Thrombin antibody or antigen binding fragment thereof and application thereof

Abstract

The invention discloses a thrombin antibody or an antigen binding fragment thereof and application thereof, and relates to the field of antibodies. The thrombin 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 thrombin detection, and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, in particular to a thrombin antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] TAT (thrombin-antithrombin complex) is an important coagulation regulatory molecule that plays a key role in the balance between blood coagulation and anticoagulation. Thrombin is the core enzyme in the coagulation cascade, primarily promoting coagulation by converting fibrinogen to fibrin. Antithrombin (AT), a serine protease inhibitor (serpin), is a major physiological anticoagulant protein. It inhibits thrombin activity by forming a stable complex (TAT) with thrombin, thereby preventing thrombus formation.

[0003] Thrombin is a serine protease composed of a light chain and a heavy chain linked by a disulfide bond. Its active site is located in the catalytic domain of the heavy chain (containing the catalytic triad His-Asp-Ser). The catalytic domain also contains a catalytic triad (His-Asp-Gly) responsible for substrate cleavage. Thrombin promotes the coagulation cascade by activating coagulation factors VIII and V and directly converts fibrinogen to fibrin, forming a blood clot. Antithrombin is a single-chain glycoprotein with an active center (reaction center loop, RCL) that binds to thrombin. The heparin binding site is located at the N-terminus. Antithrombin inhibits thrombin activity by forming a stable TAT complex with thrombin. Sulfated glycosaminoglycans such as heparin can enhance the anticoagulant effect of antithrombin.

[0004] The formation of TAT reflects the dynamic balance between the coagulation and anticoagulation systems. Under normal circumstances, TAT levels are maintained at a low level to prevent excessive coagulation. In pathological conditions, such as deep vein thrombosis, pulmonary embolism, and atherosclerosis, elevated TAT levels indicate increased coagulation activity. Abnormally elevated TAT levels may be associated with decreased anticoagulant function, while excessively low levels may indicate hypercoagulability. TAT measurement is of great value in various clinical scenarios, including: 1) Diagnosis of thrombotic diseases: elevated TAT levels indicate an increased risk of thrombosis; 2) Monitoring of anticoagulant therapy: TAT levels can help assess the effectiveness of anticoagulant therapy; 3) Cardiovascular disease risk assessment: elevated TAT levels are associated with diseases such as atherosclerosis and myocardial infarction.

[0005] Currently, TAT is primarily determined by immunological methods, such as enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, and chemiluminescence. These methods are primarily based on antigen-antibody-specific binding reactions and all require antibodies specific for thrombin. Therefore, those skilled in the art have a strong demand for high-performance thrombin antibodies. Summary of the Invention

[0006] To address the problems of the prior art, the present invention provides a monoclonal antibody, kit, and application thereof targeting thrombin. The antibody of the present invention has high binding activity and affinity for thrombin, enabling specific, sensitive, reliable, and accurate detection of trace amounts of thrombin in samples. The present invention is specifically implemented through the following technical solutions: The first object of the present invention is to provide a thrombin antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID Nos: 1-3; and LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID Nos: 4-6.

[0007] As used herein, "CDR," "CDRs," or "complementarity determining regions" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and are regions comprising one or more, or even all, of the primary amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0008] In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0009] Methods for defining CDRs are well known in the art and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but still overlap with at least a portion of the Kabat-defined CDR regions, although they may be shortened or lengthened based on predictions or experimental results for specific residues or groups of residues.

[0010] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by the Kabat system.

[0011] The second object of the present invention is to provide a thrombin antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0012] In an optional embodiment, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0013] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0014] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0015] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0016] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0017] In an alternative embodiment, the light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0018] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0019] In an alternative embodiment, the species origin of the constant region is mouse.

[0020] In an optional embodiment, the antigen-binding fragment is selected from any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0021] The third object of the present invention is to provide a thrombin antibody or an antigen-binding fragment thereof, wherein the heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.9, and the light chain amino acid sequence is shown in SEQ ID NO.10.

[0022] Antigen-binding fragments of the aforementioned antibodies generally possess the same binding specificity as the antibody from which they are derived. Based on the disclosure herein, those skilled in the art will readily appreciate that antigen-binding fragments of the aforementioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or chemical reduction to cleave disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0023] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0024] The fourth object of the present invention is to provide an antibody conjugate, which comprises the above-mentioned antibody or antigen-binding fragment thereof, and is coupled with a label or a solid phase carrier.

[0025] In an alternative embodiment, the aforementioned marker refers to a substance having properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., which can be used to achieve qualitative or quantitative detection of the corresponding target. Examples include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0026] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0027] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0028] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0029] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0030] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0031] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0032] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0033] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0034] In an optional embodiment, the colloidal metal is colloidal gold.

[0035] In an optional embodiment, the above-mentioned antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

[0036] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0037] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0038] A fifth object of the present invention is to provide a thrombin detection reagent, which comprises the above-mentioned antibody or antigen-binding fragment thereof.

[0039] For example, the above-mentioned kit can be used for immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, latex immunoturbidimetry, and other assays that utilize the specific binding properties of thrombin and its antibody. For example, in fluorescent immunochromatography, the antigen in the sample binds to the fluorescently labeled antibody on a conjugate pad. The solution undergoes chromatography due to the siphoning action of the absorbent pad, migrating toward the pad. When the complex moves to the detection line, it binds to the coated antibody on the T line, forming a "sandwich" complex that is enriched on the T line. In chemiluminescence, the antigen in the sample binds to the antibody coated on magnetic beads. After washing, it is then bound to an enzyme-labeled antibody (typically horseradish peroxidase (HRP) or alkaline phosphatase (AP)) to form a "solid-phase antibody-antigen-enzyme-labeled antibody" sandwich complex. It should be understood that the term "antibody" here does not refer to a specific sequence of the present invention. In theory, any antibody that can form a "double antibody sandwich" with the analyte, that is, antibodies targeting different epitopes of the analyte antigen, will meet the requirements.

[0040] A sixth object of the present invention is to provide use of the above-mentioned thrombin antibody or antigen-binding fragment thereof, antibody conjugate or the above-mentioned reagent in the preparation of a product for detecting thrombin. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a diagram showing the clinical relevance of detection reagents using TAT-64# antibody. DETAILED DESCRIPTION

[0042] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally 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 considered herein are standard methods. The materials, methods, and examples are illustrative and non-limiting only. In the examples, if specific conditions are not indicated, they are carried out according to conventional conditions or conditions recommended by the manufacturer, and the reagents or instruments used, if the manufacturer is not indicated, are conventional products that can be purchased commercially.

[0044] Example 1 Preparation of thrombin monoclonal antibody A. Mouse Immunization and Antibody Detection Five SPF female BALB / c mice aged 6-8 weeks were selected, and Freund's complete adjuvant and thrombin protein at a concentration of 1 mg / ml (Zhongyuan Huiji Biotechnology Co., Ltd.) were mixed and emulsified in equal volumes. The emulsified antigen was used to immunize the mice, and each mouse was injected with 20ug of antigen protein by foot injection. Two weeks after the initial immunization, the antigen protein was mixed and emulsified with Freund's complete adjuvant, and then injected subcutaneously in the back again, with each mouse injected with 30ug of antigen protein. Two weeks later, blood was collected through the jugular vein, the supernatant was collected by centrifugation, and the serum titer was tested by ELISA. Immunization was performed every two weeks and the serum titer was tested. After two immunizations, the serum titer was as high as 2.0 or above after a million-fold dilution. Screening serum titer 10 6 1E7 spleen cells were obtained from the above mice and RNA was extracted.

[0045] B. Phage display library construction RNA was used as a template for reverse transcription to obtain complementary cDNA, and then cDNA was used as a template to add mouse-specific bidirectional primers to amplify VH, V-κ and V-λ fragments respectively. The variable regions of the heavy and light chains were then amplified using overlapping PCR to produce single-chain antibodies (scFv fragments). The scFv fragments were then constructed into a phagemid vector by homologous recombination to obtain recombinant phagemids, which were transferred into Escherichia coli by electroporation and cultured overnight before centrifugation. The phage particles in the supernatant were precipitated by PEG adsorption, and the precipitate was resuspended in PBS to obtain the primary thrombin phage antibody library.

[0046] C. Specific Panning of Phage Display Libraries Thrombin native protein is used as the coating protein and gradiently diluted in a high-adsorption ELISA plate. A primary library of thrombin phage antibodies is added to capture some phage particles that can specifically bind to thrombin native protein. The unbound phage particles are then washed away with a washing solution. The remaining specific phage particles will be amplified to the secondary library under the action of Escherichia coli infection and auxiliary phage superinfection for subsequent panning.

[0047] D. Phage particle ELISA verification After specific panning, the phage particles were gradiently diluted and plated onto a resistance plate. After overnight incubation, several single colonies were clearly visible on the plate. Using a sterile pipette tip, 88 colonies were picked and placed in a deep-well plate. 600 μL of resistance culture medium was added and incubated overnight. The next day, the suspension was centrifuged at 4000 g for 10 minutes, and the phage supernatant was collected for ELISA analysis. The bacterial suspension that met the requirements was sent to a third party for sequencing to obtain the scFv base sequence. The phage was then sent for gene synthesis and expression into a complete antibody for subsequent antibody validation.

[0048] Table 1 Phage Elisa data of 6 / 88 strains

[0049] The results of Phage Elisa showed that 6 / 88 of the samples were positive. Sequence analysis revealed three scFv sequences: TAT-1#, TAT-25#, and TAT-64#.

[0050] Example 2 Performance testing of antibodies 1. Affinity Analysis Using the ELISA method, a standard curve was drawn with the antibody concentration as the horizontal axis and the OD450 value of the antigen-antibody reaction as the vertical axis. The affinity constant (KD represents the equilibrium dissociation constant, i.e., affinity) of the above-mentioned antibody was calculated based on the standard curve and the calculation formula (n[Ab1]-[Ab]) / (n-1). The test results are shown in the following table: Table 2 Affinity data Clone number KD value TAT-1# 2.24E-10 TAT-25# 5.41E-10 TAT-64# 1.87E-10

[0051] The test results show that antibodies TAT-64# and TAT-1# have better affinity with antigens.

[0052] 2. Activity Identification Dilute TAT antigen to 1 μg / mL with 50 mM carbonate buffer for microplate coating, add 100 μL per well, and incubate at 4°C overnight. The next day, wash twice with PBST and pat dry. Add the above antibodies, starting from 500 ng / mL, in a 3-fold dilution, and load at 100 μL / well at 37°C for 30 min. Wash five times with PBST and pat dry. Dilute horseradish peroxidase-labeled goat anti-mouse IgG to 1:3000 and add 100 μL per well at 37°C for 30 min. Wash five times with PBST and pat dry. Add urea peroxide (50 μL / well) and tetramethylbenzidine (50 μL / well) for 10 min. Terminate the reaction with dilute hydrochloric acid (50 μL / well). Read the OD value at 450 nm (reference 630 nm) on a microplate reader. Activity determination is shown in the following table: Table 3 Activity data Sample concentration (ng / ml) 500 16.7 55.6 18.5 6.2 2.1 0.69 0 TAT-1# 1.851 1.71 1.494 1.21 0.769 0.4 0.245 0.057 TAT-64# 2.016 1.917 1.818 1.594 1.115 0.636 0.333 0.056 The test results show that the antibodies TAT-1# and TAT-64# provided by the present invention can still be detected even when the antigen concentration is as low as 2 ng / ml, indicating that the antibodies have high activity.

[0053] 3. Stability determination The above-mentioned antibodies were heat-accelerated at 37°C for 14 days in a designated buffer (PBS, 0.05% ProClin™ 300) and then freeze-thawed five times at -20°C. The heat-accelerated stability of the antibodies was evaluated indirectly by ELISA, with the values ​​at 4°C used as a control to assess their heat-accelerated stability. The test results are presented as the deviation between the values ​​at 4°C and the values ​​after treatment. The measurement results are as follows (antibody concentration of the present invention: 1 ug / mL; TAT antigen concentration: 500 ng / mL).

[0054] Table 4 Stability study 37°C for 7 days 37°C 14 days Freeze and thaw 5 times TAT-1# -20.3% -23.2% 1.5% TAT-64# 2.4% 1.7% -2.4% The test results show that the antibody TAT-64# provided by the present invention has good accelerated stability and freeze-thaw stability.

[0055] 4. Functional testing A. Biotinylated Antibodies Weigh biotin (NHS-LC-LC-Biotin, from thermo scientific, 21343) and dissolve it in DMSO to 5.677 mg / mL. Take 0.3 mg of TAT-64# antibody (the volume of biotin solution required for every 1 mg of antibody is 3.3 μL). Add biotin to each of the six antibodies and mix well. Link the antibodies to biotin and react at 25°C in the dark for 4 h.

[0056] B. Alkaline phosphatase labeled antibody 0.3 mg of thrombin antibody (Zhongyuan Huiji Biotechnology Co., Ltd.) was exchanged into TSE (pH 8.5) buffer. 0.66 mg of ALP (Sigma, ALPI12G) was exchanged into ALP dialysis buffer (pH 7.6). Mix well and determine the concentration. Dissolve Traut's Reagent 2-Iminothiolane·HCl (2-IT, Thermo Scientific, 26101) in TSE (pH 8.5) to 13.76 mg / mL. Dissolve Sulfo-SMCC (Thermo Scientific, PG82085) in purified water to 3.7 mg / mL. Prepare both activator solutions immediately and use within 10 minutes. Calculate the required volume of 2-IT solution (5 μL per 1 mg of antibody). Add the 2-IT solution to the antibody, mix well, and incubate at (25 ± 2)°C for 20 minutes. This is antibody activation. After activation, the activated antibody was exchanged into TSE (pH 7.3) solution and the antibody concentration was determined. Simultaneously, the required volume of Sulfo-SMCC solution was calculated based on a 10 μL volume of SMCC solution per 1 mg of ALP. The Sulfo-SMCC solution was added to the desalted ALP, mixed thoroughly, and allowed to react at (25 ± 2)°C for 20 minutes. After activation, the activated ALP was exchanged into TSMZ (pH 7.3) solution and the ALP concentration was determined. The desalted antibody was diluted to 0.3 mg / mL with TSE (pH 7.3) solution, and the desalted ALP was diluted to 0.4 mg / mL with TSMZ (pH 7.3) solution. The volumes of antibody and ALP used for conjugation were calculated based on a mass ratio of 1:0.91. The antibody and ALP were mixed according to the calculated volumes and allowed to react at 2–8°C for 12–20 hours. Dissolve maleimide in DMSO to 9.7 mg / mL and dilute 10-fold with TSMZ (pH 7.3) to prepare the stop solution. Add 20 μL of the required stop solution per 1 mL of antibody-ALP conjugate to terminate the reaction.

[0057] Performance evaluation (1) Linear experiment Assay reagents, including R1, R2, and streptavidin magnetic bead solution: The R1 includes: biotin-labeled antibody, used at a concentration of 0.5 ug / mL; The R2 includes: alkaline phosphatase labeled antibody, with a concentration of 1.3ug / mL.

[0058] The specific experimental steps are as follows: a. Preparation: 5 mL each of R1 and R2 working solutions, 4 mL of magnetic bead solution, and calibrators WRS A through WRS H.

[0059] b. Place each component into the kit, then place the kit into the fully automatic chemiluminescence immunoassay analyzer EXI1800 (Zhongyuan Huiji Biotechnology Co., Ltd.). Select the anti-TAT project (sample 30uL, R1 120uL, R2 30uL, magnetic bead solution 45uL) for the experiment. The evaluation data for TAT-64# are as follows: Table 5 Calibration data

[0060] From the above data, it can be seen that TAT-64# antibody has an obvious gradient within the conventional linear range of TAT.

[0061] (2) Clinical trials The specific experimental steps are as follows: (1) Experimental preparation: Take out 100 samples of TAT-assigned reference materials, thaw them at room temperature, and mix them thoroughly on a vortex mixer; (2) Testing: 100 samples of the company's reference products were tested using the aforementioned reagents.

[0062] The test results are as attached Figure 1 As shown in the above data, the clinical measurement value of TAT-64# meets the requirements and can be used in thrombin detection reagents.

[0063] The partial amino acid sequence of the TAT-64# thrombin antibody involved in this application is shown in Table 6 (CDR region sequence defined by the Kabat system): Table 6: Amino acid sequence listing Serial number Amino acid sequence HCDR1 No.1 DYYVH HCDR2 NO.2 WIDPEDGDSECAPKFQG HCDR3 NO.3 HFFDYDVMDY LCDR1 NO.4 KSSQSLLYSRNQKNYLA LCDR2 NO.5 WASTRYS LCDR3 No. 6 QQYYSFPLT Heavy chain variable region No.7 EVQLQQSGAELVRSGASVKLSCTVSGFNIKDYYVHWVKERPEQGLEWIGWIDPEDGDSECAPKFQGKATMTADTSSNTAYLQFSSLTSEDTAVYYCNVHFFDYDVMDYWGQGTSVTVSS Light chain variable region No.8 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSRNQKNYLAWYQQKPGQSPKLLIYWASTRYSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSFPLTFGAGTKLELK Heavy chain No.9 EVQLQQSGAELVRSGASVKLSCTVSGFNIKDYYVHWVKERPEQGLEWIGWIDPEDGDSECAPKFQGKATMTADTSSNTAYLQFSSLTSEDTAVYYCNVHFFDYDVMDYWG QGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG light chain No. 10 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSRNQKNYLAWYQQKPGQSPKLLIYWAS TRYSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSFPLTFGAGTKLELKRADA APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSK DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC All data, reagents and steps herein should be understood as illustrative and non-restrictive, although described the present invention in conjunction with above-mentioned specific embodiment, many modifications and other variations are apparent to those skilled in the art.All such modifications and other variations also fall within the scheme of the present invention and protection scope.

Claims

1. A thrombin antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises: HCDR1 with the amino acid sequence shown in SEQ ID No: 1; HCDR2 with the amino acid sequence shown in SEQ ID No: 2; HCDR3 with the amino acid sequence shown in SEQ ID No: 3; LCDR1 having an amino acid sequence as shown in SEQ ID No: 4; LCDR2 having an amino acid sequence as shown in SEQ ID No: 5; The amino acid sequence of LCDR3 is shown in SEQ ID No:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

3. A thrombin antibody or an antigen-binding fragment thereof, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

4. A thrombin antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 9; the amino acid sequence of the light chain is shown in SEQ ID NO:

10.

5. An antibody conjugate, characterized in that The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The antibody conjugate according to claim 5, characterized in that The antibody conjugate includes biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.

7. The antibody conjugate according to claim 5, characterized in that The antibody conjugate comprises a label coupled to the antibody or antigen-binding fragment thereof, wherein the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents or nanoparticle labels.

8. A reagent, characterized in that The reagent comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the antibody conjugate according to any one of claims 5 to 7.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the antibody conjugate according to any one of claims 5 to 7, or the reagent according to claim 8 in the preparation of a product for detecting thrombin.

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