acid sequence of HFR1 is as shown in SEQ ID NO: 7 or has at least 80% homology therewith;
  • amino acid sequence of HFR2 is as shown in SEQ ID NO: 8 or has at least 80% homology therewith;
  • amino acid sequence of HFR3 is shown in any of SEQ ID NO:9, 21, 22, 23, or has at least 80% homology therewith;
  • the HFR4 amino acid sequence is as shown in SEQ ID NO: 10 or has at least 80% homology therewith;
  • amino acid sequence of LFR1 is as shown in any of SEQ ID NO: 11, 24, 25, 26, or has at least 80% homology therewith;
  • amino acid sequence of LFR2 is as shown in SEQ ID NO: 12 or has at least 80% homology therewith;
  • amino acid sequence of LFR3 is as shown in any of SEQ ID NO: 13, 27, 28, 29, or has at least 80% homology therewith;
  • the LFR4 amino acid sequence is as set forth in SEQ ID NO: 14 or has at least 80% homology thereto.
  • each framework region of the anti-D-dimer antibody or its functional fragment provided by the present disclosure can be the same as the above-mentioned corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14) have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
  • the antibody or its functional fragment has a KD ⁇ 10 -7 M, KD ⁇ 10 -8 M, KD ⁇ 10 -9 M, KD ⁇ 10 -10 M or KD ⁇ 10 -11 binding affinity to D-dimer.
  • the antibody or functional fragment thereof binds D-dimer with an affinity of KD ⁇ 2.14 ⁇ 10 ⁇ 8 or KD ⁇ 9.90 ⁇ 10 ⁇ 9 or 8.67 ⁇ 10 ⁇ 10 .
  • the detection of KD is carried out with reference to the method in the embodiment of the present invention.
  • the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 15, 30, 31 and 32; the light chain variable region comprises SEQ ID NO: 16 , 33, 34, 35 and 36 amino acid sequences shown.
  • the amino acid sequence of the heavy chain variable region consists of any amino acid sequence shown in SEQ ID NO: 15, 30, 31 and 32; the light chain variable region consists of SEQ ID NO : Amino acid sequence composition shown in 16, 33, 34, 35 and 36.
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 16 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:33 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:35 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:36 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:16 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:33 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:35 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:36 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:16 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:33 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 35 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:36 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:16 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:33 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:35 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:36 .
  • the antibody further comprises a constant region;
  • the constant region includes a heavy chain constant region and/or a light chain constant region; as used herein, a "heavy chain constant region” can be represented as CH; a "light chain constant region” can be represented as CL .
  • the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the ⁇ -type or ⁇ -type light chain constant region.
  • the species sources of the heavy chain constant region and the light chain constant region are cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, Deer, mink, chicken, duck, goose, turkey, gamecock or human.
  • the species origin of the heavy chain constant region and the light chain constant region is mouse.
  • the heavy chain constant region is SEQ ID NO: 17 or an amino acid sequence having more than 80% homology with SEQ ID NO: 17;
  • the light chain constant region is SEQ ID NO: 18 or an amino acid sequence with SEQ ID NO: 17 NO:18 is an amino acid sequence with more than 80% homology.
  • the amino acid sequence of the constant region of the anti-D-dimer antibody or its functional fragment provided by the present disclosure may have the same constant region (SEQ ID NO: 17, 18) as above At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98% or 99% homology.
  • the above-mentioned functional fragment is selected from VHH, F(ab')2, Fab', Fab, Fv, Fd, scFv, single chain antibody and diabody or domain antibody of the antibody any of the
  • Functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they were derived.
  • the functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to split disulfide bonds.
  • enzymatic digestion including pepsin or papain
  • chemical reduction to split disulfide bonds On the basis of the structure of the intact antibody in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.
  • amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 19, 37, 38, 39; the amino acid sequence of the light chain is as shown in SEQ ID NO: 20, 40, 41, 42, 43 either shown.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:40.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 41.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:42.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 43.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:40.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:41.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:42.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:43.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:40.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:41.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:42.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:43.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:40.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:41.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:42.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:43.
  • Some embodiments of the present disclosure also provide an anti-D-dimer antibody or a functional fragment thereof, the anti-D-dimer antibody or a functional fragment thereof comprising a heavy chain variable region and/or a light A chain variable region, the heavy chain variable region comprising the above-mentioned HCDR1-3 and the above-mentioned HFR1-4, and the light chain variable region comprising the above-mentioned LCDR1-3 and the above-mentioned LFR1-4.
  • the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 15, 30, 31 and 32; the light chain variable region comprises SEQ ID NO: 16 , 33, 34, 35 and 36 amino acid sequences shown.
  • the amino acid sequence of the heavy chain variable region consists of any amino acid sequence shown in SEQ ID NO: 15, 30, 31 and 32; the light chain variable region consists of SEQ ID NO : Amino acid sequence composition shown in 16, 33, 34, 35 and 36.
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 16 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:33 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:35 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:15; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:36 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:16 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:33 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:35 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:30; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:36 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:16 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:33 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 35 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:31; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:36 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:16 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:33 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:35 .
  • the amino acid sequence of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:32; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:36 .
  • the antibody further comprises a constant region;
  • the constant region includes a heavy chain constant region and/or a light chain constant region; as used herein, a "heavy chain constant region” can be represented as CH; a "light chain constant region” can be represented as CL .
  • the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the ⁇ -type or ⁇ -type light chain constant region.
  • the species sources of the heavy chain constant region and the light chain constant region are cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, Deer, mink, chicken, duck, goose, turkey, gamecock or human.
  • the species origin of the heavy chain constant region and the light chain constant region is mouse.
  • the heavy chain constant region is SEQ ID NO: 17 or an amino acid sequence having more than 80% homology with SEQ ID NO: 17;
  • the light chain constant region is SEQ ID NO: 18 or an amino acid sequence with SEQ ID NO: 17 NO:18 is an amino acid sequence with more than 80% homology.
  • the amino acid sequence of the constant region of the anti-D-dimer antibody or its functional fragment provided by the present disclosure may have the same constant region (SEQ ID NO: 17, 18) as above At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98% or 99% homology.
  • the present invention provides a D-dimer antibody or a functional fragment thereof, including a heavy chain and/or a light chain, and the heavy chain includes the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region ;
  • the light chain includes the above-mentioned light chain variable region and the above-mentioned light chain constant region.
  • amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 19, 37, 38, 39; the amino acid sequence of the light chain is as shown in SEQ ID NO: 20, 40, 41, 42, 43 either shown.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:40.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 41.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:19; the amino acid sequence of the light chain is shown in SEQ ID NO:42.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 43.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:40.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:41.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:42.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:37; the amino acid sequence of the light chain is shown in SEQ ID NO:43.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:40.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:41.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:42.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:38; the amino acid sequence of the light chain is shown in SEQ ID NO:43.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:20.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:40.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:41.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:42.
  • amino acid sequence of the heavy chain is shown in SEQ ID NO:39; the amino acid sequence of the light chain is shown in SEQ ID NO:43.
  • Functional fragments of the above-mentioned antibodies can also be synthesized by recombinant genetics techniques also known to those skilled in the art or by, for example, automatic peptide synthesizers such as those sold by Applied BioSystems.
  • the present disclosure also relates to nucleic acids encoding the aforementioned antibodies or functional fragments thereof.
  • Nucleic acids are usually RNA or DNA, and nucleic acid molecules can be single- or double-stranded.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • DNA nucleic acid is used when it is ligated into a vector.
  • Some embodiments of the present disclosure also relate to vectors containing the above-mentioned nucleic acids.
  • Some embodiments of the present disclosure also relate to cells containing the nucleic acids or vectors described above.
  • Some embodiments of the present disclosure also relate to an antibody conjugate, comprising the above-mentioned antibody or a functional fragment thereof and a coupling moiety coupled thereto.
  • the coupling part includes a label selected from purification tags (such as His tags); detectable labels, such as colloidal gold, radioactive labels, luminescent substances, colored substances, enzymes, such as fluorescent labels, chromophore labels, electron-dense labels, such as Radioactive isotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, carbohydrate oxidation
  • the coupling moiety is selected from a solid phase carrier.
  • the solid support is selected from microspheres, plates or membranes.
  • the solid phase support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
  • the solid phase carrier is magnetic microspheres.
  • Some embodiments of the present disclosure also provide a reagent or kit comprising the above antibody or a functional fragment thereof or an antibody conjugate, wherein:
  • Some embodiments of the present disclosure also provide the use of the above-mentioned antibodies or functional fragments thereof or antibody conjugates in the preparation of reagents or kits.
  • the above reagent or kit is used for detecting D-dimer or diagnosing D-dimer related diseases.
  • Some embodiments of the present disclosure also provide uses of the above-mentioned antibodies or functional fragments thereof, antibody conjugates or reagents or kits for detecting D-dimer or diagnosing D-dimer-related diseases.
  • the D-dimer-related diseases include myocardial infarction, cerebral infarction, preeclampsia, deep vein thrombosis, main thoracic dissection, disseminated intravascular coagulation, pulmonary hypertension, liver injury, systemic Lupus, tumor, or tissue death.
  • Some embodiments of the present disclosure also provide a method for diagnosing whether a subject has a D-dimer-related disease, comprising:
  • the D-dimer-related diseases include myocardial infarction, cerebral infarction, preeclampsia, deep vein thrombosis, main thoracic dissection, disseminated intravascular coagulation, pulmonary hypertension, liver injury, systemic Lupus, tumor, or tissue death.
  • Some embodiments of the present disclosure also provide a method of detecting D-dimer in a test sample, comprising:
  • the D-dimer antigen in the test sample is contacted with the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit to form an immune complex ;
  • the immune complex further includes a second antibody, and the second antibody binds to the above antibody or a functional fragment thereof.
  • restriction enzymes and rTaq DNA polymerase were purchased from Takara Company.
  • MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
  • BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
  • the pMD-18T vector was purchased from Takara Company.
  • Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
  • the disclosure provides an anti-D-dimer antibody, a reagent for detecting D-dimer and a kit.
  • the antibody can specifically bind to D-dimer and has a high affinity for it.
  • the antibody can be used to detect D-dimer.
  • - Dimers have better sensitivity or specificity.
  • the present disclosure provides a richer selection of antibodies for the detection of D-dimer.
  • the inventor obtained mRNA from hybridoma cell lines secreting anti-D-dimer 14D2RMb-1 monoclonal antibody through hybridoma preparation technology, and obtained DNA product by RT-PCR method, which was added with rTaq DNA polymerase After the reaction, insert it into the pMD-18T vector and transform it into DH5 ⁇ competent cells. After the colonies grow out, take 4 clones each of the Heavy Chain (heavy chain) and Light Chain (light chain) genes and send them to a gene sequencing company for sequencing.
  • VL gene sequence is 339bp, and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 363bp, which belongs to the VH1 gene family, and there is a 57bp leader peptide sequence in front of it.
  • pcDNA TM 3.4 Vector is a recombinant antibody eukaryotic expression vector constructed.
  • the expression vector has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and named it pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector in the future; according to the above pMD-18T
  • the VL and VH gene-specific primers of the 14D2RMb-1 antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, and a 0.74KB gene was amplified by PCR amplification. Light Chain gene fragment and 1.43kb Heavy Chain gene fragment.
  • the Heavy Chain and Light Chain gene fragments were respectively digested with HindIII/EcoRI double enzymes, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. 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 Recombinant expression plasmids for Heavy Chain and Light Chain.
  • Dilute D-dimer antigen (purchased from Fapon) to 2 ⁇ g/mL in coating solution (main component NaHCO 3 ), 100 ⁇ L per well, overnight at 4°C; the next day, wash solution (main component Na 2 HPO 4 +Nacl) Wash twice, pat dry; add blocking solution (20% BSA+80% PBS), 120 ⁇ L per well, 37°C, 1h, pat dry; add diluted cell supernatant, 100 ⁇ L/well, 37°C, 30min (part supernatant for 1 h); washed with washing solution for 5 times, and patted dry; added goat anti-mouse IgG-HRP, 100 ⁇ L per well, 37°C, 30 min; washed with washing solution for 5 times, patted dry; added chromogenic solution A (50 ⁇ L/well , the main component is citric acid + sodium acetate + acetanilide + carbamide peroxide), add chromogenic solution B (50 ⁇ L/well, the main component
  • Step 2 Dilute the plasmid prepared in step (2) to 40 ⁇ g/100 ⁇ L with ultrapure water, adjust the CHO cells to 1.43 ⁇ 10 7 cells/mL in a centrifuge tube, mix 100 ⁇ L of the above plasmid with 700 ⁇ L of cells, transfer to the electroporation cup, electroporation , count the next day; 25 ⁇ mol/L MSX 96-well pressurized culture for about 25 days.
  • the cells were first cultured in a 125mL shake flask with an inoculation volume of 30mL, the medium was 100% Dynamis medium, and placed in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and 8% carbon dioxide. Cultivate for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/mL. The expansion volume is calculated according to production requirements, and the medium is 100% Dynamis medium. Then every 72h expansion once. When the cell volume meets the production requirements, strictly control the inoculation density to about 500,000 cells/mL for production.
  • Shake flask parameters rotational speed 120r/min, temperature 37°C, carbon dioxide 8%.
  • Fed-batch feeding Start feeding daily after culturing in the shake flask for 72 hours.
  • HyClone TM Cell Boost TM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day , has been supplemented until the 12th day (feeding on the 12th day).
  • Glucose was supplemented with 3g/L on the sixth day.
  • Affinity purification was performed with a proteinA affinity chromatography column. The antibody was diluted to 1mg/mL for reducing SDS-PAGE, and the electropherogram is shown in Figure 1. Two bands were shown after reducing SDS-PAGE, one with a Mr of 50KD (heavy chain) and the other with a Mr of 28KD (light chain).
  • the heavy chain CDR1-3 is shown in SEQ ID NO:1-3
  • the light chain CDR1-3 is shown in SEQ ID NO:4-6, respectively.
  • the heavy chain variable region and the light chain variable region are shown in SEQ ID NO: 15, 16 in sequence, and the heavy chain and light chain are shown in SEQ ID NO: 19, 20 in turn.
  • the purified antibody was diluted to 20 ⁇ g/mL with PBST, and the D-dimer antigen was serially diluted with PBST;
  • KD means the equilibrium dissociation constant, that is, affinity
  • Kon means the association rate constant
  • Kd means the dissociation rate constant
  • Dilute D-dimer antigen (purchased from Fapon) to 2 ⁇ g/mL in coating solution (main component NaHCO 3 ), 100 ⁇ L per well, overnight at 4°C; the next day, wash solution (main component Na 2 HPO 4 +NaCl) Wash twice, pat dry; add blocking solution (20%BSA+80%PBS), 120 ⁇ L per well, 37°C, 1h, pat dry; add diluted purified antibody and control antibody, 100 ⁇ L/well, 37°C, 30min ; Wash 5 times with washing solution and pat dry; add goat anti-mouse IgG-HRP, 100 ⁇ L per well, 37°C, 30 min; wash 5 times with washing solution, pat dry; add chromogenic solution A (50 ⁇ L/well), add Color solution B (50 ⁇ L/well), 10 min; add stop solution, 50 ⁇ L/well; read OD value at 450 nm (refer to 630 nm) on a microplate reader.
  • coating solution main component NaHCO 3
  • Embodiment 4 performance evaluation
  • the 14D2RMb1 to 14D2RMb11 recombinant antibody was used as a labeled antibody, and another anti-D-dimer monoclonal antibody was used as a coating antibody.
  • the clinical relevance was determined by the reaction mode of double antibody sandwich chemiluminescence. The results showed that the detection sensitivity of 14D2RMb1 to 14D2RMb11 and Clinical sample correlation is better than control antibody. The specific results are shown in Table 6.
  • the anti-D-dimer antibody provided by the present disclosure has better affinity to D-dimer, and the detection of D-dimer by using the antibody has better sensitivity or specificity.
  • the present disclosure provides a more excellent antibody selection for the detection of D-dimer, therefore, the anti-D-dimer antibody provided by the present disclosure, the reagents and kits for detecting D-dimer all have excellent practicality. performance and broad market application prospects.

Abstract

本公开涉及一种抗D-二聚体的抗体及其制备方法和用途。本公开制备的抗D-二聚体的单克隆抗体对D-二聚体具有高亲和性、高反应活性、高灵敏度和特异性，为D-二聚体的检测提供了重要的原料来源。

Description

一种抗D-二聚体的抗体及其制备方法和用途
相关申请的交叉引用
本公开请求2021年11月20日向中国国家知识产权局提交的、专利申请号为202111410612.4的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本公开属于抗体技术领域。更具体地，涉及一种抗D-二聚体的抗体及其制备方法和用途。
背景技术
D-二聚体(D-Dimer，DD)是血液中的纤维蛋白，经过活化和水解，产生的一种特异的，最简单的降解产物，是特异性的纤溶过程标记物。在病理状态下，交联纤维蛋白在溶解过程中，释放出X’、Y’、D’、E’等碎片，并形成DD、DD/E、YD/YD、YY/DD等复合物。这些碎片进一步降解为最小的片段DD和DD/E复合物，DD分子量约62000D，在体内半衰期>3h，主要经肾脏排泄。
D-二聚体水平的升高反映了体内存在着凝血及纤溶活性增强，是急性血栓形成的一个敏感的标记物。心肌梗死、脑梗死、肺栓塞、静脉血栓形成、手术、肿瘤、弥漫性血管内凝血、感染及组织坏死等均可导致D-二聚体升高。
近年来，应用单克隆抗体和蛋白质连接技术推出了血栓导向溶栓剂，基本原理是利用抗原〔D—二聚体〕和抗体〔抗D—二聚体单抗)之间的亲和力，将抗血栓特异性成分的单抗与溶栓药物相连接，形成抗体－溶栓剂复合体。其中的单抗如同导弹一样，可携带溶栓剂特异地与血栓结合，使得血栓部位的溶栓剂高度聚集，从而增强对血栓的溶解作用。后来又发现将抗D—二聚体单抗标记放射性核素，在抗体与抗原特异性结合过程中，可将放射性核素携带到血栓局部，再用放射性核素检测仪监测体内放射性核素的分布，从而达到利用导向示踪剂定位诊断血栓的目的。
D-二聚体的检测方法有三P试验、胶乳凝集法(LATEX)、ELISA法、免疫渗滤胶体金显色反应法等。目前用的这些检测方法都是建立在特异单抗的基础上的，也就是说针对D-二聚体的单抗制备成为提高检测灵敏度和特异性的关键。
发明内容
本公开提供了一种抗D-二聚体的抗体或其功能性片段，包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1～HCDR3包括或为与SEQ ID NO:15、30、31和32任一所示重链可变区的HCDR1～HCDR3一致的氨基酸序列；所述LCDR1～LCDR3包括或为与SEQ ID NO:16、33、34、35和36任一所示轻链可变区的LCDR1～LCDR3一致的氨基酸序列。
可选地，所述CDRs由Kabat、Chothia、IMGT、AbM或Contact系统定义。
本公开提供了一种抗D-二聚体的抗体或其功能性片段，所述抗D-二聚体的抗体或其功能性片段含有以下CDRs：
HCDR1，其包含SEQ ID NO:1所示的氨基酸序列，或由其组成；
HCDR2，其包含SEQ ID NO:2所示的氨基酸序列，或由其组成；
HCDR3，其包含SEQ ID NO:3所示的氨基酸序列，或由其组成；
LCDR1，其包含SEQ ID NO:4所示的氨基酸序列，或由其组成；
LCDR2，其包含SEQ ID NO:5所示的氨基酸序列，或由其组成；
LCDR3，其包含SEQ ID NO:6所示的氨基酸序列，或由其组成。
可选地，所述的抗体或其功能性片段所述抗体或其功能性片段还具有以下的骨架区：HFR1氨基酸序列如SEQ ID NO:7所示或与其具有至少80％同源性；HFR2氨基酸序列如SEQ ID NO:8所示或与其具有至少80％同源性；HFR3氨基酸序列如SEQ ID NO:9、21、22、23任一所示，或与其具有至少80％同源性；HFR4氨基酸序列如SEQ ID NO:10所示或与其具有至少80％同源性；LFR1氨基酸序列如SEQ ID NO:11、24、25、26任一所示，或与其具有至少80％同源性；LFR2氨基酸序列如SEQ ID NO:12所示或与其具有至少80％同源性；LFR3氨基酸序列如SEQ ID NO:13、27、28、29任一所示，或与其具有至少80％同源性；LFR4氨基酸序列如SEQ ID NO:14所示或与其具有至少80％同源性。
本公开提供了一种抗D-二聚体的抗体或其功能性片段，所述抗D-二聚体的抗体或其功能性片段包含重链可变区和/或轻链可变区，所述重链可变区包含上述的HCDR1～3和上述的HFR1～4，所述轻链可变区包含上述的LCDR1～3和上述的LFR1～4。
可选地，所述重链可变区包含选自SEQ ID NO:15、30、31和32任一所示的氨基酸序列，或由其组成；所述轻链可变区包含SEQ ID NO:16、33、34、35和36任一所示的氨基酸序列，或由其组成。
可选地，所述的抗D-二聚体的抗体或其功能性片段，还包含恒定区。
可选地，所述恒定区包括重链恒定区和/或轻链恒定区。
可选地，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区。所述轻链恒定区选自κ型或λ型轻链恒定区。
可选地，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、 骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
可选地，所述恒定区的种属来源为小鼠。
可选地，所述重链恒定区为SEQ ID NO:17或与SEQ ID NO:17具有80％以上同源性的氨基酸序列；所述轻链恒定区为SEQ ID NO:18或与SEQ ID NO:18具有80％以上同源性的氨基酸序列。
可选地，所述功能性片段选自所述抗体的F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
本公开还提供了一种抗D-二聚体的抗体或其功能性片段，包括重链和/或轻链，所述重链包括上述的重链可变区和上述的重链恒定区；所述轻链包括上述的轻链可变区和上述的轻链恒定区。
可选地，所述重链的氨基酸序列如SEQ ID NO:19、37、38、39任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、40、41、42、43任一所示。
本公开还提供了一种核酸，所述核酸编码上文任一项所述的抗体或其功能性片段。
本公开还提供了一种细胞，所述细胞包含上述的核酸。
本公开还提供了一种制备上文任一项所述的抗体或其功能性片段的方法，所述方法包括培养上述的细胞。
本公开还提供了一种抗体偶联物，所述抗体偶联物包含上文任一项所述的抗体或其功能性片段以及与其偶联的偶联部分。
在可选的实施方式中，所述偶联部分选自纯化标签或可检测的标记，例如胶体金、放射性标记、发光物质、有色物质、酶，例如荧光标记、发色团标记、电子致密标记，例如放射性同位素、荧光团、罗丹明及其衍生物、荧光素酶、荧光素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记的一种或多种。
在可选的实施方式中，所述偶联部分选自磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。
本公开还提供了一种试剂或试剂盒，所述试剂或试剂盒包含上文任一项所述的抗体或其功能性片段或上述的抗体偶联物。
本公开还提供了上文任一项所述的抗体或其功能性片段或所述的抗体偶联物在制备试剂或试剂盒中的用途。
在可选的实施方式中，所述试剂或试剂盒用于检测D-二聚体或诊断D-二聚体相关疾病。
本公开还提供了上文任一项所述的抗体或其功能性片段或所述的抗体偶联物或所述的试剂或试剂盒用于检测D-二聚体或诊断D-二聚体相关疾病中的用途。
在可选的实施方式中，所述D-二聚体相关疾病包括心梗、脑梗、先兆子痫、深静脉血栓、胸主夹层、弥散性血管内凝血、肺动脉高压、肝损伤、系统性红斑狼疮、肿瘤或组织坏死。
本公开还提供了一种诊断受试者是否患有D-二聚体相关疾病中的方法，包括：
A)在足以发生结合反应的条件下，使上文任一项所述的抗体或其功能性片段，或者所述的抗 体偶联物，或者所述的试剂或试剂盒与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
在可选的实施方式中，所述D-二聚体相关疾病包括心梗、脑梗、先兆子痫、深静脉血栓、胸主夹层、弥散性血管内凝血、肺动脉高压、肝损伤、系统性红斑狼疮、肿瘤或组织坏死。
本公开还提供了一种检测测试样品中的D-二聚体的方法，其包括：
a)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的D-二聚体抗原与上文任一项所述的抗体或其功能性片段，或者所述的抗体偶联物，或者所述的试剂或试剂盒接触以形成免疫复合物；和
b)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述抗原的存在；
可选的，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述抗D-二聚体的抗体或其功能性片段结合。
附图说明
图1是14D2RMb1至14D2RMb11抗体的还原性SDS-PAGE结果(从左至右)。
具体实施方式
本公开的一些实施方式提供了一种抗D-二聚体的抗体或其功能性片段，包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1～HCDR3包括与SEQ ID NO:15、30、31和32任一所示重链可变区的HCDR1～HCDR3一致的氨基酸序列；所述LCDR1～LCDR3包括与SEQ ID NO:16、33、34、35和36任一所示轻链可变区的LCDR1～LCDR3一致的氨基酸序列。
在可选的实施方式中，所述HCDR1～HCDR3为与SEQ ID NO:15、30、31和32任一所示重链可变区的HCDR1～HCDR3一致的氨基酸序列；所述LCDR1～LCDR3为与SEQ ID NO:16、33、34、35和36任一所示轻链可变区的LCDR1～LCDR3一致的氨基酸序列。
如本文所用，术语“抗体”在最广义上使用，其可以包括全长单克隆抗体，双特异性或多特异性抗体，以及嵌合抗体，只要它们展示所需的生物学活性。
如本文所用，术语“互补性决定区”、“CDR”或“CDRs”是指免疫球蛋白的重链和轻链的高度可变区，指包含一种或多种或者甚至全部的对抗体或抗原结合片段与其识别的抗原或表位的结合亲和力起作用的主要氨基酸残基的区域。在本公开具体实施方式中，CDRs是指所述抗体的重链和轻链的高度可变区。
如本文所用，重链互补决定区用“HCDR”表示，其包括HCDR1、HCDR2和HCDR3；轻链互补决定区用“LCDR”表示，其包括LCDR1、LCDR2和LCDR3。本领域常用的CDR标示方法包括： Kabat编号方案、IMGT编号方案、Chothia和Lesk编号方案以及1997年Lefranc等人为免疫球蛋白超家族的所有蛋白质序列引入的新的标准化编号系统。Kabat等人是第一个为免疫球蛋白可变区提出标准化编号方案的人。在过去的几十年中，序列的积累导致了KABATMAN数据库的创建，Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。本公开采用Kabat注释标准标示CDR区，但其他方法标示的CDR区也属于本公开的保护范围。
对所述CDR进行定义的系统不受特别限制，本领域常规系统定义的CDR序列均在本申请的保护范围之内。例如CDR定义方法参见如Kabat等，U.S.Dept.of Health and Human Services,Sequences of Proteins of Immunological Interest(1983)或Chothia等，J Mol Biol 196：901-917(1987)。示例性的定义的CDR列于下表1中。在给定抗体的可变区氨基酸序列的情况下，本领域技术人员可以常规地确定哪些残基包含特定CDR。
表1：CDR定义 1
CDR Kabat AbM 2 IMGT
HCDR1 31-35 26-35 26-35
HCDR2 50-65 50-58 51-56
HCDR3 95-102 95-102 93-102
LCDR1 24-34 24-34 27-32
LCDR2 50-56 50-56 50-51
LCDR3 89-97 89-97 89-97
1表1中所有CDR定义的编号是依据Kabat编号系统(参见下文)。
2如表1中使用的“AbM”具有小写“b”，是指通过Oxford Molecular的“AbM”抗体建模软件定义的CDR。
Kabat等还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以明确地将该Kabat编号系统对应到任何可变区序列，而不依赖于序列本身之外的任何实验数据。如本文所述，“Kabat编号”是指Kabat等，U.S.Dept.of Health and HumanServices，“Sequence of Proteins of Immunological Interest”(1983)所述的编号系统。序列表中的多肽序列未根据Kabat编号系统编号。然而，本领域普通技术人员完全能够将序列表的序列编号转换为Kabat编号。
在可选的实施方式中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3(简称CDRs)由Kabat、Chothia、IMGT、AbM或Contact系统定义。
在可选的实施方式中，所述HCDR1、HCDR2和HCDR3依次包含重链可变区的31～35位、50～65位、95～100位氨基酸序列，或依次如重链可变区的31～35位、50～65位、95～100位氨基酸序列所示；
所述LCDR1、LCDR2和LCDR3依次包含轻链可变区的24～34位、50～56位、89～95位氨基酸序列，或依次如轻链可变区的24～34位、50～56位、89～95位氨基酸序列所示。
且，所述氨基酸位点的编号是依据Kabat编号系统。
在可选的实施方式中，，所述抗D-二聚体的抗体或其功能性片段包含以下CDRs：
HCDR1，其包含SEQ ID NO:1所示的氨基酸序列，或由其组成；
HCDR2，其包含SEQ ID NO:2所示的氨基酸序列，或由其组成；
HCDR3，其包含SEQ ID NO:3所示的氨基酸序列，或由其组成；
LCDR1，其包含SEQ ID NO:4所示的氨基酸序列，或由其组成；
LCDR2，其包含SEQ ID NO:5所示的氨基酸序列，或由其组成；
LCDR3，其包含SEQ ID NO:6所示的氨基酸序列，或由其组成。
在本发明中，“框架区”、“骨架区”或“FR”区包括重链骨架区和轻链骨架区，是指抗体重链可变区和轻链可变区中除CDR之外的区域；其中，重链骨架区可以被进一步细分成被CDR分隔开的毗邻区域，包含HFR1、HFR2、HFR3和HFR4骨架区；轻链骨架区可以被进一步细分成被CDR分隔开的毗邻区域，包含HFR1、HFR2、HFR3和HFR4骨架区。
如本文所用，重链可变区由以下编号的CDR与FR按如下组合排列连接获得：HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4；轻链可变区由以下编号的CDR与FR按如下组合排列连接获得：LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4。
如本文所用，术语“受试者”和“患者”在本文可以互换使用，是指脊椎动物，优选地是哺乳动物，最优选地是人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。
在可选的实施方式中，所述抗体还或其功能性片段还具有以下的骨架区：
HFR1氨基酸序列如SEQ ID NO:7所示或与其具有至少80％同源性；
HFR2氨基酸序列如SEQ ID NO:8所示或与其具有至少80％同源性；
HFR3氨基酸序列如SEQ ID NO:9、21、22、23任一所示，或与其具有至少80％同源性；
HFR4氨基酸序列如SEQ ID NO:10所示或与其具有至少80％同源性；
LFR1氨基酸序列如SEQ ID NO:11、24、25、26任一所示，或与其具有至少80％同源性；
LFR2氨基酸序列如SEQ ID NO:12所示或与其具有至少80％同源性；
LFR3氨基酸序列如SEQ ID NO:13、27、28、29任一所示，或与其具有至少80％同源性；
LFR4氨基酸序列如SEQ ID NO:14所示或与其具有至少80％同源性。
需要说明的是，在其他的实施方式中，本公开提供的抗D-二聚体的抗体或其功能性片段的各骨架区氨基酸序列可以与上述对应骨架区(SEQ ID NO:7、8、9、10、11、12、13或14)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、 97％、98％或99％的同源性。在可选的实施方式中，所述抗体或其功能性片段以KD≤10 -7M、KD≤10 -8M、KD≤10 -9M、KD≤10 -10M或KD≤10 -11的亲和力结合D-二聚体。
在可选的实施方式中，所述抗体或其功能性片段以KD≤2.14×10 -8或KD≤9.90×10 -9或8.67×10 -10的亲和力结合D-二聚体。
KD的检测参考本发明实施例中的方法进行。
在可选的实施方式中，所述重链可变区包含选自SEQ ID NO:15、30、31和32任一所示的氨基酸序列；所述轻链可变区包含SEQ ID NO:16、33、34、35和36所示的氨基酸序列。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15、30、31和32任一所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:16、33、34、35和36所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:16所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:33所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:34所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:35所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:36所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:16所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:33所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:34所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:35所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:36所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:16所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:33所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成； 所述轻链可变区由SEQ ID NO:34所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:35所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:36所示的氨基酸序列组成。在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:16所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:33所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:34所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:35所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:36所示的氨基酸序列组成。在可选的实施方式中，所述抗体还包含恒定区；
在可选的实施方式中，所述恒定区包括重链恒定区和/或轻链恒定区；如本文所用，“重链恒定区”可以表示为CH；“轻链恒定区”可以表示为CL。
在可选的实施方式中，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区；轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中，重链恒定区和轻链恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在可选的实施方式中，重链恒定区和轻链恒定区的种属来源为小鼠。
在可选的实施方式中，重链恒定区为SEQ ID NO:17或与SEQ ID NO:17具有80％以上同源性的氨基酸序列；轻链恒定区为SEQ ID NO:18或与SEQ ID NO:18具有80％以上同源性的氨基酸序列。
需要说明的是，在其他的实施方式中，本公开提供的抗D-二聚体的抗体或其功能性片段的恒定区氨基酸序列可以与上述对应恒定区(SEQ ID NO:17、18)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。
在可选的实施方式中，上述功能性片段选自所述抗体的VHH、F(ab’)2、Fab’、Fab、Fv、Fd、scFv、单链抗体和双价抗体或结构域抗体中的任意一种。
上述抗体的功能性片段通常具有与其来源抗体相同的结合特异性。本领域技术人员根据本公开记载的内容容易理解到，上述抗体的功能性片段可以通过比如酶消化的方法(包括胃蛋白酶或木瓜 蛋白酶)和/或通过化学还原分裂二硫键的方法获得。在本公开中完整抗体的结构基础上，本领域技术人员容易获得上述的功能性片段。
上述抗体的功能性片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪，比如Applied BioSystems等销售的自动肽合成仪合成获得。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19、37、38、39任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、40、41、42、43任一所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:40所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:41所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:42所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:43所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如SEQ ID NO:40所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如SEQ ID NO:41所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如SEQ ID NO:42所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如SEQ ID NO:43所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如SEQ ID NO:40所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如SEQ ID NO:41所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如 SEQ ID NO:42所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如SEQ ID NO:43所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:40所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:41所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:42所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:43所示。
本公开的一些实施方式还提供了一种抗D-二聚体的抗体或其功能性片段，所述抗D-二聚体的抗体或其功能性片段包含重链可变区和/或轻链可变区，所述重链可变区包含上述的HCDR1～3和上述的HFR1～4，所述轻链可变区包含上述的LCDR1～3和上述的LFR1～4。
在可选的实施方式中，所述重链可变区包含选自SEQ ID NO:15、30、31和32任一所示的氨基酸序列；所述轻链可变区包含SEQ ID NO:16、33、34、35和36所示的氨基酸序列。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15、30、31和32任一所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:16、33、34、35和36所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:16所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:33所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:34所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:35所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:15所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:36所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成； 所述轻链可变区由SEQ ID NO:16所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:33所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:34所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:35所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:30所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:36所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:16所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:33所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:34所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:35所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:31所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:36所示的氨基酸序列组成。在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:16所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:33所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:34所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:35所示的氨基酸序列组成。
在可选的实施方式中，所述重链可变区的氨基酸序列由SEQ ID NO:32所示的氨基酸序列组成；所述轻链可变区由SEQ ID NO:36所示的氨基酸序列组成。在可选的实施方式中，所述抗体还包含恒 定区；
在可选的实施方式中，所述恒定区包括重链恒定区和/或轻链恒定区；如本文所用，“重链恒定区”可以表示为CH；“轻链恒定区”可以表示为CL。
在可选的实施方式中，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区；轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中，重链恒定区和轻链恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在可选的实施方式中，重链恒定区和轻链恒定区的种属来源为小鼠。
在可选的实施方式中，重链恒定区为SEQ ID NO:17或与SEQ ID NO:17具有80％以上同源性的氨基酸序列；轻链恒定区为SEQ ID NO:18或与SEQ ID NO:18具有80％以上同源性的氨基酸序列。
需要说明的是，在其他的实施方式中，本公开提供的抗D-二聚体的抗体或其功能性片段的恒定区氨基酸序列可以与上述对应恒定区(SEQ ID NO:17、18)具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。
另一方面，本发明提供一种D-二聚体的抗体或其功能性片段，包括重链和/或轻链，所述重链包括上述的重链可变区和上述的重链恒定区；所述轻链包括上述的轻链可变区和上述的轻链恒定区。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19、37、38、39任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、40、41、42、43任一所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:40所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:41所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:42所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:19所示；所述轻链的氨基酸序列如SEQ ID NO:43所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如SEQ ID NO:40所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如 SEQ ID NO:41所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如SEQ ID NO:42所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:37所示；所述轻链的氨基酸序列如SEQ ID NO:43所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如SEQ ID NO:40所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如SEQ ID NO:41所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如SEQ ID NO:42所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:38所示；所述轻链的氨基酸序列如SEQ ID NO:43所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:40所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:41所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:42所示。
在可选的实施方式中，所述重链的氨基酸序列如SEQ ID NO:39所示；所述轻链的氨基酸序列如SEQ ID NO:43所示。上述抗体的功能性片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪，比如Applied BioSystems等销售的自动肽合成仪合成获得。
本公开还涉及核酸，核酸编码上述抗体或其功能性片段。
核酸通常是RNA或DNA，核酸分子可以是单链或双链的。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至上述编码序列。当其连入载体时采用DNA核酸。
本公开的一些实施方式还涉及载体，载体含有上述核酸。
本公开的一些实施方式还涉及细胞，细胞含有上述核酸或载体。
本公开的一些实施方式还涉及一种抗体偶联物，包含上述抗体或其功能性片段以及与其偶联的偶联部分。
所述偶联部分包括选自纯化标签(如His标签)；可检测的标记，例如胶体金、放射性标记、发光物质、有色物质、酶，例如荧光标记、发色团标记、电子致密标记，例如放射性同位素、荧光团、罗丹明及其衍生物、荧光素酶、荧光素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记、药物(如血栓导向溶栓剂)中的一种或多种。
在可选的实施方式中，所述偶联部分选自固相载体。
在可选的实施方式中，所述固相载体选自微球、板或膜。
在可选的实施方式中，所述固相载体包括但不限于磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。
在可选的实施方式中，所述固相载体为磁性微球。
本公开的一些实施方式还提供一种试剂或试剂盒，其包含上述抗体或其功能性片段或抗体偶联物，其中：
本公开的一些实施方式还提供了上述抗体或其功能性片段或抗体偶联物在制备试剂或试剂盒中的用途。
在可选的实施方式中，上述试剂或试剂盒用于检测D-二聚体或诊断D-二聚体相关疾病。
本公开的一些实施方式还提供了上述抗体或其功能性片段、抗体偶联物或试剂或试剂盒用于检测D-二聚体或诊断D-二聚体相关疾病中的用途。
在可选的实施方式中，所述D-二聚体相关疾病包括心梗、脑梗、先兆子痫、深静脉血栓、胸主夹层、弥散性血管内凝血、肺动脉高压、肝损伤、系统性红斑狼疮、肿瘤或组织坏死。
本公开的一些实施方式还提供了一种诊断受试者是否患有D-二聚体相关疾病的方法，包括：
A)在足以发生结合反应的条件下，使上述的抗体或其功能性片段、抗体偶联物、试剂或试剂盒与来自受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
在可选的实施方式中，所述D-二聚体相关疾病包括心梗、脑梗、先兆子痫、深静脉血栓、胸主夹层、弥散性血管内凝血、肺动脉高压、肝损伤、系统性红斑狼疮、肿瘤或组织坏死。
本公开的一些实施方式还提供了一种检测测试样品中的D-二聚体的方法，其包括：
a)在足以发生抗体/抗原结合反应的条件下，使测试样品中的D-二聚体抗原与上述的抗体或其功 能性片段、抗体偶联物、试剂或试剂盒接触以形成免疫复合物；和
b)检测免疫复合物的存在，复合物的存在指示上述测试样品中上述抗原的存在。
可选的，在步骤a)中，免疫复合物中还包括第二抗体，第二抗体与上述抗体或其功能性片段结合。
为使本发明公开实施例实施方式的目的、技术方案和优点更加清楚，下面将对本发明公开实施例实施方式中的技术方案进行清楚、完整地描述。实施例实施方式中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明，否则实践本发明公开将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(MolecμLar Cloning:A Laboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编，1984)；《动物细胞培养(Animal Cell CμLture)》(R.I.Freshney编，1987)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《哺乳动物细胞用基因转移载体(Gene Transfer Vectors for Mammalian Cells)》(J.M.Miller和M.P.Calos编，1987)；《当代分子生物学方法(Current Protocols in MolecμLar Biology)》(F.M.Ausubel等人编，1987)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(MμLlis等人编，1994)；以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编，2011)，所述文献中的每个文献均通过引用明确并入本文中。
除非特别说明，以下实施例所用试剂和材料均为市购。
以下实施例中，限制性内切酶、rTaq DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMART TMRACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。
本公开提供了抗D-二聚体的抗体、检测D-二聚体的试剂和试剂盒，该抗体可以特异性结合D- 二聚体，对其具有较高的亲和力，用该抗体检测D-二聚体具有较好的灵敏度或特异性。本公开为D-二聚体的检测提供了更为丰富的抗体选择。
实施例
以下结合实施例对本发明公开的特征和性能作进一步的详细描述。
实施例1 14D2RMb-1抗体的制备
1、表达质粒构建
(1)14D2RMb-1抗体基因制备
发明人前期通过杂交瘤制备技术获得分泌抗D-二聚体14D2RMb-1单克隆抗体的杂交瘤细胞株中提取mRNA，通过RT-PCR方法获得DNA产物，该产物用rTaq DNA聚合酶进行加A反应后插入到pMD-18T载体中，转化到DH5α感受态细胞中，长出菌落后分别取Heavy Chain(重链)及Light Chain(轻链)基因克隆各4个克隆送基因测序公司进行测序。
(2)14D2RMb-1抗体可变区基因的序列分析
将上述测序得到的基因序列放在Kabat抗体数据库中进行分析，并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的，其中Light Chain扩增出的基因片段中，VL基因序列为339bp，其前方有57bp的前导肽序列；Heavy Chain引物对扩增出的基因片段中，VH基因序列为363bp，属于VH1基因家族，其前方有57bp的前导肽序列。
(3)重组抗体表达质粒的构建
pcDNA TM3.4
Figure PCTCN2022132982-appb-000001
vector为构建的重组抗体真核表达载体，该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点，并命名为pcDNA3.4A表达载体，后续简称3.4A表达载体；根据上述pMD-18T中抗体可变区基因测序结果，设计14D2RMb-1抗体的VL和VH基因特异性引物，两端分别带有HindIII、EcoRI酶切位点和保护碱基，通过PCR扩增方法扩出0.74KB的Light Chain基因片段和1.43kb的Heavy Chain基因片段。Heavy Chain和Light Chain基因片段分别采用HindIII/EcoRI双酶切，3.4A载体采用HindIII/EcoRI双酶切，将片段和载体纯化回收后Heavy Chain基因和Light Chain基因分别连接3.4A表达载体中，得到Heavy Chain和Light Chain的重组表达质粒。
2、稳定细胞株筛选
(1)重组抗体表达质粒瞬时转染CHO细胞，确定表达质粒活性
将步骤1-(3)步骤制备得到的质粒用超纯水稀释至40μg/100μL，调节CHO细胞1.43×10 7cells/mL于离心管中，100μL上述质粒与700μL细胞混合，转入电转杯，电转，第3、5、7天取样计数，第 7天收样检测。
包被液(主要成分NaHCO 3)稀释D-二聚体抗原(购自菲鹏)至2μg/mL，每孔100μL，4℃过夜；次日，洗涤液(主要成份Na 2HPO 4+Nacl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，37℃，1h，拍干；加入稀释后的细胞上清，100μL/孔，37℃，30min(部分上清1h)；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100μL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔，主要成份柠檬酸+醋酸钠+乙酰苯胺+过氧化脲)，加入显色液B液(50μL/孔，主要成份柠檬酸+EDTA·2Na+TMB+浓HCL)，10min；加入终止液(50μL/孔，EDTA·2Na+浓H 2SO 4)；酶标仪上450nm(参考630nm)处读OD值。
结果显示细胞上清稀释1000倍后反应OD仍大于1.0，未加细胞上清孔反应OD小于0.1，表明质粒瞬转后产生的抗体对D-二聚体抗原有活性。
(2)重组抗体表达质粒线性化
准备下述试剂：Buffer 50μL、步骤1-(3)步骤制备得到的质粒100μg/管、PvuⅠ酶10μL、无菌水补至500μL，37℃水浴酶切过夜；先用等体积酚/氯仿/异戊醇(下层)25:24:1，再用氯仿(水相)依次进行抽提；0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀，70％乙醇漂洗沉淀，去除有机溶剂，待乙醇挥发完全用适量的灭菌水进行复融，最后进行浓度的测定。
(3)重组抗体表达质粒稳定转染，加压筛选稳定细胞株
步骤2-(2)步骤制备得到的质粒用超纯水稀释至40μg/100μL，调节CHO细胞1.43×10 7cells/mL于离心管中，100μL上述质粒与700μL细胞混合，转入电转杯，电转，次日计数；25μmol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔，并记录汇合度；取培养上清，送样检测；挑选抗体浓度、相对浓度高的细胞株转24孔，3天左右转6孔；3天后保种批培，调整细胞密度0.5×10 6cells/mL，2.2mL进行批培养，细胞密度0.3×10 6cells/mL，2mL进行保种；7天6孔批培上清送样检测，挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
3、重组抗体生产
(1)细胞扩培
细胞复苏之后先在125mL规格的摇瓶中培养，接种体积为30mL，培养基为100％Dynamis培养基，放置于转速120r/min，温度为37℃，二氧化碳为8％的摇床中。培养72h，以50万cells/mL接种密度接种扩培，扩培体积根据生产需求进行计算，培养基为100％Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时，严格控制接种密度为50万cells/mL左右进行生产。
(2)摇瓶生产及纯化
摇瓶参数：转速120r/min，温度为37℃，二氧化碳为8％。流加补料：在摇瓶中培养至72h时开始每天补料，HyClone TM Cell Boost TMFeed 7a每天流加初始培养体积的3％，Feed 7b每天流加量为初始培养体积的千分之一，一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用proteinA亲和层析柱进行亲和纯化。将抗体稀释到1mg/mL进行还原性SDS-PAGE，电泳图如图1所示。在还原性SDS-PAGE后显示两条带，1条Mr为50KD(重链)，另一条Mr为28KD(轻链)。
经上述步骤获得的14D2RMb-1抗体，经测序及Kabat分析，重链CDR1-3分别如SEQ ID NO:1-3所示，轻链CDR1-3分别如SEQ ID NO:4-6所示，重链可变区及轻链可变区依次如SEQ ID NO:15、16所示，重链及轻链依次如SEQ ID NO:19、20所示。
对14D2RMb-1的结构进行分析，并进行突变引物设计，重复步骤1-(3)至3-(2)，获得10个突变抗体，命名为14D2RMb-2至11。14D2RMb-1至11的重链和轻链氨基酸序列分别如下表所示：
表2
Figure PCTCN2022132982-appb-000002
实施例2抗体亲和力分析及活性鉴定
1、亲和力分析
利用AMC传感器，将纯化出来的抗体用PBST稀释到20μg/mL，D-二聚体抗原用PBST进行梯度稀释；
运行流程：缓冲液1(PBST)中平衡60s，抗体溶液中固化抗体300s，缓冲液2(PBST)中孵育180s，抗原溶液中结合420s，缓冲液2中解离1200s，用10mM pH 1.69 GLY溶液及缓冲液3进行传感器再生，输出数据。
表3
抗体名称 KD(M) Kon(1/Ms) Kdis(1/s)
对照抗体 2.14E-08 6.17E+03 1.32E-04
14D2RMb-1 9.90E-09 5.50E+05 5.45E-03
14D2RMb-2 1.22E-09 6.75E+04 8.21E-05
14D2RMb-3 2.24E-09 5.71E+04 1.28E-04
14D2RMb-4 8.90E-09 5.10E+05 4.54E-03
14D2RMb-5 8.67E-10 6.02E+04 5.22E-05
14D2RMb-6 7.40E-09 5.00E+05 3.70E-03
14D2RMb-7 3.02E-09 5.93E+04 1.79E-04
14D2RMb-8 2.08E-09 6.12E+04 1.27E-04
14D2RMb-9 6.31E-09 6.10E+05 3.85E-03
14D2RMb-10 1.86E-09 5.61E+04 1.04E-04
14D2RMb-11 2.08E-09 6.16E+04 1.28E-04
注：KD表示平衡解离常数即亲和力；Kon表示结合速率常数；Kdis表示解离速率常数。
2、活性鉴定
包被液(主要成分NaHCO 3)稀释D-二聚体抗原(购自菲鹏)至2μg/mL，每孔100μL，4℃过夜；次日，洗涤液(主要成份Na 2HPO 4+NaCl)清洗2次，拍干；加入封闭液(20％BSA+80％PBS)，每孔120μL，37℃，1h，拍干；加入稀释后的纯化抗体和对照抗体，100μL/孔，37℃，30min；洗涤液清洗5次，拍干；加入羊抗鼠IgG-HRP，每孔100μL，37℃，30min；洗涤液清洗5次，拍干；加入显色液A液(50μL/孔)，加入显色液B液(50μL/孔)，10min；加入终止液，50μL/孔；酶标仪上450nm(参考630nm)处读OD值。
表4
Figure PCTCN2022132982-appb-000003
Figure PCTCN2022132982-appb-000004
实施例3稳定性考核
将自产最优抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天，取7天、14天、21天抗体样品进行状态观察，并对21天抗体样品进行活性检测，结果显示三种考核条件下抗体样品放置21天均未见明显蛋白状态变化，活性也未随考核温度的升高呈下降越势，说明自产抗体稳定。下表为考核21天的酶免活性检测OD结果。
表5
抗体浓度(ng/mL) 62.5 15.625 0
4℃，21天样品 0.861 0.389 0.061
-80℃，21天样品 0.911 0.339 0.073
37℃，21天样品 0.991 0.339 0.077
实施例4性能评价
14D2RMb1至14D2RMb11重组抗体作为标记抗体，另一株抗D-二聚体单克隆抗体作为包被抗体，通过双抗体夹心化学发光的反应模式测定临床相关性，结果显示：14D2RMb1至14D2RMb11的检测灵敏度及临床样本相关性优于对照抗体。其具体结果见表6。
表6
Figure PCTCN2022132982-appb-000005
Figure PCTCN2022132982-appb-000006
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
工业实用性
本公开提供的抗D-二聚体的抗体，其对D-二聚体的亲和力较好，使用该抗体检测D-二聚体具有较好的灵敏度或特异性。本公开为D-二聚体的检测提供了更为优秀的抗体选择，因此，本公开提供的抗D-二聚体的抗体、检测D-二聚体的试剂和试剂盒均具备优异的实用性能和广阔的市场应用前景。

Claims (17)

  1. 一种抗D-二聚体的抗体或其功能性片段，包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，其特征在于，所述HCDR1～HCDR3包括与SEQ ID NO:15、30、31和32任一所示重链可变区的HCDR1～HCDR3一致的氨基酸序列；所述LCDR1～LCDR3包括与SEQ ID NO:16、33、34、35和36任一所示轻链可变区的LCDR1～LCDR3一致的氨基酸序列。
  2. 根据权利要求1所述的抗体或其功能性片段，其特征在于，所述CDRs由Kabat、Chothia、IMGT、AbM或Contact系统定义。
  3. 根据权利要求1所述的抗体或其功能性片段，其特征在于，所述抗体或其功能性片段包含以下CDRs：
    HCDR1，其包含SEQ ID NO:1所示的氨基酸序列，或由其组成；
    HCDR2，其包含SEQ ID NO:2所示的氨基酸序列，或由其组成；
    HCDR3，其包含SEQ ID NO:3所示的氨基酸序列，或其组成；
    LCDR1，其包含SEQ ID NO:4所示的氨基酸序列，或由其组成；
    LCDR2，其包含SEQ ID NO:5所示的氨基酸序列，或由其组成；
    LCDR3，其包含SEQ ID NO:6所示的氨基酸序列，或由其组成。
  4. 根据权利要求1至3任一项所述的抗体或其功能性片段，其特征在于，所述抗体或其功能性片段还具有以下的骨架区：
    HFR1氨基酸序列如SEQ ID NO:7所示或与其具有至少80％同源性；
    HFR2氨基酸序列如SEQ ID NO:8所示或与其具有至少80％同源性；
    HFR3氨基酸序列如SEQ ID NO:9、21、22、23任一所示，或与其具有至少80％同源性；
    HFR4氨基酸序列如SEQ ID NO:10所示或与其具有至少80％同源性；
    LFR1氨基酸序列如SEQ ID NO:11、24、25、26任一所示，或与其具有至少80％同源性；
    LFR2氨基酸序列如SEQ ID NO:12所示或与其具有至少80％同源性；
    LFR3氨基酸序列如SEQ ID NO:13、27、28、29任一所示，或与其具有至少80％同源性；
    LFR4氨基酸序列如SEQ ID NO:14所示或与其具有至少80％同源性。
  5. 一种抗D-二聚体的抗体或其功能性片段，其特征在于，所述抗体或其功能性片段包含重链可变区和/或轻链可变区，所述重链可变区包含权利要求1至3任一项所述的HCDR1～3和权利要求4所述的HFR1～4，所述轻链可变区包含权利要求1至3任一项所述的LCDR1～3和权利要求4所述的LFR1～4；
    可选地，所述重链可变区包含选自SEQ ID NO:15、30、31和32任一所示的氨基酸序列，或由其组成；
    所述轻链可变区包含SEQ ID NO:16、33、34、35和36任一所示的氨基酸序列，或由其组成。
  6. 根据权利要求1至5任一所述的抗体或其功能性片段，其特征在于，还包含恒定区；
    可选地，所述恒定区包括重链恒定区和/或轻链恒定区；
    可选地，所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区；所述轻链恒定区选自κ型或λ型轻链恒定区；
    可选地，所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人；可选地，所述恒定区的种属来源为小鼠；
    可选地，所述重链恒定区为SEQ ID NO:17或与SEQ ID NO:17具有80％以上同源性的氨基酸序列；所述轻链恒定区为SEQ ID NO:18或与SEQ ID NO:18具有80％以上同源性的氨基酸序列。
  7. 根据权利要求1至6任一所述的抗体或其功能性片段，其特征在于，所述功能性片段选自所述抗体的F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
  8. 一种抗D-二聚体的抗体或其功能性片段，包括重链和/或轻链，其特征在于，所述重链包括权利要求5所述的重链可变区和权利要求6所述的重链恒定区；所述轻链包括权利要求5所述的轻链可变区和权利要求6所述的轻链恒定区；
    可选地，所述重链的氨基酸序列如SEQ ID NO:19、37、38、39任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、40、41、42、43任一所示。
  9. 一种核酸，其特征在于，所述核酸编码权利要求1至8任一所述的抗体或其功能性片段。
  10. 一种细胞，其特征在于，所述细胞包含权利要求9所述的核酸。
  11. 一种制备权利要求1至8任一项所述的抗体或其功能性片段的方法，其特征在于，所述方法包括培养权利要求10所述的细胞。
  12. 一种抗体偶联物，其特征在于，所述抗体偶联物包含权利要求1至8任一所述的抗体或其功能性片段以及与其偶联的偶联部分；
    可选地，所述偶联部分选自纯化标签或可检测的标记，例如胶体金、放射性标记、发光物质、有色物质、酶，例如荧光标记、发色团标记、电子致密标记，例如放射性同位素、荧光团、罗丹明及其衍生物、荧光素酶、荧光素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记的一种或多种；
    可选地，所述偶联部分选自磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。
  13. 一种试剂或试剂盒，其特征在于，所述试剂或试剂盒包含权利要求1至8任一项所述的抗体或其功能性片段或权利要求12所述的抗体偶联物。
  14. 权利要求1至8任一所述的抗体或其功能性片段或权利要求12所述的抗体偶联物在制备试剂或试剂盒中的用途；
    可选地，所述试剂或试剂盒用于检测D-二聚体或诊断D-二聚体相关疾病。
  15. 权利要求1至8任一所述的抗体或其功能性片段、权利要求12所述的抗体偶联物或权利要求13所述的试剂或试剂盒用于检测D-二聚体或诊断D-二聚体相关疾病中的用途。
  16. 根据权利要求14或15所述的用途，其特征在于，所述D-二聚体相关疾病包括心梗、脑梗、先兆子痫、深静脉血栓、胸主夹层、弥散性血管内凝血、肺动脉高压、肝损伤、系统性红斑狼疮、肿瘤或组织坏死。17.一种诊断受试者是否患有D-二聚体相关疾病的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1至8任一项所述的抗体或其功能性片段，或者权利要求12所述的抗体偶联物，或者权利要求13所述的试剂或试剂盒与来自所述受试者的样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物；
    可选地，所述D-二聚体相关疾病包括心梗、脑梗、先兆子痫、深静脉血栓、胸主夹层、弥散性血管内凝血、肺动脉高压、肝损伤、系统性红斑狼疮、肿瘤或组织坏死。
  17. 一种检测测试样品中的D-二聚体的方法，其包括：
    a)在足以发生抗体/抗原结合反应的条件下，使所述测试样品中的D-二聚体抗原与权利要求1至8任一项所述的抗体或其功能性片段，或者权利要求12所述的抗体偶联物，或者权利要求13所述的试剂或试剂盒接触以形成免疫复合物；和
    b)检测所述免疫复合物的存在，所述复合物的存在指示所述测试样品中所述抗原的存在；
    可选的，在步骤a)中，所述免疫复合物中还包括第二抗体，所述第二抗体与所述抗D-二聚体的抗体或其功能性片段结合。
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