A plasmin alpha 2 plasmin inhibitor complex antibody and use thereof

Abstract

The application provides a plasmin alpha2 plasmin inhibitor complex antibody 4C1 and 2D5, the sequence of the heavy chain variable region of the antibody 4C1 is shown as SEQ ID NO:1, and the light chain variable region is shown as SEQ ID NO:2; the sequence of the heavy chain variable region of the antibody 2D5 is shown as SEQ ID NO:9, and the light chain variable region is shown as SEQ ID NO:10. The detection sensitivity of the antibody is high.

Description

Technical Field

[0001] The present invention relates to a plasmin α2 plasmin inhibitor complex antibody and application thereof, belonging to the technical field of medical detection. Background Art

[0002] Disseminated intravascular coagulation (DIC) is an intermediate link in the complex pathophysiological process of various diseases. Its common underlying diseases or triggers include sepsis, malignant tumors, trauma, and surgery, which can cause a large amount of procoagulants to appear in the circulating blood in a short period of time, causing extensive thrombosis in the blood vessels, followed by consumption of coagulation factors and large-scale activation of the fibrinolytic system, resulting in an increase in fibrin degradation products that hinder normal fibrin polymerization and the binding of fibrinogen to platelets, interfering with fibrin clot formation and platelet aggregation, and causing secondary bleeding tendency.

[0003] A thrombus is a small mass of blood formed when blood flows onto the surface of a vessel in the cardiovascular system where the inner surface of the vessel is broken or repaired. In the variable fluid-dependent type, a thrombus is composed of insoluble fibrin, deposited platelets, accumulated white blood cells, and trapped red blood cells.

[0004] The main causes of thrombosis are: damage to the endothelium of the heart and blood vessels; changes in blood flow; changes in blood properties. Abnormalities in the coagulation system and fibrinolytic system lead to excessive thrombosis, causing cardiovascular and cerebrovascular diseases, tissue ischemia, vascular infarction, etc. Currently, thrombosis detection is included in various blood tests, mainly involving coagulation and hemostasis tests. Currently, there is a lack of laboratory indicators for early diagnosis of clinical DIC and thrombosis. Impaired endothelial system function cannot be detected in the laboratory. Surgeons also lack laboratory basis for early diagnosis of postoperative anticoagulant treatment to prevent thrombosis. For this reason, the laboratory department has carried out four tests, including thrombosis TAT, to assist in the early diagnosis and monitoring of clinical DIC and postoperative thrombosis. The four coagulation tests are for the assessment of the body's coagulation function, while the four thrombosis tests are a means of further exploring the risk of abnormal thrombosis in the body and feedback on thrombosis treatment.

[0005] Plasminogen (PLG) is the inactive form of plasmin (P), primarily synthesized in the liver and also expressed in major organs and tissues. Under physiological conditions, plasminogen PLG is converted to plasmin P via cleavage of the active loop. Activated plasmin has a broad substrate profile, including fibrin, fibrinogen, complement component 3, and complement component 5. Plasmin P can also target the key prothrombin activators tPA and uPA, thereby generating a positive feedback loop. Active plasmin P plays an important role in physiological and pathological processes, including fibrinolysis and hemostasis, extracellular matrix degradation, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation, and tumor cell migration. The full-length plasminogen PLG consists of 791 amino acid residues and seven structural domains. Plasminogen PLG has two distinct conformations: closed and open. Glycosylated plasminogen PLG exists in the bloodstream in a closed conformation and is resistant to activation by tPA and uPA. The half-life of activated plasmin P is extremely short and cannot be directly measured in vivo.

[0006] α2-Antiplasmin (α2-AP) is a single-chain glycoprotein synthesized and secreted by the liver. It contains 452 amino acids and has a molecular weight of 70,000. It is a member of the serine enzyme inhibitor family. In human plasma, α2-AP is the main plasmin inhibitor and plays a key role in controlling fibrinolysis.

[0007] Plasminogen (PLG) and antiplasmin (AP) are abundant in the blood, but plasmin P is absent. Plasmin P is immediately captured by antiplasmin AP in the blood, forming the α2-plasmin inhibitor-Plasmin complex (PIC). The ratio of plasmin P to α2-antiplasmin AP is 1:1, making it a biomarker that directly reflects the degree of fibrinolytic system activation. Formation of this complex involves the formation of new covalent bonds, making it highly stable. PIC has a plasma half-life of approximately 6 hours and can be directly measured. A normal plasma value is <0.8 μg / mL, and a PIC >0.8 μg / mL often indicates fibrinolytic system activation. PIC measurement has the following clinical implications: it is suitable for the early diagnosis of DIC and pre-DIC states, guiding DIC treatment; it serves as a specific diagnostic marker for common thrombotic embolism (VTE); its elevated level can provide a warning of thrombosis induced by major surgery or malignancy, and can predict disease progression; and it can be used to monitor anticoagulant and thrombolytic therapy. Therefore, PIC measurement is of great significance.

[0008] However, since plasminogen PLG in the blood also forms a non-covalent complex PLG-AP with antiplasmin AP, although the complex PLG-AP is not stable enough and can be destroyed by weaker means, if PIC antibodies are used for PIC detection, the PIC antibodies will not only bind to PIC but also to the complex PLG-AP. In this way, the complex PLG-AP will interfere with the detection of the complex PIC, resulting in a certain impact on the detection sensitivity.

[0009] In summary, there are currently no PIC detection reagents on the market that meet clinical needs, especially the key raw material PIC antibodies. Summary of the Invention

[0010] The present invention provides a plasmin α2 plasmin inhibitor complex antibody and its application, which can effectively solve the above problems.

[0011] The present invention is achieved in that:

[0012] A plasmin α2 plasmin inhibitor complex antibody 4C1, the sequence of its heavy chain variable region is shown in SEQ ID NO: 1, and the sequence of its light chain variable region is shown in SEQ ID NO: 2.

[0013] In some embodiments, the heavy chain of the plasmin α2 plasmin inhibitor complex antibody 4C1 includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and their amino acid sequences are shown in SEQ ID NOs: 3-5; the light chain includes light chain CDR1, light chain CDR2 and light chain CDR3, and their amino acid sequences are shown in SEQ ID NOs: 6-8.

[0014] In some embodiments, the antibody is a monoclonal antibody prepared using plasmin α2 plasmin inhibitor complex as an immunogen.

[0015] A plasmin α2 plasmin inhibitor complex antibody 2D5, the sequence of its heavy chain variable region is shown in SEQ ID NO: 9, and the sequence of its light chain variable region is shown in SEQ ID NO: 10.

[0016] In some embodiments, the heavy chain of the plasmin α2 plasmin inhibitor complex antibody 2D5 includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and their amino acid sequences are shown in SEQ ID NOs: 11-13; their light chain includes light chain CDR1, light chain CDR2 and light chain CDR3, and their amino acid sequences are shown in SEQ ID NOs: 14-16.

[0017] In some embodiments, the antibody is a monoclonal antibody prepared using plasmin α2 plasmin inhibitor complex as an immunogen.

[0018] A use of the antibody 4C1 in preparing a plasmin α2 plasmin inhibitor complex detection reagent.

[0019] A use of the antibody 2D5 in preparing a plasmin α2 plasmin inhibitor complex detection reagent.

[0020] A detection kit for plasmin α2 plasmin inhibitor complex comprises the above-mentioned antibody 4C1 and / or antibody 2D5.

[0021] A storage solution for a plasmin α2 plasmin inhibitor complex comprises the following components: 20 mM PBS 7.4, 150 mM NaCl, 1 wt% BSA, 10 wt% sucrose, and 10 wt% NBS.

[0022] The beneficial effects of the present invention are:

[0023] Existing PIC antibodies generally have strong reactivity only toward PIC itself, with weak reactivity toward AP or P, and also weak reactivity toward PLG. Using such antibodies for PIC detection can lead to the following problems: when the coated antibody, due to its weak binding ability, binds to one of the antigen monomers, AP or PLG, this new structure acquires the ability to bind to the other antigen monomer, PLG or AP. The strength of the binding is no longer determined solely by the affinity of the two antigen monomers, AP or PLG, because the antibody's strong binding ability helps stabilize it. As a result, the originally unstable PLG-AP is firmly bound by the antibody, resulting in detection and interference with the actual target, PIC.

[0024] The antibody 4C1 of the present invention exhibits strong reactivity with both α2-antiplasmin AP and the plasmin-α2 plasmin inhibitor complex PIC. Antibody 2D5 also exhibits strong reactivity with both plasmin P and the plasmin-α2 plasmin inhibitor complex PIC. When these antibodies are used for PIC detection, they lack the ability to consolidate PLG-AP, which can be removed during the detection process through various methods, such as washing. Therefore, the detected value is closer to the actual PIC level of the subject being tested, avoiding interference from PLG-AP in PIC detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is an SDS-PAGE identification diagram of the three antigens provided in Example 1 of the present invention.

[0027] Figure 2 This is a diagram showing the antibody SDS-PAGE purity identification results provided in Example 2 of the present invention.

[0028] Figure 3 This is the standard curve of the detection sensitivity of the antibody provided in Example 6 of the present invention to the PIC antigen. DETAILED DESCRIPTION

[0029] In order 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 will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention for which protection is sought, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0031] Example 1 Preparation of PIC Antigen Complex

[0032] 1. Preparation: Plasmin (plasmin P) and Alpha-2 Antiplasmin (AP) were obtained from Lee Biosolutions (Cat. Nos. 491-41 and 107-50, respectively). Plasmin and Alpha-2 Antiplasmin antigens were dissolved in 20 mM PBS (7.4% PBS) to a concentration of 2 mg / mL. The reconstituted plasmin and Alpha-2 Antiplasmin solutions are referred to below as Antigen 1 and Antigen 2, respectively.

[0033] 2. Synthesis of plasmin α2 plasmin inhibitor complex (PIC) antigen: 500 μL of antigen 1 and antigen 2 were mixed and reacted at 37°C for 10 minutes. The mixture was then designated as antigen 3. The concentration of the complex was 2 mg / mL. The three antigens were identified by 12% SDS-PAGE. The results were as follows: Figure 1 shown.

[0034] Example 2 Preparation of PIC monoclonal antibody

[0035] 1. Preparation of the Immunogen: The immunogen was antigen 3 prepared in Example 1. Antigen 3 was diluted to 0.4 mg / mL with 10 mmol / L PBS. An equal volume of the corresponding antigen was mixed with Freund's adjuvant to form a water-in-oil emulsion, achieving a final concentration of 200 μg / mL. Freund's complete adjuvant was used for the primary immunization, and Freund's incomplete adjuvant was used for the booster immunization.

[0036] 2. Basic immunization: 6-8 week old BALB / c female mice were selected for subcutaneous multi-point immunization. The immunogen injection dose was 500 μL / mouse / time. The immunization interval was 2 weeks. The complete immunization program consisted of 4 injections. Two weeks after the 2nd, 3rd, and 4th immunizations, serum was collected and separated by orbital blood collection for indirect ELISA to determine the immune titer. After determining that the mouse serum titer reached the plateau phase, spleen immunization was performed with 100 μg / mouse of antigen 3 72 hours before cell fusion.

[0037] 3. Hybridoma Cell Screening

[0038] 3.1 Preparation of feeder macrophage cells:

[0039] (i) BALB / c mice, approximately 6 weeks old, were sacrificed by cervical dislocation and soaked in 75% alcohol solution for 5 min. The mice were removed and placed in a sterile dish pre-placed on a clean bench. Hemostats were used to adjust the mouse's posture so that its abdomen was facing upward, allowing for a more relaxed posture. The mice were then placed sideways. Hemostats were used to clamp the skin near the lower abdomen of the mouse. The skin was then suddenly pulled apart in the opposite direction to fully expose the abdomen.

[0040] (ii) Use sterile ophthalmic curved forceps to lift the peritoneum. Then, use a 5 mL syringe to inject an appropriate amount of culture medium into the abdominal cavity, ensuring that the abdomen is fully inflated. Place the syringe down, hold the hemostatic forceps with both hands, and lift the contralateral limbs of the mouse, gently shaking them to allow sufficient infiltration of macrophages in the peritoneal cavity. Afterwards, use the same syringe to aspirate the macrophages and inject them into the pre-prepared 1640HT medium containing 20% ​​fetal bovine serum for later use.

[0041] 3.2 Preparation of thymocyte feeder cells:

[0042] (i) Three-week-old BALB / c mice were sacrificed by cervical dislocation and soaked in 75% alcohol solution for 5 min. The mice were removed and placed in a sterile dish pre-placed on a clean bench. Hemostats were used to adjust the mouse's posture so that its abdomen was facing upward, allowing for a more relaxed posture. The mice were then placed sideways. Hemostats were used to clamp the skin just below the chest. The skin was then suddenly pulled apart in the opposite direction to fully expose the chest and abdomen.

[0043] (ii) Use sterile curved forceps in your left hand to grasp the sternal tip through the chest and abdominal skin. Cut along the outer edge of the sternum to fully expose the thoracic diaphragm. Keep grasping the sternal tip to expose the chest cavity as much as possible.

[0044] (iii) Using clean ophthalmic scissors in the right hand, cut open the thoracic diaphragm to fully expose the thoracic cavity. Using curved forceps, reach into the thoracic cavity, grasp the thymus at the base, and remove the thymus intact. Grind the thymus through a 200-mesh sieve that has been pre-placed on a plate and soaked in culture medium to obtain thymic feeder cell fluid. Transfer the entire thymus to the aforementioned 1640HT medium supplemented with 20% fetal bovine serum for later use.

[0045] 3.3 Preparation of mouse myeloma cells: 5 days before fusion, the mouse myeloma cells were revived. Each fusion required approximately 6 bottles of 35 cm2 mouse myeloma cells at a density of 90%-100%.

[0046] 3.4 Preparation of splenocytes:

[0047] (i) Orbital blood was collected from BALB / c mice to be fused. After bleeding stopped, the mice were killed by cervical dislocation. The mice were immersed in 75% alcohol solution for 5 minutes and then placed on a sterile plate in a clean bench in the right lateral decubitus position.

[0048] (ii) The abdominal cavity was exposed using the method described in 3.1. The spleen was removed by aseptic surgery and ground through a 200-mesh sieve pre-placed on a plate and soaked in culture medium. The spleen cell suspension was prepared and transferred to a 50 mL sterile centrifuge tube.

[0049] (iii) Add an appropriate amount of RPMI-1640 culture medium (30-35 mL). After removing obvious fat aggregates and other impurities with a curved pipette, collect spleen cells by centrifugation at 1500 rpm for 5 min each time. Remove the supernatant by centrifugation and add 30-35 mL of new culture medium. Repeat this process twice to wash the spleen cells.

[0050] (iv) Resuspend the cells in RPMI-1640 medium and count them.

[0051] 3.5 Cell Fusion:

[0052] (i) Before fusion, pre-warm 1 mL of PEG-1450, 35 mL of RPMI-1640 serum-free medium, and 200 mL of HAT complete medium containing 20% ​​fetal bovine serum to 37°C.

[0053] (ii) Prepared myeloma cells and spleen cells were mixed at a ratio of 1×10 8 Splenocytes + 1×10 7 Myeloma cells were mixed in a 50 mL centrifuge tube at a ratio of approximately 10:1 and centrifuged at 1500 rpm for 5 min. After centrifugation, the supernatant was discarded as much as possible and the bottom of the tube was gently tapped to loosen the cells into a paste.

[0054] (iii) Pipette 1 mL of PEG into the centrifuge tube with a 1 mL pipette and gently pipette to mix thoroughly for approximately 60 s. Immediately, add 35 mL of pre-warmed RPMI-1640 complete culture medium to terminate the fusion reaction.

[0055] (iv) After standing for 1-5 minutes, centrifuge at 1000 rpm for 5 minutes. Carefully discard the supernatant. Gently flick the cells with your fingers. Add the above complete medium to fully resuspend the cells. Transfer all cells to complete medium that has been supplemented with feeder cells. After mixing, plate 200 μL / well into a 96-well cell culture plate and culture in a CO2 incubator.

[0056] (v) After 7 days, 100% of the cell supernatant in the wells was replaced with 15% HT complete medium; after 7 days, the supernatant was aspirated for detection.

[0057] 3.6 Hybridoma screening: Differential screening was performed using antigens 1 and 2 prepared in Example 1. Indirect ELISA screening was performed. 300 ng / mL of antigen was coated, 0.1 mL was coated per well, 50 μL of cell supernatant was added for detection, and positive clone wells were selected.

[0058] 3.7 Cloning of Hybridoma Cells: Using the limiting dilution method, cells are first diluted to a certain concentration and then inoculated into each well of a 96-well cell culture plate, with the goal of ensuring that only one cell grows in each well. Hybridoma monoclonal positive cell lines are cloned at least three times, and a 100% positive result is required to confirm the line as a stable clone.

[0059] 3.8 Antibody typing: Add 50 μL / well of the stable cell line supernatant to the plate coated with antigen 3 in parallel. After placing in a 37°C incubator for 30 min, wash the plate five times, pat dry, add 50 μL / well of IgG1, IgG2a, IgG2b, IgG3, or IgM typing enzyme, react for 30 min, wash the plate, add 100 μL / well of colorimetric solution, and stop reading with stop solution after 15 min. Determine the antibody subtype based on the results.

[0060] 4. Monoclonal Antibody Preparation

[0061] 4.1 Production of Monoclonal Antibody Ascites BALB / c mice were sensitized. One week later, the stable hybridoma cells in the logarithmic growth phase were resuspended and centrifuged at 1500 rpm for 5 min to collect the cells. The pelleted cells were suspended in serum-free culture medium and the cell count was adjusted to (1-2) × 10 6 / mL, and 0.5mL was injected intraperitoneally into each mouse to induce ascites. After 7–10 days, when the mouse abdomen was noticeably distended, the ascites was collected. The collected ascites was placed in a centrifuge tube and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and set aside.

[0062] 4.2 Purification of mAb Ascites

[0063] After ammonium sulfate precipitation and protein A affinity chromatography purification (purchased from GE, USA), high-purity monoclonal antibodies (named 1D2, 2D5, 2F4, 11F1, 9A8, 4C1, 1D5, 5B3, 8G7, 5F11) were obtained. The results of antibody 12% SDS-PAGE purity identification are as follows: Figure 2 shown.

[0064] 4.3 Indirect monoclonal antibody activity determination

[0065] The reactivity of purified antibodies with antigens was evaluated using the EIA method. Antigens 1, 2, and 3 were coated separately at 300 ng / mL. 100 μL of each solution was coated into each well. The antibody to be evaluated was diluted to 0.1 μg / mL in 20 mM PBS and 100 μL of each solution was added to three pre-coated antigen plates. The plates were incubated at 37°C for 30 minutes. The plates were washed five times, and GAM-HRP (1 / 5000 dilution) was added and incubated at 37°C for 30 minutes. The plates were washed five times, and the substrate was added and incubated for 15 minutes. The microplate reader read the values ​​at wavelengths of 450 / 620 nm, as shown in Table 1.

[0066] Table 1

[0067]

[0068]

[0069] From the analysis of the results in Table 1, it can be seen that antibodies can be divided into two types, one type reacts strongly with antigen 1 and antigen 3, and the other type reacts strongly with antigen 2 and antigen 3.

[0070] Example 3 Monoclonal antibody paired ELISA evaluation

[0071] 1. Five monoclonal antibodies (4C1, 1D5, 5B3, 8G7, and 5F11) were diluted to 5 μg / mL in phosphate buffer (20 mmol / L PBS, pH 7.4) and coated onto polyvinyl chloride plates. Five monoclonal antibodies (1D2, 2D5, 2F4, 11F1, and 9A8) were labeled with horseradish peroxidase. PIC quality control was diluted to 5 μg / mL in diluent. A 50 μL portion of a pooled positive serum (contained at 5 μg / mL) and a 20% NBS blank were added to the corresponding wells and incubated at 37°C for 40 minutes. The plates were washed five times, and horseradish peroxidase-labeled antibodies from each of the ten monoclonal antibodies (1 / 500 dilution) were added and incubated for 40 minutes. The plates were washed five times, and substrate was added and incubated for 15 minutes. Readings were performed on a microplate reader at a wavelength of 450-620 nm. Data analysis is shown in Table 2 below.

[0072] Table 2

[0073]

[0074] From the results analysis in Table 2, it can be seen that the preliminary enzyme immunoassay orthogonal pairing showed that the better coating antibodies were 1D5, 4C1, and 5B3, and the better labeling antibodies were 1D2, 2D5, and 11F1.

[0075] 1. Five monoclonal antibodies (1D2, 2D5, 2F4, 11F1, and 9A8) were diluted to 5 μg / mL in phosphate buffer (20 mmol / L PBS, pH 7.4) and coated onto polyvinyl chloride plates. Five monoclonal antibodies (4C1, 1D5, 5B3, 8G7, and 5F11) were labeled with horseradish peroxidase. PIC quality control samples were diluted to 5 μg / mL in diluent. A 50 μL portion of a pooled positive serum (contained at 5 μg / mL) and a 20% NBS blank control were added to the corresponding wells and incubated at 37°C for 40 minutes. The plates were washed five times, and horseradish peroxidase-labeled antibodies from each of the ten monoclonal antibodies (1 / 500 dilution) were added and incubated for 40 minutes. The plates were washed five times, and substrate was added and incubated for 15 minutes. Readings were performed on a microplate reader at wavelengths of 450-620 nm. Data analysis is shown in Table 3 below.

[0076] Table 3

[0077]

[0078] From the results in Table 3, we can see that the better coating antibodies are 1D2, 2D5, and 11F1 according to the preliminary enzyme immunoassay orthogonal pairing, and the better labeling antibodies are 1D5, 4C1, and 5B3.

[0079] In summary, antibodies that react to antigen 1 / antigen 3 and antibodies that react to antigen 2 / antigen 3 have better mutually compatible antibody pairs when they are orthogonally matched.

[0080] 3. Three of the top-performing monoclonal antibodies (4C1, 1D5, and 5B3) and three (1D2, 2D5, and 11F1) were coated onto polyvinyl chloride plates diluted to 5 μg / mL in phosphate buffer (20 mmol / L PBS, pH 7.4). Three of the top-performing labeled monoclonal antibodies (1D2, 2D5, and 2F4) and three of the top-performing monoclonal antibodies (4C1, 1D5, and 5B3) were labeled with horseradish peroxidase. Seven clinical samples were collected for evaluation; 50 μL was added to the corresponding wells and incubated at 37°C for 40 minutes. The plates were washed five times, and then horseradish peroxidase-labeled with each of the three monoclonal antibodies (1 / 500 dilution) was added and incubated for 40 minutes. The plates were washed five times, and substrate was added and incubated for 15 minutes. Readings were performed on a microplate reader at a wavelength of 450-620 nm. Data analysis is shown in Tables 4 and 5 below.

[0081] Table 4

[0082]

[0083] Table 5

[0084]

[0085]

[0086] From the analysis of the results in Table 4 and Table 4: Antibodies that specifically react with antigen 1 / antigen 3 and antibodies that specifically react with antigen 2 and antigen 3 have good reaction effects when paired with each other, among which antibody 4C1 paired with antibody 2D5 has the best serum correlation.

[0087] After sequencing, the heavy chain variable region sequence of antibody 4C is shown in SEQ ID NO: 1 (SDVQLQESGPGLVKPSQSLSLTCSVTGDSITSGYYWNWIRQFPENKLEWMAYINYDGVNKYNPSLKNRISITRDTSKNQFFLRLNSVTIEDTATYFCTREGVHDGYLDFWGQGTTLTF); the light chain variable region sequence is shown in SEQ ID NO: 2

[0088] The heavy chain comprises a heavy chain CDR1 (GDSITSGYY), a heavy chain CDR2 (INYDGVN), and a heavy chain CDR3 (TREGVHDGYLDF), and the amino acid sequences thereof are shown in SEQ ID NOs: 3-5; the light chain comprises a light chain CDR1 (QDVKTA), a light chain CDR2 (WAS), and a light chain CDR3 (QQHYNTPWT), and the amino acid sequences thereof are shown in SEQ ID NOs: 6-8.

[0089] After sequencing, the heavy chain variable region sequence of antibody 2D5 was shown in SEQ ID NO: 9 (EVQLQQSGPELVKPGTSMKISCKTSGYSFSGYTMNWVKESHGKNLEWIGLMNPYNGGTHYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCAREGDYGGYFDCWGQGTTLTF).

[0090] The light chain variable region sequence is set forth in SEQ ID NO: 10 (DVQITQSPSYLAASPGETITINCRTSKNISKYLVWYQAKPGKTNKLLIYSGSALQSGVPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHDEYPLTFGTGTKLEL). The heavy chain comprises a heavy chain CDR1 (GYSFSGYT), a heavy chain CDR2 (MNPYNGGT), and a heavy chain CDR3 (AREGDYGGYFDC), the amino acid sequences of which are set forth in SEQ ID NOs: 11-13. The light chain comprises a light chain CDR1 (KNISKY), a light chain CDR2 (SGS), and a light chain CDR3 (QQHDEYPLT), the amino acid sequences of which are set forth in SEQ ID NOs: 14-16.

[0091] Example 4 Quality Control Product Stability Optimization Experiment

[0092] In Example 3, it was found that the reactivity of the PIC quality control product at room temperature decreased over time after preparation. To improve this phenomenon and explore the related causes, the following experimental process was carried out:

[0093] 4.1 The PIC enzyme immunoassay pairing in the following examples is carried out in Example 3, and its features are exactly the same as those in Example 3.

[0094] 4.2 The storage conditions of the quality control products were optimized and compared with different schemes, including the following 5 schemes:

[0095] (i) 20 mM PBS 7.4

[0096] (ii) 20 mM PBS 7.4, 150 mM NaCl, 1 wt% BSA, 10 wt% sucrose, 10 wt% NBS

[0097] (iii) 20 mM PBS 7.4, 1 wt% BSA, 10 wt% sucrose

[0098] (iv) 20mM Tris-HCl 8.0, 150mM NaCl, 0.4wt% BSA, 0.1% TritonX-100

[0099] (v) 50 mM Tris-HCl 8.0, 150 mM NaCl, 10 wt% sucrose

[0100] 4.3 The preparation method for the above protocol is as follows: Prepare the above solutions and add Antigen 1 and Antigen 2 at a 1:1 mass ratio to the solutions prepared in the five conditions above to produce 20 μg / mL of antigen. These are designated Sample 1, Sample 2, Sample 3, Sample 4, and Sample 5 in the order described above. For each of the five numbered samples, 100 μL of each sample was collected in chronological order, for a total of 25 samples. Five original samples and 25 samples collected at different time points were tested. The deviations in OD450 values ​​between the samples collected at different time points and the original samples are shown in Table 6 below.

[0101] Table 6

[0102] Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 7h -9％ 5％ 9％ 6％ -26％ 24h -13％ -4％ 9％ -7％ -23％ 36h -19％ 5％ 7％ -38％ -53％ 48h -20％ 0％ 3％ -39％ -49％ 52h -21％ -5％ 8％ -52％ -57％

[0103] From the results analysis in Table 6, it can be seen that: by enzyme immunoassay, sample 5 is confirmed to be the best condition for preparing the buffer solution, that is, condition (ii) is the optimal condition, which can improve the stability of the PIC index sample.

[0104] Example 5 A plasmin α2 plasmin inhibitor complex detection kit

[0105] 5.1 PIC detection kit, comprising the following components: M reagent, containing 0.5-1 mg / ml of magnetic microparticles coated with a primary antibody, wherein the primary antibody (4C1) is coated at a concentration of 20-80 μg / ml. The magnetic microparticles are purchased from Thermo Fisher Scientific. The reagent is prepared by mixing the primary antibody and magnetic microparticles in 2-morpholineethanesulfonic acid buffer (pH 5.0-6.0), coating at 25°C-37°C for 1-3 hours, then adding phosphate buffer (pH 8.0-9.0) containing 0.1%-0.5% bovine serum albumin for 1-3 hours, separating the coated magnetic microparticles and dispersing them in phosphate buffer (pH 7.0-8.0), followed by the addition of casein and bovine serum albumin. Reagent R2 contains an acridinium ester coated with a secondary antibody, 0.5-1% casein, and 0.5-1% bovine serum albumin, in a phosphate buffer solution at pH 7.0-8.0. The coating concentration of the secondary antibody is 5-15 μg / mL of acridinium ester. The preparation method is as follows: the secondary antibody (2D5) and the acridinium ester are mixed in a phosphate buffer solution at pH 8.0-9.0, and the coating is carried out at 25°C-37°C for 1-3 hours. The coating is terminated by adding Tris buffer at pH 8.0-9.0 containing 0.1%-0.5% bovine serum albumin for 1-3 hours. The resulting mother solution is diluted 1-500 times with phosphate buffer at pH 7.0-8.0 before use. The pre-challenge solution is a 1% (w / v) hydrogen peroxide solution. The challenge solution is a 1 mol / L sodium hydroxide solution.

[0106] 5.2 The PIC detection kit detection method comprises the following steps: adding 20 μL of sample M reagent and 50 μL of sample M reagent to the reaction well in a volume ratio of 2:5, incubating for 15-20 minutes, washing with phosphate buffer containing 0.05-0.08% Tween 20, adding 20-25 μL of R2 reagent, incubating for 10-15 minutes, washing with phosphate buffer containing 0.05-0.08% Tween 20, adding 100-200 μL of pre-excitation solution for pre-excitation, removing the pre-excitation solution, adding 100-200 μL of excitation solution, exciting, and measuring the luminescence value.

[0107] Example 6 Analysis of the Detection Range and Sensitivity of a Detection Kit for Plasmin α2 Plasmin Inhibitor Complex

[0108] Using the kit described in Example 5, the PIC preparation antigen was diluted to 62.5 μg / mL in 20 mM PBS 7.4, 150 mM NaCl, 1 wt% BSA, 10 wt% sucrose, and 10 wt% NBS. Eleven dilutions were then performed in a 2-fold gradient. A blank solution containing no PIC preparation antigen (20 mM PBS 7.4, 150 mM NaCl, 1 wt% BSA, 10 wt% sucrose, and 10 wt% NBS) was used as a negative control. Testing was performed according to the method described in Example 5. The results are shown in Table 7.

[0109] Table 7

[0110]

[0111] The data in Table 7 above show that the detection sensitivity of PIC antigen is 0.06 μg / ml, which meets the clinical detection requirements. The standard curve is established as follows: Figure 3 shown.

[0112] Example 7 Evaluation of the Detection Accuracy of a Detection Kit for Plasmin α2 Plasmin Inhibitor Complex

[0113] Using the kit of Example 5, samples collected from a hospital were tested. Currently, 48 serum samples with a PIC background were collected. Of these, 24 had normal PIC and 24 had abnormal PIC. Testing each serum sample using the kit of Example 5 revealed 26 normal PIC and 22 abnormal PIC. The abnormality compliance rate was calculated as: the number of abnormalities in this example / the number of abnormalities detected by the hospital; this was 91.7%. The normality compliance rate was calculated as: the number of normal cases in this example / the number of normal cases detected by the hospital; this was 100%. The total compliance rate = (abnormality compliance rate + normality compliance rate) / 2 = 95.8%.

[0114] The overall compliance rate of this embodiment is high, indicating that the performance of the kit of this embodiment meets clinical needs.

[0115] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A detection kit for plasmin α2 plasmin inhibitor complex, characterized in that: including plasmin α2 plasmin inhibitor complex antibody 4C1 and plasmin α2 plasmin inhibitor complex antibody 2D5; The sequence of the heavy chain variable region of the plasmin α2 plasmin inhibitor complex antibody 4C1 is shown in SEQ ID NO: 1, and the sequence of the light chain variable region is shown in SEQ ID NO: 2; The sequence of the heavy chain variable region of the plasmin α2 plasmin inhibitor complex antibody 2D5 is shown in SEQ ID NO: 9, and the sequence of the light chain variable region thereof is shown in SEQ ID NO:

10.

2. A plasmin α2 plasmin inhibitor complex antibody 4C1, characterized in that: The sequence of its heavy chain variable region is shown in SEQ ID NO: 1, and the sequence of its light chain variable region is shown in SEQ ID NO:

2.

3. The plasmin α2 plasmin inhibitor complex antibody 4C1 according to claim 2, characterized in that: The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown in SEQ ID NOs: 3-5; the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 6-8.

4. The plasmin α2 plasmin inhibitor complex antibody 4C1 according to claim 2, characterized in that: The antibody is a monoclonal antibody prepared by taking plasmin α2 plasmin inhibitor complex as immunogen.

5. A plasmin α2 plasmin inhibitor complex antibody 2D5, characterized in that: The sequence of its heavy chain variable region is shown in SEQ ID NO: 9, and the sequence of its light chain variable region is shown in SEQ ID NO:

10.

6. The plasmin α2 plasmin inhibitor complex antibody 2D5 according to claim 5, characterized in that: The amino acid sequences of its heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown in SEQ ID NOs: 11-13; the amino acid sequences of its light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NOs: 14-16.

7. The plasmin α2 plasmin inhibitor complex antibody 2D5 according to claim 5, characterized in that The antibody is a monoclonal antibody prepared by taking plasmin α2 plasmin inhibitor complex as immunogen.

8. Use of the antibody according to any one of claims 2 to 4 in the preparation of a plasmin α2 plasmin inhibitor complex detection reagent.

9. Use of the antibody according to any one of claims 5 to 7 in the preparation of a plasmin α2 plasmin inhibitor complex detection reagent.

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