An antibody against heparin-antithrombin complex and its use in a method for detecting the anticoagulant effect of heparin

Abstract

The application discloses an anti-heparin-antithrombin complex antibody and application thereof in a heparin drug anticoagulation effect detection method, and comprises an antibody molecule capable of combining with a heparin-antithrombin complex, a light chain variable region amino acid sequence of the antibody molecule is shown as SEQ ID NO:1, and a heavy chain variable region amino acid sequence is shown as SEQ ID NO:2. The antibody molecule can be prepared into a kit for detecting the anticoagulation effect of the heparin drug, and has the characteristics of simple operation, high sensitivity, good repeatability, wide linearity and the like, and is suitable for high-throughput testing of the anticoagulation effect of the heparin drug.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an anti-heparin-antithrombin complex antibody and its application in a method for detecting the anticoagulant effect of heparin. Background Technology

[0002] Thromboembolic diseases are a leading cause of death from cardiovascular and cerebrovascular diseases in humans, characterized by high morbidity, high disability rates, and high mortality rates. Thrombotic diseases primarily result from the formation of thrombi that block local blood flow or detach and become emboli that obstruct blood vessels, causing ischemia, necrosis, and even functional impairment of local tissues and organs. They often occur alongside diseases of various systems, significantly increasing the difficulty of diagnosing and treating the primary disease. Thrombosis is also a key factor contributing to poor prognosis and recurrent symptoms. In the coagulation system, thrombin plays a crucial role in thrombus formation; therefore, anticoagulation is the primary clinical treatment for thromboembolic diseases.

[0003] Heparin is one of the most widely used anticoagulants in clinical practice, including unfractionated heparin (UFH) and low molecular weight heparin (LMWH). Both UFH and LMWH are commonly used anticoagulants for the treatment or prevention of thromboembolism. UFH is usually the first-line drug in hemodialysis, percutaneous transluminal angioplasty, and cardiopulmonary bypass; however, due to its unstable pharmacokinetics, individual anticoagulant efficacy varies considerably; adverse reactions include bleeding, thrombocytopenia, and long-term use can lead to osteoporosis; the predictability of treatment response is poor, requiring continuous dosage adjustments. Therefore, monitoring is recommended when using UFH to achieve better anticoagulation and reduce or avoid complications such as bleeding. LMWH, due to its long half-life and high safety profile, is widely used in the prevention of deep vein thrombosis during orthopedic or general surgery, acute coronary syndrome, acute myocardial infarction, and anticoagulation therapy during pregnancy. However, monitoring is also recommended for obese patients, patients with renal insufficiency, children, and pregnant patients. Anti-Xa activity monitoring is the gold standard for monitoring heparin anticoagulant therapy. It is not affected by factors such as coagulation factor deficiency, lupus anticoagulants, and inflammation, and has higher methodological specificity than APTT, which can more directly and reliably reflect the concentration of heparin in the body.

[0004] Patent publications CN117191724A, CN117106856A, CN116026761A, and CN115372351A disclose an anti-Xa factor detection kit. This kit is based on a chromogenic substrate method. Heparin in the sample forms a complex with thrombin, which inhibits excess Xa factor added to the kit. The remaining uninhibited Xa factor is inversely proportional to the effective drug concentration in the sample. The remaining uninhibited Xa factor reacts with its specific substrate to produce p-nitroaniline. The absorbance of p-nitroaniline (pNA) is measured at a wavelength of 405 nm. The actual anticoagulant effect of the drug is calculated by determining the activity of this remaining Xa factor. Because this method detects whole blood samples, it has low sensitivity, narrow linearity, and the results are easily affected by abnormal factors such as hemolysis and lipemia.

[0005] Patent publication CN117309966A discloses an electrochemical method for detecting the content of factor Xa inhibitors in blood samples. After the blood sample undergoes a coagulation reaction with a detection reagent containing factor Xa, the oxidizable substances produced are oxidized to obtain an oxidation current signal. The oxidation current signal is collected to obtain a current curve that changes over time. Feature points are obtained from the current curve. The content of factor Xa inhibitors in the blood sample is detected based on the feature points. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-heparin-antithrombin complex antibody and its application in the detection method of the anticoagulant effect of heparin, avoiding the defects of low sensitivity, narrow linear range and low detection throughput of current detection kits.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A first aspect of the present invention provides an antibody molecule comprising a light chain and a heavy chain, wherein the complementarity-determining regions of the light chain include LCDR1, LCDR2, and LCDR3, and the complementarity-determining regions of the heavy chain include HCDR1, HCDR2, and HCDR3; wherein, The amino acid sequence of LCDR1 is shown in SEQ ID NO:3; The amino acid sequence of LCDR2 is shown in SEQ ID NO:4; The amino acid sequence of LCDR3 is shown in SEQ ID NO:5; The amino acid sequence of HCDR1 is shown in SEQ ID NO:10; The amino acid sequence of HCDR2 is shown in SEQ ID NO:11; The amino acid sequence of HCDR3 is shown in SEQ ID NO:12.

[0008] As a further aspect of the present invention, the framework region of the light chain includes LFR1, LFR2, LFR3 and LFR4, wherein the amino acid sequence of LFR1 is shown in SEQ ID NO:6.

[0009] As a further embodiment of the present invention, the amino acid sequence of the LFR2 is shown in SEQ ID NO:7.

[0010] As a further embodiment of the present invention, the amino acid sequence of the LFR3 is shown in SEQ ID NO:8.

[0011] As a further embodiment of the present invention, the amino acid sequence of the LFR4 is shown in SEQ ID NO:9.

[0012] As a further aspect of the present invention, the complementary determining region and the frame region of the light chain are arranged in the order of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0013] As a further embodiment of the present invention, the framework region of the heavy chain includes HFR1, HFR2, HFR3 and HFR4, wherein the amino acid sequence of HFR1 is shown in SEQ ID NO:13.

[0014] As a further embodiment of the present invention, the amino acid sequence of HFR2 is shown in SEQ ID NO:14.

[0015] As a further embodiment of the present invention, the amino acid sequence of the HFR3 is shown in SEQ ID NO:15.

[0016] As a further embodiment of the present invention, the HFR4 amino acid sequence is shown in SEQ ID NO:16.

[0017] As a further aspect of the present invention, the complementary determination region and the frame region of the heavy chain are arranged in the order of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0018] As a further aspect of the present invention, the amino acid sequence of the light chain variable region of the antibody molecule is shown in SEQ ID NO:1.

[0019] As a further embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the antibody molecule is shown in SEQ ID NO:2.

[0020] As a further aspect of the present invention, the antibody molecule is capable of binding to the heparin-antithrombin complex.

[0021] As a further aspect of the present invention, 1 to 3 amino acids are substituted in the light chain variable region and / or the heavy chain variable region.

[0022] As a further aspect of the present invention, the antibody molecule includes a murine monoclonal antibody.

[0023] As a further aspect of the present invention, the antibody molecule is capable of specifically binding to the heparin-antithrombin complex, rather than binding to heparin or antithrombin alone.

[0024] A second aspect of the present invention provides a nucleic acid molecule that encodes an antibody molecule as described in any of the preceding claims.

[0025] As a further embodiment of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:17.

[0026] As a further embodiment of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:18.

[0027] A third aspect of the present invention provides a kit comprising an antibody molecule encoded by an antibody molecule as described in any of the preceding claims or an antibody molecule encoded by a nucleic acid molecule as described in any of the preceding claims.

[0028] As a further aspect of the present invention, the antibody molecules are labeled to form antibody markers.

[0029] As a further aspect of the present invention, the antibody molecule is labeled with at least one of acridine ester, alkaline phosphatase, ruthenium tripyridine, and horseradish peroxidase to form an antibody marker.

[0030] As a further aspect of the present invention, the kit also includes antithrombin.

[0031] As a further aspect of the present invention, the antithrombin is immobilized using at least one of magnetic microparticles and an enzyme-labeled plate.

[0032] As a further embodiment of the present invention, the kit further includes at least one reagent selected from 4-morpholine ethanesulfonic acid, sodium chloride, Tween, bovine serum albumin, sodium azide, and tris(hydroxymethyl)aminomethane.

[0033] As a further aspect of the present invention, the kit includes an antibody marker reagent and an antithrombin reagent, wherein the antibody marker reagent includes the antibody molecule described in any one of the above.

[0034] As a further aspect of the present invention, the antibody marker reagent further includes at least one of 4-morpholine ethanesulfonic acid, sodium chloride, Tween-20, bovine serum albumin, and sodium azide.

[0035] As a further embodiment of the present invention, the antibody labeling reagent comprises 9.5~11.5 g / L of 4-morpholine ethanesulfonic acid, 8~10 g / L of sodium chloride, 0.2~0.8 g / L of Tween-20, 8~12 g / L of bovine serum albumin, 0.7~1.1 g / L of sodium azide, and 10~30 μL / L of the antibody molecule label.

[0036] As a further embodiment of the present invention, the antithrombin reagent comprises 2-4 g / L of tris(hydroxymethyl)aminomethane, 8-10 g / L of sodium chloride, 0.2-0.8 g / L of Tween-20, 3-7 g / L of bovine serum albumin, 0.7-1.1 g / L of sodium azide, and 30-70 μL / L of antithrombin magnetic microparticles.

[0037] As a further aspect of the present invention, the kit also includes a chemiluminescent substrate.

[0038] As a further aspect of the present invention, the reaction principle of the kit includes: reacting heparin drug with antithrombin to form a heparin-antithrombin complex, then adding the antibody molecule to detect the content of the heparin-antithrombin complex, thereby obtaining the anticoagulant effect of the heparin drug.

[0039] As a further aspect of the present invention, the reaction principle of the kit includes: the anticoagulant effect of heparin in the test sample is directly proportional to the content of the heparin-antithrombin complex formed, and the content of the heparin-antithrombin complex is directly proportional to the intensity of the luminescent signal generated by the antibody marker.

[0040] The fourth aspect of the present invention provides the application of the antibody molecule or the nucleic acid molecule encoded by any of the above-described antibody molecules or the kit described in any of the above-described reagents in a method for detecting the anticoagulant effect of heparin.

[0041] As a further aspect of the present invention, the detection method uses chemiluminescent immunoassay.

[0042] As a further aspect of the present invention, the detection method obtains the anticoagulant effect of the heparin drug by detecting the content of the heparin-antithrombin complex using the antibody molecules.

[0043] As a further aspect of the present invention, the detection method includes: The test sample containing the heparin drug is mixed with antithrombin to form a heparin-antithrombin complex. The anticoagulant effect of the heparin drug is obtained by detecting the content of the heparin-antithrombin complex by detecting the marker signal of the antibody molecule.

[0044] The present invention has at least the following beneficial effects: The antibody molecule method for detecting the anticoagulant effect of heparin provided by this invention is mainly based on the specific binding of antibody molecules to the complex formed by the specific binding of heparin and antithrombin. The anticoagulant effect of heparin is detected by reading the luminescence signal generated by the chemiluminescent substrate (including but not limited to AMPPD, APS-5, acridinium ester, etc.). It has higher sensitivity, wider linearity and better repeatability.

[0045] The kit provided by this invention for detecting the anticoagulant effect of heparin is based on chemiluminescence immunoassay. It can be used with a fully automated chemiluminescence analyzer, offering simple operation, high sensitivity, wide linearity, and good repeatability, making it suitable for high-throughput testing. Other methodologies, based on chromogenic substrates, have low reagent throughput and narrow linear ranges, making them unsuitable for high-throughput testing. Attached Figure Description

[0046] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0047] Figure 1 This is a calibration curve of the reagent kit of the present invention; Figure 2 This is a linear range graph of the reagent kit of the present invention; Figure 3 This is a linear range graph for commercially available reagent kits. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0049] Therefore, the following detailed description of embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0050] In this invention, "antibody molecule" should be understood as an immunoglobulin or a portion thereof, or any polypeptide containing a binding domain homologous to an antibody binding domain. Antibody molecules include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies and fragments thereof, as well as chimeric antibodies containing an immunoglobulin binding domain fused to another polypeptide.

[0051] A complete antibody comprises an immunoglobulin molecule consisting of a heavy chain and a light chain, each carrying a variable region designated VH and VL, respectively. The variable region consists of three complementarity-determining regions (CDRs, also known as hypervariable regions) and four framework regions (FRs). The CDRs form a complementary stereostructure to the antigen molecule and determine the antibody's specificity.

[0052] Antibody fragments can retain the binding ability of whole antibodies and can be used in place of whole antibodies. Therefore, for the purposes of this invention, unless the context otherwise requires, the term "antibody molecule" should be understood to encompass antibody fragments.

[0053] In some embodiments of the present invention, the amino acid sequence of the light chain variable region of the antibody molecule includes a complementarity-determining region and a framework region, wherein the complementarity-determining region includes LCDR1, LCDR2, and LCDR3. The frame region includes LFR1, LFR2, LFR3, and LFR4. And arranged in the order of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0054] In some embodiments of the present invention, the amino acid sequence of LCDR1 is shown in SEQ ID NO:3; The amino acid sequence of LCDR2 is shown in SEQ ID NO:4; The amino acid sequence of LCDR3 is shown in SEQ ID NO:5; The amino acid sequence of LFR1 is shown in SEQ ID NO:6; The amino acid sequence of LFR2 is shown in SEQ ID NO:7; The amino acid sequence of LFR3 is shown in SEQ ID NO:8; The amino acid sequence of LFR4 is shown in SEQ ID NO:9.

[0055] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody molecule includes a complementarity-determining region and a framework region, wherein the complementarity-determining region includes HCDR1, HCDR2, and HCDR3. The frame region includes HFR1, HFR2, HFR3, and HFR4. And arranged in the order of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0056] In some embodiments of the present invention, the HCDR1 amino acid sequence is shown in SEQ ID NO:10; The amino acid sequence of HCDR2 is shown in SEQ ID NO:11; The amino acid sequence of HCDR3 is shown in SEQ ID NO:12; The HFR1 amino acid sequence is shown in SEQ ID NO:13; The HFR2 amino acid sequence is shown in SEQ ID NO:14; The HFR3 amino acid sequence is shown in SEQ ID NO:15; The HFR4 amino acid sequence is shown in SEQ ID NO:16.

[0057] In some embodiments of the present invention, the antibody molecule is capable of binding to a heparin-antithrombin complex, and the amino acid sequence of the light chain variable region of the antibody molecule is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2.

[0058] The amino acid sequences involved in this invention are shown in Table 1 below.

[0059] Table 1 Amino acid sequence listing

[0060] The nucleotide sequences involved in this invention are shown in Table 2 below.

[0061] Table 2 Nucleotide Sequence List

[0062] In some embodiments of the present invention, the kit includes markers formed by antibody molecules as described in the present invention, magnetic microparticles coated with antithrombin, and chemiluminescent substrate components, which have fewer components than conventional kits, resulting in higher sensitivity and a wider linear range.

[0063] In some embodiments of the present invention, the reaction principle of the reagent kit includes: The solid phase coated with antithrombin is mixed with a human blood sample, and the heparin in the sample combines with the solid phase coated with antithrombin to form a heparin-antithrombin complex. Then, a marker formed by the antibody molecules is added to the system, which can specifically bind to the heparin-antithrombin complex; A chemiluminescent substrate was then added, and the luminescence signal of the labeled substance was tested. The intensity of the luminescence signal was directly proportional to the anticoagulant effect of heparin.

[0064] The stronger the anticoagulant effect of heparin in the sample, the more heparin-antithrombin complexes are formed, and the stronger the luminescent signal. By establishing a standard curve, the instrument can automatically calculate the anticoagulant effect of heparin in the sample.

[0065] Example 1: Preparation of the reagent kit In this embodiment, the light chain variable region of the antiheparin-antithrombin complex monoclonal antibody is encoded by the nucleotide sequence shown in SEQ ID NO:17, and its amino acid sequence is shown in SEQ ID NO:1; The heavy chain variable region is encoded by the nucleotide sequence shown in SEQ ID NO:18, and its amino acid sequence is shown in SEQ ID NO:2.

[0066] The conserved region sequence of the antiheparin-antithrombin complex monoclonal antibody is the conventional conserved region sequence of murine monoclonal antibodies and can be obtained using existing technologies.

[0067] In some embodiments of the present invention, the conserved amino acid sequence of the antiheparin-antithrombin complex monoclonal antibody is disclosed in the following literature: Bourgois A, Fougereau M, Rocca-Serra J. Determination of the primary structure of a mouse IgG2a immunoglobulin: amino-acid sequence of the Fc fragment. Implications for the evolution of immunoglobulin structure and function. Eur J Biochem. 1974 Apr 16;43(3):423-35. doi: 10.1111 / j.1432-1033.1974.tb03428.x. PMID: 4831970.

[0068] (1) Preparation of antiheparin-antithrombin complex monoclonal antibody markers Antiheparin-antithrombin complex monoclonal antibody markers were prepared by the following method: a) Weigh 50 mg of acridine ester NSP-DMAE-NHS (purchased from Shanghai Maituowei Chemical New Material Technology Co., Ltd.), and dissolve it in 50 ml of anhydrous N,N-dimethylformamide (DMF); b) Using a suitable reaction tube, add 50 μl of the solution prepared in step a), then add 100 μg of antiheparin-antithrombin complex monoclonal antibody, shake for 30 seconds, and wrap the reaction tube with aluminum foil to protect it from light. c) Place the reaction tube at 37°C and react in the dark for 6 hours; d) After the reaction is complete, add 50 μl of 10 g / L lysine and react at 37°C in the dark for 1 h. e) Transfer the above liquid to a 30k ultrafiltration tube, then place the ultrafiltration tube in a centrifuge, set the speed to 9000 rpm / min, and centrifuge for 25 min; d) After centrifugation, add 1 ml of 50 mM phosphate buffer (pH 7.4, containing 5% BSA) and store at 2-8 ℃ in the dark for later use.

[0069] The composition of the antiheparin-antithrombin complex monoclonal antibody marker reagent is shown in Table 3 below.

[0070] Table 3. Composition of reagents for antiheparin-antithrombin complex monoclonal antibody markers

[0071] Add the materials shown in Table 3 to 1000 mL of deionized water, stir thoroughly to dissolve, and adjust the pH to 6.20 ± 0.10 to obtain the anti-heparin-antithrombin complex monoclonal antibody marker reagent.

[0072] (2) Preparation of magnetic microparticle reagent coated with antithrombin The magnetic microparticles coated with antithrombin were prepared by the following method: a) Using a suitable reaction tube, add 20 mg of carboxyl magnetic microparticles, add 50 mM phosphate buffer (pH 7.4) to make up to 1 ml, vortex for 30 seconds, place the reaction tube in a magnetic separator, let stand for 2 minutes, and then discard the supernatant; b) Add 50 μL of 10 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 10 μL of 10 mg / mL N-hydroxysuccinimide (NHS), and add 50 mM phosphate buffer (pH 7.4) to a final volume of 500 μL. Vortex for 30 seconds. c) Place the above liquid on a rotary mixer (set the rotation speed to 60 rpm) and mix it at 35°C for 1 hour. d) After the reaction is complete, place the reaction tube in the magnetic separator and let it stand for 2 minutes, then remove the supernatant; e) Add 100 μg of antithrombin (purchased from Merck Chemical Technology (Shanghai) Co., Ltd., catalog number SRP6316), add 50 mM phosphate buffer (pH 7.4) to make up to 500 μL, and vortex for 30 seconds; f) Place the above liquid on a rotary mixer (set the rotation speed to 60 rpm) and mix it at 35°C for 3 hours. g) After the reaction is complete, place the reaction tube in the magnetic separator and let it stand for 2 minutes, then remove the supernatant; h) Add 1 ml of 50 mM phosphate buffer (pH 7.4, containing 5% BSA) and store at 2-8 ℃ protected from light for later use.

[0073] The composition of the magnetic microparticle reagent coated with antithrombin is shown in Table 4 below.

[0074] Table 4 Composition of the magnetic microparticle reagent coated with antithrombin

[0075] Add the materials shown in Table 4 to 1000 mL of deionized water, stir thoroughly to dissolve, and adjust the pH to 7.00 ± 0.10.

[0076] (3) Assembly: Assemble the above reagent components into a box and store at 2~8℃.

[0077] Example 2: Test method for the reagent kit (1) Testing process: The test was conducted on the Shine i1910 fully automated chemiluminescence immunoassay analyzer manufactured by Shenzhen Yingkai Biotechnology Co., Ltd. The instrument parameters were set as follows: a) Reaction mode: one-step sandwich process; b) Sample volume: 50 μl; c) Antiheparin-antithrombin complex monoclonal antibody marker reagent sample volume: 50 μl; d) Sample volume of magnetic microparticle reagent coated with antithrombin: 50 μl; e) Incubation time: 20 minutes; f) Number of cleaning cycles: 3 times; g) Add 200 μL of chemiluminescent substrate (purchased from Shenzhen Junhe Biotechnology Co., Ltd.); h) Read the light emission signal.

[0078] In some embodiments of the present invention, the reaction principle of the kit includes: mixing the solid phase coated with antithrombin with the sample to be tested; the heparin in the sample combines with the solid phase coated with antithrombin to form a complex; then, an antiheparin-antithrombin complex monoclonal antibody marker is added for reaction; then, a chemiluminescent substrate is added, and the luminescence signal is tested. The intensity of the luminescence signal is directly proportional to the anticoagulant effect of heparin. That is, the stronger the anticoagulant effect of heparin in the sample, the more complexes are formed, and the stronger the luminescence signal. By establishing a standard curve, the instrument automatically calculates the anticoagulant effect of heparin in the sample. The kit includes an antiheparin-antithrombin complex monoclonal antibody marker, magnetic microparticles coated with antithrombin, and a chemiluminescent substrate component, which has fewer components than traditional kits, higher sensitivity, and a wider linear range.

[0079] In some embodiments of the present invention, the detection method involves mixing a solid phase coated with antithrombin with the sample to be tested. Heparin in the sample to be tested combines with the solid phase coated with antithrombin to form a complex. Then, an antiheparin-antithrombin complex monoclonal antibody marker is added to react, followed by the addition of a chemiluminescent substrate. The luminescence signal is then tested, and the intensity of the luminescence signal is directly proportional to the anticoagulant effect of heparin.

[0080] In some embodiments of the present invention, the linear range of the kit is 0~10.00 U / mL.

[0081] In some embodiments of the present invention, the sensitivity of the kit is 0.002~0.005 U / mL.

[0082] In some embodiments of the present invention, the coefficient of variation of the kit is 1% to 3%.

[0083] In some embodiments of the present invention, the linear range, sensitivity, and repeatability test results of the kit are shown in Table 5.

[0084] Table 5 Performance test results of the reagent kit of the present invention

[0085] The detection performance of the kit in this embodiment was tested using the following methods: a) Calibration: Calibrators were prepared with heparin sample concentrations of 0.00 U / mL, 0.50 U / mL, 1.00 U / mL, 2.00 U / mL, 4.00 U / mL, and 8.00 U / mL. Calibration tests were performed using the kit of this invention on a fully automated chemiluminescence immunoassay analyzer, Shine i1910, manufactured by Shenzhen Yingkai Biotechnology Co., Ltd. Each calibrator was tested twice, and the average values ​​were used to plot a calibration curve. The calibration data are shown in Table 6, and the calibration curve is shown in the figure below. Figure 1 As shown.

[0086] Table 6 Calibration data of the reagent kit of the present invention

[0087] b) Linear Range: Nine linear samples were prepared by serially diluting the high-value heparin sample (concentration of 8.00 U / ml) and the zero-value sample (concentration of 0.00 U / ml) at a volume ratio of 1:1. The linear range was tested using the kit of this invention on a fully automated chemiluminescence immunoassay analyzer, Shine i1910, manufactured by Shenzhen Yingkai Biotechnology Co., Ltd. Each sample was tested twice. The linear range graph is shown below. Figure 2 .

[0088] The linear equation is y = 0.9884x + 0.027, and the correlation R0 is... 2=0.9998. This indicates that the kit of the present invention exhibits good linearity within the concentration range of 0~8.00 U / ml.

[0089] Figure 3 Linearity data were obtained from testing a commercially available reagent kit (Anti-Xa assay kit manufactured by Shanghai Zhenyuan Diagnostic Supplies Co., Ltd.). The results show that the kit of this invention has better linearity compared to the commercially available kit.

[0090] c) Sensitivity: A blank heparin sample (concentration 0.00 U / ml) was repeatedly tested 20 times using the kit of this invention on a fully automated chemiluminescence immunoassay analyzer (Shine i1910) manufactured by Shenzhen Yingkai Biotechnology Co., Ltd. The data are shown in Table 7. The mean + 2SD was calculated, and the results were substituted into the curves fitted by calibrators S1 and S2 to calculate the kit sensitivity to be 0.002 U / ml. Data from commercially available kits are shown in Table 8, with a calculated sensitivity of 0.08 U / ml. The results show that the kit of this invention has higher sensitivity compared to commercially available kits.

[0091] Table 7 Sensitivity data of the reagent kit of the present invention

[0092] Table 8 Sensitivity data for commercially available reagent kits

[0093] d) Reproducibility: Heparin samples (concentration 1.00 U / ml) were repeatedly tested 20 times using the kit of this invention on a fully automated chemiluminescence immunoassay analyzer, Shine i1910, manufactured by Shenzhen Yingkai Biotechnology Co., Ltd. The data are shown in Table 9. The calculated coefficient of variation was 2.46%, indicating good reproducibility of the kit of this invention. Test data from commercially available kits are shown in Table 10, with a calculated coefficient of variation of 5.48%, indicating even better reproducibility of the kit of this invention.

[0094] Table 9. Repeatability data of the reagent kit of the present invention

[0095] Table 10. Reproducibility data of commercially available reagent kits

[0096] 2) Correlation analysis: Forty-eight clinical plasma samples were tested using the kit of the present invention and a commercially available kit (anti-Xa assay kit produced by Shanghai Zhenyuan Diagnostic Supplies Co., Ltd., chromogenic substrate method). The test results are shown in Table 11. The negative concordance rate, positive concordance rate and total concordance rate were all 100%, indicating that the kit of the present invention has good clinical accuracy.

[0097] Table 11. Reagent kit accuracy test data

[0098] The results above show that the heparin drug anticoagulation effect detection kit of the present invention has good repeatability, wide linearity, and high sensitivity.

[0099] The above description is merely an example and illustration of the structure of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. An antibody molecule, characterized in that, The antibody molecule comprises a light chain and a heavy chain, wherein the complementarity-determining regions of the light chain include LCDR1, LCDR2, and LCDR3, and the complementarity-determining regions of the heavy chain include HCDR1, HCDR2, and HCDR3; wherein, The amino acid sequence of LCDR1 is shown in SEQ ID NO:3; The amino acid sequence of LCDR2 is shown in SEQ ID NO:4; The amino acid sequence of LCDR3 is shown in SEQ ID NO:5; The amino acid sequence of HCDR1 is shown in SEQ ID NO:10; The amino acid sequence of HCDR2 is shown in SEQ ID NO:11; The amino acid sequence of HCDR3 is shown in SEQ ID NO:

12.

2. The antibody molecule according to claim 1, characterized in that, The framework region of the light chain includes LFR1, LFR2, LFR3, and LFR4, wherein, The amino acid sequence of the LFR1 is shown in SEQ ID NO:6; And / or, the amino acid sequence of the LFR2 is as shown in SEQ ID NO:7; And / or, the amino acid sequence of the LFR3 is as shown in SEQ ID NO:8; And / or, the amino acid sequence of the LFR4 is as shown in SEQ ID NO:9; And / or, the complementary determining regions and frame regions of the light chain are arranged in the order LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

3. The antibody molecule according to claim 1, characterized in that, The frame region of the heavy chain includes HFR1, HFR2, HFR3, and HFR4, wherein, The amino acid sequence of HFR1 is shown in SEQ ID NO:13; And / or, the amino acid sequence of the HFR2 is shown in SEQ ID NO:14; And / or, the amino acid sequence of the HFR3 is shown in SEQ ID NO:15; And / or, the HFR4 amino acid sequence is as shown in SEQ ID NO:16; And / or, the complementary determining regions and frame regions of the heavy chain are arranged in the order HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

4. The antibody molecule according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the antibody molecule is shown in SEQ ID NO:1; And / or, the amino acid sequence of the heavy chain variable region of the antibody molecule is shown in SEQ ID NO:2; And / or, the antibody molecule is capable of binding to the heparin-antithrombin complex.

5. A nucleic acid molecule, characterized in that, The antibody molecule is encoded as described in any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:17; And / or, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

18.

7. A reagent kit, characterized in that, It includes antibody molecules encoded by the antibody molecules of any one of claims 1 to 4 or the nucleic acid molecules of any one of claims 5 to 6.

8. The reagent kit according to claim 7, characterized in that, The antibody molecules are labeled to form antibody markers; And / or, the antibody molecule is labeled with at least one of acridine ester, alkaline phosphatase, ruthenium tripyridine, and horseradish peroxidase to form an antibody marker.

9. The use of the antibody molecule as described in any one of claims 1 to 4, or the antibody molecule encoded by the nucleic acid molecule as described in any one of claims 5 to 6, or the kit as described in any one of claims 7 to 8, in the method for detecting the anticoagulant effect of heparin.

10. The application according to claim 9, characterized in that, The detection method uses the antibody molecules to detect the content of the heparin-antithrombin complex to obtain the anticoagulant effect of the heparin drug.

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