Preparation method of macromolecular d-dimer and application thereof

Abstract

The application discloses a preparation method and application of macromolecular D-dimer, and first finds that tannic acid or glutaraldehyde is used as a coagulation accelerator, and EDTA or aprotinin is used as a fibrinolysis terminator, so that the macromolecular D-dimer can be efficiently prepared in vitro; and a stable, repeatable and large-scale preparation process for the macromolecular D-dimer is first established. The application provides key raw materials and technical support for precise diagnosis and dynamic monitoring of thrombus, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a macromolecular D-dimer and its application. Background Technology

[0002] D-dimer is a key biomarker for the activation of the coagulation and fibrinolytic systems. Based on molecular weight and structural characteristics, it can be divided into two categories: large molecular weight D-dimer and small molecular weight D-dimer. Large molecular weight D-dimer is the complete major product of early, in-situ degradation of cross-linked fibrin by plasmin. It is generated in large quantities only when pathological thrombi form in vivo and secondary hyperfibrinolysis occurs, and can directly correspond to fresh, active thrombus states. Small molecular weight D-dimer is the terminal fragment of further degradation of large molecules and can also originate from the degradation of old thrombi. It is not an exclusive biomarker for active thrombi, and the two have significantly different values ​​in clinical diagnosis.

[0003] Currently, clinical testing for D-dimer measures the total concentration of all D-dimer epitope fragments in the blood. Detection methods primarily include double-antibody sandwich ELISA and latex immunoturbidimetry, both of which calculate concentration by measuring the total amount of antigen-antibody complexes and cannot distinguish between large and small molecule components. Total D-dimer concentration is easily affected by various factors such as renal insufficiency, infection, inflammation, tumors, and pregnancy, leading to a high likelihood of false positives. It can only serve as a diagnostic indicator for thrombosis exclusion; its specificity for confirming thrombosis is extremely low, failing to meet the needs of precise diagnosis and treatment.

[0004] Compared to total D-dimer, large molecular weight D-dimer offers significant clinical advantages in thrombosis diagnosis and treatment: its levels are unaffected by clearance barriers of small molecules, accurately reflecting newly formed thrombosis and demonstrating significant value in monitoring recurrent thrombosis; when anticoagulation therapy is effective, large molecular weight D-dimer levels decrease rapidly, allowing for direct assessment of treatment efficacy. Therefore, successfully preparing and obtaining large molecular weight D-dimer is a core prerequisite for developing specific detection antibodies and establishing dedicated detection methods for large molecular weight D-dimer, and it is also a key technical problem that urgently needs to be solved in the field of precision thrombosis diagnosis. Summary of the Invention

[0005] In view of this, in order to solve the above-mentioned technical problems existing in the field, the purpose of this invention is to provide a method for preparing macromolecular D-dimers and their applications.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution: The first aspect of the present invention provides a method for preparing a macromolecular D-dimer.

[0007] Furthermore, the method includes the following steps: (1) Coagulation: Plasma is subjected to a cross-linking reaction under coagulation activation conditions to obtain cross-linked fibrin; (2) Coagulation: A coagulating agent is added to the cross-linked fibrin to carry out a coagulation reaction to obtain fixed cross-linked fibrin. The coagulating agent is selected from tannic acid solution or glutaraldehyde solution. (3) Fibrinolysis: Add streptokinase and plasminogen to the fixed cross-linked fibrin to carry out the fibrinolysis reaction and obtain fibrinolytic products; (4) Termination: A terminator is added to the fibrinolytic product to terminate the fibrinolytic reaction and obtain a concentrated fibrinolytic solution. The terminator is selected from EDTA solution or aprotinin. The concentrated fibrinolytic solution contains a large molecular D-dimer.

[0008] Furthermore, the coagulation activation conditions described in step (1) are to mix plasma, calcium chloride and thrombin, and incubate in a 37°C water bath for 1-5 hours; Optionally, the coagulation activation conditions in step (1) are to mix plasma, calcium chloride and thrombin and incubate in a 37°C water bath for 2 hours; Optionally, the amounts of plasma, calcium chloride, and thrombin used are 1-10 mL of plasma, 1-10 mL of 10-100 mmol / L calcium chloride, and 0.1-10 kU of thrombin, respectively. Optionally, the amounts of plasma, calcium chloride, and thrombin used are 5 mL of plasma, 5 mL of 30 mmol / L calcium chloride, and 1 kU of thrombin, respectively. Optionally, step (1) further includes centrifuging the product after the water bath to obtain cross-linked fibrin.

[0009] Furthermore, the reaction concentration of the tannic acid solution in step (2) is 0.01%-0.5% (w / v). Optionally, the reaction concentration of the glutaraldehyde solution in step (2) is 0.005%-0.2% (w / v). Optionally, the conditions for the coagulation reaction in step (2) are 10 min-2 h at room temperature; Optionally, the reaction concentration of the tannic acid solution in step (2) is 0.02% (w / v). Optionally, the reaction concentration of the glutaraldehyde solution in step (2) is 0.01% (w / v). Optionally, the conditions for the coagulation reaction in step (2) are a reaction at room temperature for 30 min; Optionally, step (2) further includes removing the supernatant from the reaction product to obtain the fixed cross-linked fibrin.

[0010] Furthermore, when the coagulant is tannic acid, the conditions for the fibrinolytic reaction in step (3) are: adding 1-125 U of streptokinase and 0.0002-0.05 U of plasminogen, and reacting at room temperature for 1-18 h; Optionally, when the coagulant is glutaraldehyde, the conditions for the fibrinolytic reaction in step (3) are: adding 1-125 U of streptokinase and 0.0002-0.05 U of plasminogen, and reacting at room temperature for 2-5 h; Optionally, when the coagulant is tannic acid, the conditions for the fibrinolytic reaction in step (3) are: adding 12.5 U of streptokinase and 0.0025 U of plasminogen, and reacting at room temperature for 3 h; Optionally, when the coagulant is glutaraldehyde, the fibrinolytic reaction conditions in step (3) are: adding 12.5 U of streptokinase and 0.0025 U of plasminogen, and reacting at room temperature for 3 h.

[0011] Furthermore, the reaction concentration of the EDTA solution in step (4) is 1-15 mM; Optionally, the reaction concentration of the EDTA solution is 5 mM; Optionally, the reaction concentration of the aprotinin in step (4) is 0.1-5 kU / mL; Optionally, the reaction concentration of the aprotinin in step (4) is 0.5 kU / mL.

[0012] Furthermore, the method also includes purifying the fibrinolytic concentrate in step (4) to obtain purified macromolecular D-dimer; Optionally, the purification includes affinity purification using antibodies that specifically recognize macromolecular D-dimers, and / or separation using molecular sieves.

[0013] In some implementations, after steps (1) and (2) and before the addition of plasminogen, a step of washing the coagulated cross-linked fibrin is included. Specifically, after the coagulation reaction is completed with tannic acid or glutaraldehyde solution in a buffer such as PBS, the supernatant is removed, and the fixed cross-linked fibrin is washed at least once with fresh PBS buffer to remove residual coagulant and other soluble impurities, providing a purer reaction system for subsequent fibrinolysis steps. For example, after the coagulation reaction is completed, the fibrin is washed twice with 5 mL of 0.01 M PBS solution.

[0014] In some embodiments, step (4), after adding the terminator, further includes a step of concentrating the reaction mixture and / or replacing the buffer. Specifically, this may include concentrating the reaction solution containing the large D-dimer using an ultrafiltration tube (e.g., with a molecular weight cutoff of 100 kDa) to enrich the target product, and during this process, repeatedly washing with a buffer such as PBS to remove small molecule impurities, salts, and residual terminator, ultimately obtaining a concentrated fibrinolytic concentrate with a more homogeneous composition. For example, the reaction solution may be concentrated to approximately 300 μL or 100 μL and washed with PBS.

[0015] In some embodiments, the fibrinolytic concentrate containing macromolecular D-dimers obtained in step (4) can be used directly or after further separation and purification to prepare standards, quality control products, or to immunize animals to prepare specific antibodies. The separation and purification method, in addition to affinity purification (such as magnetic bead antibody purification) and molecular sieve chromatography using antibodies that specifically recognize D-dimers, may also include conventional protein purification techniques such as ion exchange chromatography and hydrophobic chromatography to obtain macromolecular D-dimers with higher purity.

[0016] In some embodiments, the method further includes a step of identifying the final macromolecular D-dimer product. This identification step may include: verifying the apparent molecular weight (approximately 228 kDa) of the target product by denaturing non-reducing SDS-PAGE electrophoresis; and / or identifying whether it contains D-dimer-specific peptide sequences (e.g., cross-linking sequences of Gamma-A or Gamma-B proteins) by mass spectrometry analysis (e.g., liquid chromatography-tandem mass spectrometry).

[0017] In a specific embodiment of the present invention, the final product is determined to be a macromolecular D-dimer through the following two methods: First, denaturing non-reducing SDS-PAGE electrophoresis verifies that the target product presents a single main band in the electrophoretic pattern, with an apparent molecular weight of approximately 228 kDa, located below the 250 kDa standard protein band, which is consistent with the estimated molecular weight range of macromolecular D-dimers (DDE complexes). This confirms from a physical molecular weight perspective that the product is different from small molecule degradation fragments. Second, mass spectrometry analysis (such as liquid chromatography-tandem mass spectrometry) identifies the gel strip. The results show that the band cut at approximately 228 kDa contains a specific peptide sequence of the D-dimer, particularly detecting cross-linking characteristic peptides from Gamma-A and / or Gamma-B proteins, thus confirming from a primary structural perspective that it contains a DD-dimer motif. Combining its large molecular weight (approximately 228 kDa) and characteristic peptide sequence, it can be finally confirmed that the prepared product is a structurally complete macromolecular D-dimer containing a DDE structure.

[0018] In some embodiments, the purification step includes: incubating the fibrinolytic concentrate with magnetic beads conjugated with anti-human D-dimer antibody, washing and eluting to obtain the purified antibody product, and then further separating it by molecular sieve chromatography to obtain high-purity macromolecular D-dimer.

[0019] In some embodiments, the macromolecular D-dimer has a molecular weight of approximately 228 kDa and a structure of a DDE complex.

[0020] In some embodiments, the macromolecular D-dimer comprises a D-dimer-specific sequence and Gamma. AD D-dimer characteristic peptides.

[0021] In some embodiments, the macromolecular D-dimer prepared by the method contains little or no small molecule D-dimer, and can be specifically used for the detection of active thrombosis-related disorders.

[0022] In some implementations, the preparation method is an in vitro preparation method, which does not rely on metabolism in the human body or animal, and can be stably repeated and is suitable for scale-up production.

[0023] In a specific embodiment of the present invention, the macromolecule D Dimer refers to the DD / E complex formed by the incomplete early degradation of cross-linked fibrin by plasmin. Its structure is DDE, and its molecular weight is approximately 228 kDa. It is a specific marker of fresh, active thrombi, distinguishing it from the smaller molecule D produced by further degradation. Dimer.

[0024] A second aspect of the present invention provides a macromolecular D-dimer prepared by the method described in the first aspect of the present invention.

[0025] In some embodiments, the macromolecular D-dimer prepared by the method described in the first aspect of the present invention has a molecular weight of approximately 228 kDa. This can be verified by non-reducing SDS-PAGE electrophoresis, in which its position in the electrophoretic pattern should be below the 250 kDa standard protein band. This characteristic molecular weight is one of the key physical indicators that distinguishes it from smaller molecule D-dimer fragments.

[0026] In some embodiments, the macromolecular D-dimer contains a basic DD (D-dimer) structure, on which an E fragment is covalently bonded, with the structure DDE. This structural feature is the core basis for its designation as a macromolecular D-dimer, and also provides direct evidence that it is the complete major product of the early, in-situ degradation of cross-linked fibrin by plasmin.

[0027] In some embodiments, the macromolecular D-dimer can be identified by mass spectrometry to determine the specific peptide sequence of the D-dimer. Specifically, the mass spectrometry results should show cross-linking characteristic peptides from Gamma-A and / or Gamma-B proteins, which confirms the presence of its D-dimer motif at the primary structural level.

[0028] In some embodiments, the macromolecular D-dimer can be used to prepare antibodies that specifically recognize it. Because this product is a structurally defined antigen prepared by a specific method, immunizing animals with it as an immunogen can produce antibodies that bind to the macromolecular D-dimer (rather than all fragments containing D-dimer epitopes) with high specificity and high affinity. Such antibodies are a core ingredient for developing subsequent specific detection kits.

[0029] In some implementations, the macromolecular D-dimer is a specific marker for fresh thrombi and active thrombi, unaffected by small molecule D-dimers, and has higher clinical diagnostic specificity.

[0030] In some implementation schemes, the macromolecular D-dimer has clear clinical application value in monitoring anticoagulation therapy, predicting thrombosis recurrence, and dynamically monitoring thrombosis in very high-risk patients.

[0031] The third aspect of the invention provides any of the following products: (1) An antibody for specifically recognizing a macromolecular D-dimer, wherein the antibody is prepared using the macromolecular D-dimer described in the second aspect of the present invention as an immunogen; (2) A kit for detecting macromolecular D-dimer, the kit comprising the antibody.

[0032] In some implementations, the antibody used to specifically recognize the large D-dimer can be a polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody, fully human antibody, or its antigen-binding fragment. The antigen-binding fragment can be selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, single-chain antibodies (scFv), or nanobodies. These different forms of antibodies and their fragments provide a variety of options to meet the sensitivity, specificity, and stability requirements of different detection platforms (such as enzyme-linked immunosorbent assay, chemiluminescence, latex immunoturbidimetry, etc.) or diagnostic applications.

[0033] In some embodiments, the kit for detecting macromolecular D-dimers includes labeled antibodies. The label may be, but is not limited to, enzymes, fluorescent substances, chemiluminescent substances, radioactive isotopes, biotin, latex particles, or gold particles. Specifically, when the antibody is a monoclonal antibody, it can be used as a capture antibody immobilized on a solid-phase carrier, while a monoclonal antibody labeled with the same or different epitopes of a reporter molecule can be used as the detection antibody, thereby constructing a double-antibody sandwich assay kit with higher detection specificity.

[0034] In some embodiments, the kit may further comprise one or more of the following components: a sample diluent for diluting or dissolving the sample, a wash solution for rinsing unbound substances, a stop solution for terminating the reaction, a substrate solution for color development or signal generation, and calibrators containing a known concentration of macromolecular D-dimers and / or quality control materials for quality control. These components together constitute a complete detection system, ensuring accuracy, repeatability, and ease of operation.

[0035] In some implementations, the kit may be in the form of an enzyme-linked immunosorbent assay (ELISA) plate, a chemiluminescent immunoassay plate, a latex-enhanced immunoturbidimetric assay card, a lateral flow immunochromatographic test strip, or a reagent kit suitable for a fully automated immunoassay analyzer. These different product forms can meet a variety of application needs, from manual laboratory operations to rapid clinical emergency testing, and high-throughput fully automated analysis.

[0036] The fourth aspect of the present invention provides the use of the macromolecular D-dimer prepared by the method described in the first aspect of the present invention in the preparation of detection reagents or detection products for thrombosis detection, dynamic monitoring of thrombosis, evaluation of the effect of anticoagulation therapy or prediction of thrombosis recurrence.

[0037] Furthermore, the testing products are selected from test kits, test strips, test chips, automated testing instruments, or automated testing systems.

[0038] In some implementations, the thrombosis detection can be an auxiliary diagnostic test for venous thromboembolism. Specifically, a highly specific antibody or detection kit developed based on the macromolecular D-dimer prepared by this method is used to detect the concentration of macromolecular D-dimer in patient plasma or serum samples. Since the macromolecular D-dimer prepared by this invention is a specific marker of fresh, active thrombi, compared with conventional total D-dimer detection, its detection results can effectively exclude false-positive interference caused by non-thrombotic factors such as renal insufficiency, inflammation, and pregnancy, thereby significantly improving the specificity of venous thrombosis diagnosis and providing more accurate auxiliary diagnostic evidence for clinical practice.

[0039] In some implementations, the dynamic thrombosis monitoring can be conducted periodically or irregularly on high-risk groups for thrombosis, such as cancer patients, patients with thrombophilia, and patients after major orthopedic surgery. By using a detection product containing a specific antibody against the large molecular weight D-dimer of this invention, specific high-risk patients can be continuously tested at multiple time points. If a significant increase in the level of the large molecular weight D-dimer is detected, it can indicate the risk of new thrombosis or increased thrombotic activity more promptly and accurately than total D-dimer, thereby supporting timely clinical intervention and achieving early warning of thrombosis.

[0040] In some implementations, the assessment of anticoagulation therapy efficacy can be a monitoring of the treatment's effectiveness in thrombotic patients receiving anticoagulation therapy (such as heparin, warfarin, or novel oral anticoagulants). Since large molecular weight D-dimer originates directly from active thrombi, its level should decrease rapidly when anticoagulation therapy is effective. Monitoring the trend of large molecular weight D-dimer levels in patients after treatment can help physicians more accurately determine the effectiveness of anticoagulation therapy, assess thrombolysis, and provide objective evidence based on specific molecular markers for adjusting treatment regimens (such as drug dosage and duration), rather than relying solely on nonspecific observations of a decrease in total D-dimer.

[0041] In some implementations, the prediction of thrombotic recurrence can be a prognostic assessment for patients who have completed a phase of anticoagulation therapy but remain at high risk of recurrence. After patients discontinue medication, periodic monitoring of their D-dimer levels is used. If these levels rise again, it strongly suggests a risk of thrombotic recurrence or reformation, even if the total D-dimer level may not yet be significantly elevated. This provides clinicians with a more sensitive and specific early warning signal to assess the risk of patient discontinuation and develop individualized prevention strategies (such as prolonged anticoagulation or enhanced monitoring).

[0042] In addition, the present invention provides a method for detecting macromolecular D-dimers in a sample, the method comprising the following steps: detecting the sample using the antibody or the kit described above.

[0043] In some implementations, the detection is an immunoassay, including but not limited to: enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, or latex immunoturbidimetric assay.

[0044] Furthermore, the present invention also provides a method for thrombosis detection, dynamic monitoring of thrombosis, evaluation of anticoagulation therapy efficacy, or prediction of thrombosis recurrence, the method comprising the following steps: (1) Obtain biological samples from the subjects; (2) Detect the level of macromolecular D-dimer in the biological sample using the antibody or kit described above; (3) Assess the thrombosis status, anticoagulation therapy efficacy, or risk of thrombosis recurrence based on the level of the macromolecular D-dimer.

[0045] In some implementations, the biological sample from the subject may be a clinical sample collected for thrombosis risk screening, thrombotic event diagnosis, anticoagulation therapy monitoring, or prognostic assessment. Specifically, the biological sample includes, but is not limited to, plasma, serum, or whole blood. Plasma obtained from anticoagulated whole blood is preferred because it minimizes the formation of additional D-dimer during coagulation, thus most accurately reflecting the level of large-molecule D-dimer already present in the body due to thrombus fibrinolysis, ensuring the in vivo relevance of the test results. After collection, the sample can be processed and stored according to standard clinical laboratory procedures for testing.

[0046] In some implementations, the step of assessing the level of macromolecular D-dimer can be specified as a comparison with a pre-established threshold or dynamic trend. For thrombosis detection, a clinically determined threshold can be set; a value higher than this threshold suggests a high probability of active thrombosis. For evaluating the effectiveness of anticoagulation therapy, the magnitude and rate of decrease in macromolecular D-dimer levels before and after treatment (e.g., key time points such as 24 hours, 3 days, and 7 days after the start of treatment) can be compared; a rapid and significant decrease usually indicates effective treatment. For predicting thrombosis recurrence, after the patient completes a phase of anticoagulation therapy, an observation period can be entered, with regular monitoring (e.g., every 1-3 months). If the level consistently or intermittently increases from a post-treatment low, it suggests a high risk of recurrence. This method utilizes macromolecular D-dimer as a specific marker of active thrombosis, providing a more precise quantitative basis for clinical decision-making than total D-dimer.

[0047] In some implementation schemes, the subjects refer to individuals who require or are undergoing thrombosis-related assessment or monitoring, including but not limited to: patients suspected of having venous thromboembolism (such as deep vein thrombosis or pulmonary embolism); individuals at high risk of thrombosis, such as patients with malignant tumors, patients with thrombophilia, patients who have recently undergone major surgery (especially orthopedic or oncological surgery), and patients who are bedridden or inactive for extended periods; thrombotic patients currently receiving anticoagulation therapy (such as using heparin, warfarin, or direct oral anticoagulants) and requiring monitoring of efficacy; and post-treatment patients who have completed a phase of anticoagulation therapy, are in the observation period after drug withdrawal, but still have a risk of recurrence. By performing macromolecular D-dimer detection on biosamples from these specific populations, accurate diagnosis of thrombosis, dynamic risk warning, treatment efficacy evaluation, and recurrence prediction can be achieved.

[0048] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention is the first to discover that by using tannic acid or glutaraldehyde as a coagulant accelerator, and in combination with EDTA or aprotinin as a fibrinolysis terminator, it is possible to efficiently prepare large molecular weight D in vitro. Dimers; and for the first time, a macromolecular D that can be stably and reproducibly prepared and is suitable for large-scale production was established. In vitro preparation process of dimer, which solves the technical problem that the prior art cannot obtain macromolecular D dimer directionally. The macromolecular D dimer prepared by the present invention The dimer has a molecular weight of about 228 kDa and a structure of D D E, containing D Dimer specific sequence and Gamma A D D dimer characteristic peptide segment, which can be used for specific antibody screening and development of detection reagents. Compared with the detection of total D dimer, the macromolecular D dimer prepared by the present invention can effectively avoid false positive interference caused by factors such as renal function, inflammation, and tumor, and has higher specificity and clinical value in the judgment of new thrombosis, monitoring of anticoagulation efficacy, and prediction of recurrent thrombosis, providing key raw materials and technical support for accurate diagnosis and dynamic monitoring of thrombosis. Brief Description of the Drawings

[0049] Figure 1 : Schematic diagram of the D-dimer formation process; Figure 2 : Electrophoresis result diagram of macromolecular D-dimer; Figure 3 : Mass spectrometry result diagram of mass spectrometry detection of the cut macromolecular D-dimer gel strip; Figure 4 : Electrophoresis result diagram corresponding to Comparative Example 1; Figure 5 : Electrophoresis result diagram corresponding to Comparative Example 2; Figure 6 : Electrophoresis result diagram corresponding to Comparative Example 3; Figure 7 : Electrophoresis result diagram corresponding to Comparative Example 4; Figure 8 : Electrophoresis result diagram corresponding to Comparative Example 5; Figure 9 : Electrophoresis result diagram corresponding to Example 2. Detailed Embodiments

[0050] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommended conditions.

[0051] D-dimer is a general term referring to multiple peptide fragments produced by plasmin-mediated cross-linked fibrin degradation, with molecular weights ranging from 160 to over 10,000 kDa. Its presence reflects the simultaneous activation of the coagulation and fibrinolytic systems. Only fibrin polymers cross-linked via coagulation factor XIII will produce fragments containing covalent bonds between two adjacent D domains (i.e., D-dimers).

[0052] The macromolecular D-dimer is the main complete product formed during the early, in-situ degradation of cross-linked fibrin under the action of plasmin. It belongs to the larger molecular weight fragments in the D-dimer family, with a molecular weight of 228 kDa. Structurally, it contains the most basic DD (D-dimer) unit, which is covalently bonded to an E fragment, thus forming a complex with the structure DDE. This ternary complex structure of DDE makes its molecular weight significantly larger than that of the small molecule D-dimer (DD) (with a molecular weight of 160 kDa), which consists of only two D domains, and it contains the specific sequence of the D-dimer.

[0053] The initial reaction in D-dimer formation involves thrombin catalyzing the conversion of fibrinogen into fibrin monomers. Human fibrinogen is a soluble plasma glycoprotein composed of three pairs of distinct polypeptide chains (Aa, Bβ, and Cγ chains). The fibrin monomers then bind together to form a soluble network. Simultaneously, the complex formed by the soluble fibrin polymer, thrombin, and plasma factor XIII promotes the generation of factor XIIIa, which catalyzes the covalent cross-linking of the fibrin polymer via intermolecular bonds between lysine and glutamine residues, thereby forming a stable and insoluble clot. Subsequently, the fibrinolytic pathway leads to the degradation of the stable clot through plasmin activation. Plasmin is activated from plasminogen on the fibrin surface by tissue plasminogen activator (t-PA) and cleaves fibrin at specific sites. Continued degradation produces a fragment D-dimer / fragment E complex (DD / E, or DDE), which has two covalently bound D domains and an E fragment. Due to variations in the degree of plasmin-mediated proteolysis, plasma samples contain a mixture of fibrin fragment complexes comprising one or more D-dimer motifs, with molecular weights ranging from 228 kDa (DD / E) to several thousand kDa (X-oligomers). The D-dimer formation process is as follows... Figure 1 As shown.

[0054] Example 1: Preparation and Verification of Macromolecular D-Dimers 1. Experimental materials Fresh human plasma (source: Peking University International Hospital), thrombin (9002-04-4, Yusen Biotechnology), plasminogen (16-16-161200, Athens), streptokinase (YC0002, Yawei), physiological saline, 30 mmol / L calcium chloride solution, 0.01 M PBS solution, 0.5 M EDTA solution, freshly prepared 20% (w / v) tannic acid solution (solvent: physiological saline), freshly prepared 20% (w / v) glutaraldehyde solution (solvent: physiological saline), aprotinin (10820, Sigma), D-dimer antibody (DD(1H6), Wantai Biopharmaceutical), magnetic bead purification reagents, denaturing non-reducing SDA-PAGE electrophoresis materials, small molecule D-dimer antigen (8D70, Hytest).

[0055] 2. Experimental Methods 2.1 Solidification Take a 50 mL centrifuge tube and add 20 mL of physiological saline, 5 mL of 30 mmol / L calcium chloride, and 1 kU of porcine thrombin in sequence. Mix well, then add 5 mL of fresh plasma. Mix well and incubate at 37°C for 2 h. Centrifuge at 1500 g for 10 min. At this point, the test tube will contain white, gelatinous cross-linked fibrin. Discard the supernatant, leaving the white, gelatinous cross-linked fibrin.

[0056] 2.2 Coagulation promoters (tannic acid group and glutaraldehyde group) Add the cross-linked fibrin prepared in the previous step, 2 mL of 0.01 M PBS solution, and 2 μL of freshly prepared 20% tannic acid solution (the 20% tannic acid solution is a concentrated solution, with an actual reaction concentration of 0.02% (w / v)) or 1 μL of freshly prepared 20% glutaraldehyde solution (the 20% glutaraldehyde solution is a concentrated solution, with an actual reaction concentration of 0.01% (w / v)) to a 50 mL centrifuge tube. Mix well and react at room temperature for 30 min. After the reaction is complete, discard the supernatant and retain the solid cross-linked fibrin. Wash twice with 5 mL of 0.01 M PBS solution. Take another 50 mL centrifuge tube, add the washed cross-linked fibrin and 2 mL of 0.01 M PBS solution, and set aside for later use.

[0057] 2.3 Fibrinolysis and Fibrinolysis Termination Tannic acid-induced coagulation group: 12.5 U of streptokinase (i.e., streptococcal streptokinase, enzyme activity unit defined as: the amount of enzyme required to liquefy a standard clot composed of fibrinogen, plasminogen, and thrombin within 10 minutes under conditions of 37°C and pH 7.5) and 0.0025 U of plasminogen (i.e., human plasma plasminogen, enzyme activity unit defined as: the amount of plasminogen required to completely activate plasminogen at 37°C and pH 7.5 and hydrolyze 1 μmol of lysine equivalent substrate per minute under conditions of 37°C and pH 7.5) were added to the prepared cross-linked fibrin and 2 mL of 0.01 M PBS solution, mixed well, and fibrinolysed at room temperature for about 3 hours. After fibrinolysis, pipette 1 mL of the supernatant into a centrifuge tube. Add 10 μL of 0.5 M EDTA solution (0.5 M is the concentrate; the actual reaction concentration is 5 mM) or 0.5 kU aprotinin (reaction volume is 1 mL, aprotinin dosage is 0.5 kU, aprotinin reaction concentration is 0.5 kU / mL; enzyme activity unit definition: 1 U of aprotinin that can completely inhibit 1 unit of trypsin at 25℃ and pH 8.0) to terminate the fibrinolysis reaction. Concentrate using a 100 KD ultrafiltration tube, and wash three times with 0.01 M PBS solution. At this point, the ultrafiltration tube contains approximately 300 μL of concentrated fibrinolytic solution. Pipette this solution into a centrifuge tube, and wash the ultrafiltration tube with 200 μL of 0.01 M PBS solution, adding the washings to the concentrate. Finally, approximately 500 μL of concentrated fibrinolytic solution is obtained.

[0058] Glutaraldehyde-induced coagulation group: 12.5 U of streptokinase and 0.0025 U of plasminogen were added to the prepared cross-linked fibrin and 2 mL of 0.01 M PBS solution. The mixture was incubated and fibrinolysed at room temperature for approximately 3 hours. After fibrinolysis, 1 mL of the supernatant was pipetted into a centrifuge tube. 10 μL of 0.5 M EDTA solution (0.5 M is the concentrate; the actual reaction concentration is 5 mM) or 0.5 kU aprotinin (reaction volume 1 mL, aprotinin dosage 0.5 kU, aprotinin reaction concentration 0.5 kU / mL) was added to terminate the fibrinolysis reaction. The solution was concentrated using a 100 KD ultrafiltration tube and washed three times with 0.01 M PBS solution. The ultrafiltration tube contained approximately 100 μL of the concentrated fibrin solution. This was pipetted into a centrifuge tube, and the ultrafiltration tube was washed with 200 μL of 0.01 M PBS solution. The washings were added to the concentrate. The final result is approximately 300 μL of concentrated fiber solution.

[0059] 2.4 Magnetic bead antibody purification The concentrated fiber solution was incubated with magnetic beads conjugated with mouse anti-human D-dimer antibody at room temperature for 2 h. After removing the supernatant by placing the solution on a magnetic rack, the magnetic beads were washed with PBS and pure water, followed by elution twice with elution buffer. The elution buffer was concentrated in a 100 kDa ultrafiltration tube, and the solution was changed twice with 0.01 M PBS. Approximately 100 μL of purified D-dimer mixture was finally obtained.

[0060] 2.5 Protein Identification SDS-PAGE electrophoresis: Denaturing non-reducing electrophoresis was used for preliminary verification. The loading buffer and electrophoresis buffer did not contain reducing agents but contained SDS.

[0061] Mass spectrometry: The macromolecular D-dimer strips obtained after SDS-PAGE electrophoresis were cut off and identified by mass spectrometry. For the mass spectrometry detection method, please refer to the article: DOI: 10.1021 / acs.jproteome.0c00148.

[0062] 2.6 Molecular sieve purification Macromolecular D-dimers are separated from a mixed solution of D-dimers using molecular sieves.

[0063] 3. Experimental Results Electrophoresis results are shown in the figure. Figure 2 As shown, the molecular weight was determined to be approximately 228 kDa, which falls within the molecular weight range of macromolecular D-dimers. Therefore, the prepared solution is indeed a macromolecular D-dimer solution. The macromolecular D-dimer strip was cut and analyzed by mass spectrometry. The mass spectrometry results are shown below. Figure 3As shown, the results revealed the presence of a D-dimer-specific sequence, confirming that the band is a large D-dimer. (The band at approximately 228 kDa (consistent with the estimated molecular weight of a large D-dimer) contained a unique and specific amino acid sequence characteristic of D-dimers, confirming that the banded substance is a D-dimer and not some other unrelated protein. Combined with its large molecular weight (228 kDa, excluding small D-dimers), it can be confirmed that it is a structurally complete large D-dimer containing a DDE structure.) Furthermore, the observation of secondary fragment ion information of the Gamma-A DD dimer peptide confirms the presence of the DD dimer.

[0064] Comparative Example 1: Effect of no coagulation accelerator and no termination step on the preparation method 1. Experimental Methods Except for the absence of coagulation accelerator and termination step, the other experimental conditions are basically the same as those in Example 1. The specific experimental conditions are shown in Table 1 below.

[0065] Table 1. Experimental conditions for Comparative Example 1 (except for the experimental conditions in the table, all other conditions are the same as in Example 1).

[0066] 2. Experimental Results Electrophoresis results are shown in the figure. Figure 4 As shown, the molecular weight of the macromolecule DD (macromolecule D-dimer) is 228 kDa, which should be below 250 kDa in the Marker. However, it does not appear in the gel image. This indicates that only coagulation and fibrinolysis are present, without coagulation promotion and termination. Only small-molecule D-dimers can be obtained, and macromolecule D-dimers cannot be obtained. Whether the fibrinolysis conditions are fixed or the coagulation conditions (concentration, temperature, time) are changed, or the coagulation conditions are fixed and the fibrinolysis conditions (concentration, temperature, time) are changed, macromolecule D-dimers cannot be obtained.

[0067] Comparative Example 2: Effect of different coagulants on the preparation method 1. Experimental Methods Except for the type of coagulant, the other experimental conditions were basically the same as in Example 1. The specific experimental conditions are shown in Table 2 below.

[0068] Table 2 Experimental conditions for Comparative Example 2 (except for the experimental conditions in the table, all other conditions are the same as in Example 1)

[0069] 2. Experimental Results Electrophoresis results are shown in the figure. Figure 5As shown, the results indicate that although proanthocyanidins, tannic acid, and glutaraldehyde are all commonly used coagulants in biological experiments, not all coagulants can produce large molecular D-dimers. Only tannic acid and glutaraldehyde can successfully prepare large molecular D-dimers as coagulants. This result is a technical effect that would not have been expected by those skilled in the art based on the existing technology.

[0070] Comparative Example 3: Effect of different terminators on the preparation method (using glutaraldehyde as a coagulant). 1. Experimental Methods Except for the type of terminator, the other experimental conditions were basically the same as in Example 1. The specific experimental conditions are shown in Table 3 below.

[0071] Table 3 Experimental conditions for Comparative Example 3 (except for the experimental conditions in the table, all other conditions are the same as in Example 1)

[0072] 2. Experimental Results Electrophoresis results are shown in the figure. Figure 6 As shown, the results indicate that under fixed coagulation and fibrinolysis conditions, when using glutaraldehyde as a coagulant accelerator, aprotinin and EDTA as terminators can successfully prepare macromolecular D-dimers. However, using other terminators (PMSF, tranexamic acid, or 6-aminoacetic acid) or without terminators cannot yield macromolecular D-dimers. This demonstrates that not all types of terminators can successfully prepare macromolecular D-dimers using the preparation method described in this invention. This result is also a technical effect that would not have been anticipated by those skilled in the art based on existing technology.

[0073] Comparative Example 4: Effect of different terminators on the preparation method (using tannic acid as a coagulant) 1. Experimental Methods Except for the type of terminator, the other experimental conditions were basically the same as in Example 1. The specific experimental conditions are shown in Table 3 below.

[0074] Table 4. Experimental conditions for Comparative Example 4 (except for the experimental conditions in the table, all other conditions are the same as in Example 1).

[0075] 2. Experimental Results Electrophoresis results are shown in the figure. Figure 7As shown, the results indicate that under fixed coagulation and fibrinolysis conditions, when using tannic acid as a coagulant accelerator, aprotinin and EDTA as terminators can successfully prepare macromolecular D-dimers. However, using other terminators (PMSF, tranexamic acid, or 6-aminoacetic acid) or without terminators cannot produce macromolecular D-dimers. This demonstrates that not all types of terminators can successfully prepare macromolecular D-dimers using the preparation method described in this invention. This result is also a technical effect that would not have been anticipated by those skilled in the art based on existing technology.

[0076] Comparative Example 5: Effect of different coagulation-promoting conditions on the preparation method 1. Experimental Methods Except for the coagulation-promoting conditions, the other experimental conditions are basically the same as those in Example 1. The specific experimental conditions are shown in Table 3 below.

[0077] Table 5. Experimental conditions for Comparative Example 5 (except for the experimental conditions in the table, all other conditions are the same as in Example 1).

[0078] 2. Experimental Results Electrophoresis results are shown in the figure. Figure 8 As shown in the figure, the results indicate that, in addition to the normal coagulation and fibrinolysis processes, adding coagulation-promoting and termination steps can successfully prepare macromolecular D-dimers.

[0079] Example 2: Confirmation of final preparation conditions and results Based on the results of the above comparative examples, the final preparation conditions and results of this application are as follows: 1. Experimental Methods The final preparation conditions are shown in Table 6 below.

[0080] Table 6. Optimal preparation conditions for preparing macromolecular D-dimer solutions, as determined by the final analysis.

[0081] 2. Experimental Results Electrophoresis results are shown in the figure. Figure 9 As shown, the results indicate that, using the optimal preparation conditions described above, the prepared macromolecular D-dimer solution, after antibody purification, was subjected to SDS-PAGE electrophoresis together with the purchased small-molecule D-dimer antigen. The results demonstrate that the prepared macromolecular D-dimer solution contains a significant proportion of macromolecular D-dimers, which is suitable for further separation and purification.

Claims

1. A method for preparing a macromolecular D-dimer, characterized in that, The method includes the following steps: (1) Coagulation: Plasma is subjected to a cross-linking reaction under coagulation activation conditions to obtain cross-linked fibrin; (2) Coagulation: A coagulating agent is added to the cross-linked fibrin to carry out a coagulation reaction to obtain fixed cross-linked fibrin. The coagulating agent is selected from tannic acid solution or glutaraldehyde solution. (3) Fibrinolysis: Add streptokinase and plasminogen to the fixed cross-linked fibrin to carry out the fibrinolysis reaction and obtain fibrinolytic products; (4) Termination: A terminator is added to the fibrinolytic product to terminate the fibrinolytic reaction and obtain a concentrated fibrinolytic solution. The terminator is selected from EDTA solution or aprotinin. The concentrated fibrinolytic solution contains a large molecular D-dimer.

2. The method according to claim 1, characterized in that, The coagulation activation conditions described in step (1) are to mix plasma, calcium chloride and thrombin, and incubate in a 37°C water bath for 1-5 hours; Optionally, the coagulation activation conditions in step (1) are to mix plasma, calcium chloride and thrombin and incubate in a 37°C water bath for 2 hours; Optionally, the amounts of plasma, calcium chloride, and thrombin used are 1-10 mL of plasma, 1-10 mL of 10-100 mmol / L calcium chloride, and 0.1-10 kU of thrombin, respectively. Optionally, the amounts of plasma, calcium chloride, and thrombin used are 5 mL of plasma, 5 mL of 30 mmol / L calcium chloride, and 1 kU of thrombin, respectively. Optionally, step (1) further includes centrifuging the product after the water bath to obtain cross-linked fibrin.

3. The method according to claim 1, characterized in that, The reaction concentration of the tannic acid solution in step (2) is 0.01%-0.5% (w / v); Optionally, the reaction concentration of the glutaraldehyde solution in step (2) is 0.005%-0.2% (w / v). Optionally, the conditions for the coagulation reaction in step (2) are 10 min-2 h at room temperature; Optionally, the reaction concentration of the tannic acid solution in step (2) is 0.02% (w / v). Optionally, the reaction concentration of the glutaraldehyde solution in step (2) is 0.01% (w / v). Optionally, the conditions for the coagulation reaction in step (2) are a reaction at room temperature for 30 min; Optionally, step (2) further includes removing the supernatant from the reaction product to obtain the fixed cross-linked fibrin.

4. The method according to claim 1, characterized in that, When the coagulant is tannic acid, the conditions for the fibrinolytic reaction in step (3) are: adding 1-125 U of streptokinase and 0.0002-0.05 U of plasminogen, and reacting at room temperature for 1-18 h; Optionally, when the coagulant is glutaraldehyde, the conditions for the fibrinolytic reaction in step (3) are: adding 1-125 U of streptokinase and 0.0002-0.05 U of plasminogen, and reacting at room temperature for 2-5 h; Optionally, when the coagulant is tannic acid, the conditions for the fibrinolytic reaction in step (3) are: adding 12.5 U of streptokinase and 0.0025 U of plasminogen, and reacting at room temperature for 3 h; Optionally, when the coagulant is glutaraldehyde, the fibrinolytic reaction conditions in step (3) are: adding 12.5 U of streptokinase and 0.0025 U of plasminogen, and reacting at room temperature for 3 h.

5. The method according to claim 1, characterized in that, The reaction concentration of the EDTA solution mentioned in step (4) is 1-15 mM; Optionally, the reaction concentration of the EDTA solution in step (4) is 5 mM; Optionally, the reaction concentration of the aprotinin in step (4) is 0.1-5 kU / mL; Optionally, the reaction concentration of the aprotinin in step (4) is 0.5 kU / mL.

6. The method according to claim 1, characterized in that, The method further includes purifying the fibrinolytic concentrate in step (4) to obtain purified macromolecular D-dimer; Optionally, the purification includes affinity purification using antibodies that specifically recognize macromolecular D-dimers, and / or separation using molecular sieves.

7. The macromolecular D-dimer prepared by the method according to any one of claims 1-6.

8. Any of the following products: (1) An antibody for specifically recognizing macromolecular D-dimers, characterized in that, The antibody was prepared using the macromolecular D-dimer as described in claim 7 as an immunogen; (2) A kit for detecting macromolecular D-dimer, characterized in that the kit contains the antibody.

9. The use of the macromolecular D-dimer prepared by any one of claims 1-6 in the preparation of diagnostic reagents or products for thrombosis detection, dynamic monitoring of thrombosis, evaluation of anticoagulation therapy efficacy, or prediction of thrombosis recurrence.

10. The application according to claim 9, characterized in that, The testing products are selected from test kits, test strips, test chips, automated testing instruments, or automated testing systems.

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