Soluble fibrin sf antibodies and uses thereof

Abstract

The application relates to biotechnology, and particularly discloses a soluble fibrin SF antibody and application thereof. For the first time, a single B cell antibody preparation technology and a flow cytometry sorting method are applied to the development of a coagulation in-vitro diagnosis fibrinolysis system detection antibody, a mammalian cell expression system (293F) is adopted, three strains of SF monoclonal antibodies with biological activity (antibody numbers SF-1, SF-2 and SF-3) are successfully expressed and obtained. The three strains of antibodies have specific reactions with SF and have no cross reactions with primary fibrinolysis substances (X, Y and D), can be applied to the fields of SF indirect ELISA and Western blotting detection and screening, and can make up for the vacancy of domestic SF antibodies on the market, and realize the integrity of the fibrinolysis system detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, more particularly, it relates to a soluble fibrin SF antibody and application thereof. BACKGROUND

[0002] Fibrinogen (FIB) is the target protein of the coagulation cascade. Fibrinogen is a 340 kDa soluble plasma protein composed of three pairs of α, β and γ chains connected by disulfide bonds, and its structure is shown in Figure 1 The activation of the coagulation system leads to thrombin generation, which converts fibrinogen into fibrin by catalyzing proteolytic removal of fibrinopeptide A and fibrinopeptide B. These cleavages expose two polymerization sites in the E domain of fibrinogen, forming soluble fibrin (SF) to which D domains from fibrin (ogen) molecules can bind. Soluble fibrin spontaneously polymerizes into double-stranded fibrils, with the fibrin monomers in each strand arranged in an end-to-end manner, while the cross-strand fibrins are arranged in a half-staggered overlap manner, as shown in the combination of a chain and b chain in Figure 2 Factor XIIIa catalyzes the covalent cross-linking of adjacent D domains through isopeptide bonds, forming cross-linked stable fibrin.

[0003] The basic process of fibrinolysis (i.e., fibrinolysis) can be divided into two stages: activation of plasminogen (PLG) and degradation of fibrin. Plasmin degrades fibrinogen to produce X, Y, D and E fragments (see Figure 1 ). When plasmin degrades cross-linked stable fibrin, due to different enzyme cleavage sites, various fragments are produced (see Figure 2 ), such as DD / E, DY / YD, DXY / YXD and DXD / YY, where DD / E is the smallest fragment of degradation (D-dimer). All the above fragments are collectively referred to as fibrin (ogen) degradation products (FDP).

[0004] At present, there are many studies and related kits on D-dimer, but there are few types of kits for detecting soluble fibrin, mainly based on immunoturbidimetry, and relying on imports. The development of SF antibodies necessary for the formation of kits is the rate-limiting step for the development of kits. However, there is no SF antibody on the market at present.

[0005] Furthermore, most existing fibrinolytic degradation products are mouse monoclonal antibodies prepared using traditional hybridoma technology. Hybridoma technology has drawbacks, including limited species development (most monoclonal antibodies produced using hybridoma technology are murine), long preparation cycles, cumbersome procedures, a tendency to generate human anti-mouse antibody (HAMA) reactions, and rapid clearance from the human circulatory system. For monoclonal antibodies related to fibrinolysis system detection, D-dimer, FDP, and SF are all obtained from fibrinogen through different enzyme catalysis, and their structures are highly similar to fibrinogen, posing significant challenges to the screening of hybridoma mouse monoclonal antibodies.

[0006] Therefore, there is an urgent need to develop an SF antibody that can effectively bind to SF and is easy to screen and obtain. Summary of the Invention

[0007] To address the aforementioned technical problems, this application provides a soluble fibrin SF antibody and its applications.

[0008] The technical solution adopted in this application is as follows:

[0009] In a first aspect, this application provides a soluble fibrin SF antibody, wherein the SF antibody includes any one of a first antibody SF-1, a second antibody SF-2, or a third antibody SF-3;

[0010] The complementarity-determining regions (CDRs) of the heavy chain H-SF-1 of the first antibody SF-1 are as follows: CDR1 shown in SEQ ID NO.01, CDR2 shown in SEQ ID NO.02, and CDR3 shown in SEQ ID NO.03; the complementarity-determining regions (CDRs) of the light chain L-SF-1 of the first antibody SF-1 are as follows: CDR1 shown in SEQ ID NO.06, CDR2 shown in SEQ ID NO.07, and CDR3 shown in SEQ ID NO.08.

[0011] The complementarity-determining regions (CDRs) of the heavy chain H-SF-2 of the second antibody SF-2 are as follows: CDR1 shown in SEQ ID NO.11, CDR2 shown in SEQ ID NO.12, and CDR3 shown in SEQ ID NO.13; the complementarity-determining regions (CDRs) of the heavy chain L-SF-2 of the second antibody SF-2 are as follows: CDR1 shown in SEQ ID NO.16, CDR2 shown in SEQ ID NO.17, and CDR3 shown in SEQ ID NO.18.

[0012] The complementarity-determining regions (CDRs) of the heavy chain H-SF-3 of the third antibody SF-3 are as follows: CDR1 shown in SEQ ID NO.21, CDR2 shown in SEQ ID NO.22, and CDR3 shown in SEQ ID NO.23; the complementarity-determining regions (CDRs) of the heavy chain L-SF-3 of the third antibody SF-3 are as follows: CDR1 shown in SEQ ID NO.26, CDR2 shown in SEQ ID NO.27, and CDR3 shown in SEQ ID NO.28.

[0013] Secondly, this application provides a soluble fibrin SF antibody, wherein the SF antibody is a first antibody SF-1, the amino acid sequence of the heavy chain variable region of the first antibody SF-1 is shown in SEQ ID NO.04, and the amino acid sequence of the light chain variable region of the first antibody SF-1 is shown in SEQ ID NO.09.

[0014] Thirdly, this application provides a soluble fibrin SF antibody, wherein the SF antibody is a second antibody SF-2, the amino acid sequence of the heavy chain variable region of the second antibody SF-2 is shown in SEQ ID NO.14, and the amino acid sequence of the light chain variable region of the second antibody SF-2 is shown in SEQ ID NO.19.

[0015] Fourthly, this application provides a soluble fibrin SF antibody, wherein the SF antibody is a third antibody SF-3, the amino acid sequence of the heavy chain variable region of the third antibody SF-3 is shown in SEQ ID NO.24, and the amino acid sequence of the light chain variable region of the third antibody SF-3 is shown in SEQ ID NO.29.

[0016] Fifthly, this application provides a biomaterial related to the above-mentioned soluble fibrin SF antibody, wherein the biomaterial is any one of the following:

[0017] (a) Nucleic acid molecules encoding the aforementioned SF antibody;

[0018] (b) Expression cassette containing the nucleic acid molecules from (a);

[0019] (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b);

[0020] (d) Recombinant cells containing the nucleic acid molecules in (a), the expression cassette in (b), or the recombinant vector in (c).

[0021] Sixthly, this application provides a method for preparing the above-mentioned soluble fibrin SF antibody, comprising:

[0022] (1) Use D-dimer as an immunogen to immunize rabbits and collect lymphocytes from the spleen of the immunized rabbits.

[0023] (2) B lymphocytes were fluorescently labeled with D-dimer as a screening agent and sorted by flow cytometry to obtain positive clone cell lines.

[0024] (3) Extract the antibody gene from the positive clone cell line, construct an expression vector, and transfect it into 293F cells to amplify and express the positive clone;

[0025] (4) Collect cell culture medium, purify antibodies, and detect antibodies using an ELISA platform coated with fibrinogen degradation fragments (SF).

[0026] Furthermore, the fibrinogen degradation fragments include primary fibrinolysin (X, Y, and D), secondary fibrinolysin (D-dimer), and soluble fibrin SF.

[0027] Seventhly, this application provides a kit for detecting soluble fibrin SF, the kit containing the SF antibody as described above.

[0028] Eighthly, this application provides a conjugate comprising an SF antibody, as described above, covalently linked to a chemical or biological marker.

[0029] Ninthly, this application provides a conjugate formed by coupling the above-mentioned SF antibody and / or the above-mentioned conjugate with a solid or semi-solid medium.

[0030] Tenthly, this application provides the use of the above-mentioned soluble fibrin SF antibody, conjugate or conjugate in the preparation of products for detecting SF expression.

[0031] In one aspect, this application provides a method for detecting soluble fibrin (SF), comprising:

[0032] The above-mentioned SF antibody, kit, conjugate or conjugate is used to contact the sample to be tested to form an immune complex; based on the signal of the immune complex, it is determined whether the sample to be tested contains SF or the amount of SF.

[0033] In summary, this application has the following beneficial effects:

[0034] 1. Compared with traditional mouse hybridoma cell antibody screening technology, this invention optimizes multiple aspects such as immune species (rabbit source), screening scheme and antibody expression. For the first time, it applies single B cell antibody preparation technology and flow cytometry to the development of antibodies for fibrinolysis system detection, achieving accurate and high-throughput screening.

[0035] 2. This application involves isolating antigen-specific B cells from immunized animal tissues or peripheral blood, amplifying the IgG heavy and light chain variable region genes from individual antibody-secreting B cells using single-cell PCR technology, and then expressing them in mammalian cells to obtain biologically active monoclonal antibodies. This technology preserves the natural pairing of the light and heavy chain variable regions and has the advantages of good gene diversity, high efficiency, and a small number of cells required.

[0036] 3. This application obtains three SF antibodies with high specificity and high affinity through a single immunization screening, which can achieve high affinity, high accuracy and high precision determination of SF.

[0037] 4. The monoclonal antibodies screened in this application were expressed in mammalian cells (293F) to obtain bioactive monoclonal antibodies SF, independent of animals. The mammalian expression system allows for post-translational modifications such as phosphorylation and glycosylation, resulting in recombinant proteins with correct conformation and higher-order structures, closely resembling natural proteins. This system also allows for the direct secretion of exogenous recombinant proteins into the culture medium, facilitating purification and application. Furthermore, the mammalian expression system possesses advantages such as high genetic stability and good reproducibility, making it widely used in biomedical research. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the structure of fibrinogen and its degradation products.

[0039] Figure 2 A schematic diagram illustrating the formation and degradation of fibrin;

[0040] Figure 3 This is the immunogen electrophoresis image provided in Example 1 of this application;

[0041] Figure 4 This is the original electrophoresis image provided in Embodiment 2 of this application; Detailed Implementation

[0042] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0043] The technical solution of this invention is as follows:

[0044] In this implementation method, mammals were used as immunogens. The specific sequences of the three specific SF antibodies obtained through a single immunization are shown in Tables 1, 2, and 3.

[0045] The terms "complementarity-determining region" or "CDR" refer to the highly variable region of the heavy and light chains of immunoglobulins, which contains one or more or even all of the major amino acid residues that affect the binding affinity of antibodies to the antigens or epitopes they recognize.

[0046] Table 1. Heavy and light chain sequences of the primary antibody SF-1

[0047]

[0048]

[0049]

[0050]

[0051] Table 2. Heavy and light chain sequences of the secondary antibody SF-2

[0052]

[0053]

[0054]

[0055]

[0056] Table 3. Heavy and light chain sequences of the third antibody SF-3

[0057]

[0058]

[0059]

[0060]

[0061] Rabbits are the preferred mammalian immunogen. The reason for choosing rabbits as the immunogen is that they are superior to mice in several aspects, especially in monoclonal antibody technology and antibody drug development, where they have the following advantages:

[0062] (1) Diversity of immune response: Rabbits have large spleens and rich antibody profiles, which can produce immune responses stronger than those of humans and mice. They can recognize some antigens that mice cannot recognize, thereby producing antibodies that recognize more unique epitopes.

[0063] (2) Immune response characteristics: Rabbits produce obvious immune responses to small molecules and haptens, while rodents do not produce obvious immune responses. This provides a better model for studying the immune responses of small molecules and drugs.

[0064] (3) Antibody diversity: Rabbit B cell and antibody libraries exhibit greater diversity during individual development, providing more options for developing specific antibodies against a variety of diseases.

[0065] (4) Antibody properties: Rabbit IgG antibodies are structurally simpler than mouse IgG antibodies, with fewer amino acids and additional disulfide bonds, which makes rabbit monoclonal antibodies more stable and crucial for the long-term stability and efficacy of antibody drugs.

[0066] (5) Application advantages: Because rabbits are relatively large, more B cells can be obtained from a single rabbit, which is very beneficial for large-scale monoclonal antibody production.

[0067] This embodiment also provides a nucleic acid molecule, an expression cassette, a recombinant vector, and recombinant cells:

[0068] In the process of preparing or obtaining the first antibody SF-1, the second antibody SF-2, or the third antibody SF-3, nucleic acid molecules expressing these antibodies can be linked to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0069] The nucleic acid molecules used to encode the aforementioned antibodies include DNA and RNA. It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is usually shown herein, the complementary strand is also disclosed. Furthermore, the molecular sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0070] Preferably, the nucleic acid molecules encoding the variable regions of the first antibody SF-1, the second antibody SF-2, or the third antibody SF-3 are shown in Table 4:

[0071] Table 4. Base sequences encoding antibody variable regions

[0072]

[0073]

[0074]

[0075] Preferably, the vector in the recombinant vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0076] Preferably, the recombinant cells are obtained by introducing the above-mentioned vector into cells. The cells are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. Preferably, the cells are mammalian cells.

[0077] Based on the amino acid sequence of the antibody disclosed herein, those skilled in the art will readily conceive of preparing the antibody using genetic engineering or other techniques (chemical synthesis, recombinant expression), such as isolating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody disclosed herein, it falls within the protection scope of this disclosure.

[0078] This embodiment also provides a conjugate, a coupling compound, and a reagent kit:

[0079] The SF antibody in this application can be used in combination with any detection reagent or therapeutic agent, such as diagnostic radionuclides, nanomaterials, etc., to detect the target site through the radioactivity of the radionuclide and thus obtain information about the target site; it can also be used in combination with therapeutic radionuclides to specifically kill target cells, tissues, etc. using the radioactivity of the radionuclide.

[0080] The conjugate in this application is a chemically or biologically labeled covalently linked SF antibody. The chemical label includes one or more selected from radioactive isotopes, fluorophores, rhodamine and its derivatives, luciferase, and luciferin; the biological label includes one or more selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase.

[0081] The conjugates in this application include solid or semi-solid media that bind to SF antibodies, including substances capable of being suspended or dispersed in a liquid phase (e.g., solid-phase carriers such as particles and magnetic beads), or solid phases capable of containing or carrying a liquid phase (e.g., supports such as plates, membranes, and test tubes, as well as containers such as well plates, microfluidic paths, glass capillaries, nanopillars, and monolithic columns). Preferably, the conjugate portion includes at least one selected from magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

[0082] The kit described in this application includes the aforementioned SF antibody, conjugate, or conjugate. This kit can effectively perform qualitative or quantitative detection of SF. As mentioned above, the antibodies in some specific embodiments or examples of this invention have higher binding activity to SF; therefore, reagents or kits containing these SF antibodies have higher detection sensitivity or specificity.

[0083] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0084] Example

[0085] Example 1: In vitro degradation and purification of immunogen

[0086] 1) Add 2 mL of reconstituted quality control plasma and 2 mL of 25 mM CaCl2 buffer to a 7 mL siliconization bottle and mix well.

[0087] 2) Add 2 μL of 120 IU / mL thrombin (CAS No.: 9002-04-4, manufacturer: Hubei Bello Biotechnology Co., Ltd., catalog number: EP1204-1000KU), mix well, and incubate at 37℃ without stirring for 1 hour to promote coagulation. Then, add a magnetic stir bar and 2 μL of 150 mU / mL plasmin (CAS No.: 9001-91-6, manufacturer: SIGMA, catalog number: P1867), and stir at 800 rpm for 72 hours to degrade plasmin.

[0088] 3) The degradation products were separated and purified using polyacrylamide gel electrophoresis (manufacturer: Solarbio, catalog number: P1200) and protein gel extraction (manufacturer: Solarbio, catalog number: G7200) to obtain D-dimer fragments with a purity >90% (e.g., Figure 3 (As shown).

[0089] Example 2: Screening of the original in vitro degradation and purification

[0090] Fibrinogen degradation fragments include X, Y, and D, and their preparation methods include:

[0091] 1. Degradation of fibrinogen

[0092] Fibrinogen was a commercially available product (CAS No.: 9001-32-5, manufacturer: Sigma; product number: F3879). 20 μl of plasmin solution (150 μg / mL) was added to 1 mL of fibrinogen solution, and the mixture was incubated at 37°C for 30 min to degrade it. The fibrinolytic reaction was then terminated by adding 10 μL of aprotinin solution (1 mg / mL), yielding a mixed FDP.

[0093] 1. Preparation of X, Y, and D fragments

[0094] The resulting FDP mixture was subjected to SDS-PAGE using an 8% separating gel and a 5% stacking gel. The 5% stacking gel was electrophoresed at 80V, and the 8% separating gel was electrophoresed at 120V. After 1 hour of electrophoresis, the gel was stained for 30 minutes, and the results were observed after destaining.

[0095] like Figure 4 The SDS-PAGE results shown have molecular weights of 250 kDa, 150 kDa, and 100 kDa for fragments X, Y, and D, respectively.

[0096] 3) SF preparation method

[0097] Add 2 μL of 120 IU / mL thrombin to 1 mL of fibrinogen solution with a concentration of 1 mg / mL, mix well, and incubate at 37°C without stirring for 1 hour to promote clotting.

[0098] Example 3 Animal Immunization

[0099] Rabbits were used as immunized animals. Pre-immunization serum from 8 rabbits was collected for immunogen detection, and 4 rabbits with lower background levels were selected for immunization.

[0100] During the immunization process, the immunogen (D-dimer fragment prepared in Example 1) was used for serum ELISA detection. Table 5 shows the serum ELISA results of the final selected rabbit (number E14143) for 3rd, 4th, and 5th immunizations. After the standard 63-day immunization program, the rabbits were euthanized, and their spleens were collected for monoclonal antibody screening.

[0101] Table 5. Rabbit serum ELISA detection

[0102]

[0103] Example 4: Flow Cytometry Cell Sorting and Positive Clone Amplification

[0104] In this embodiment, fluorescently labeled multi-parameter flow cytometry is used for individual B cell sorting during the B cell sorting stage, achieving precise and high-throughput screening. Fluorescently labeled multi-parameter flow cytometry sorting involves adding fluorescent antibodies to label B cell surface markers and fluorescently labeled target antigens (used to bind to the BCR on the B cell surface), thereby sorting out target antigen-specific antibodies. This enables rapid, accurate, and high-throughput separation of B cells and simultaneous multi-parameter analysis.

[0105] The specific sorting process is as follows:

[0106] B lymphocytes were isolated from the spleen and fluorescently labeled with FITC-labeled D-dimer selector. These cells were then enriched using flow cytometry and sorted into 96-well plates, one cell per well, yielding a total of 2000 single cells. The individual B cells were then cultured primaryly in the 96-well plates. The cultured B cells secreted a small amount of antibody supernatant. After 10–14 days, the cell supernatant was collected for ELISA verification and cross-antibody screening.

[0107] Antibody genes were extracted from selected cell lines, and an expression module pcDNA 3.4 was constructed. This module was then transfected into 293F cells for positive clone amplification and expression.

[0108] Example 5: 293F cell transfection

[0109] This embodiment enables the expression of the screened monoclonal antibodies in mammalian cells (293F) to obtain biologically active monoclonal antibodies without animal dependence. Because mammalian expression systems can perform post-translational modifications, such as phosphorylation and glycosylation, the expressed recombinant proteins possess correct conformation and higher-order structure, closely resembling natural proteins. Furthermore, this mammalian expression system allows exogenous recombinant proteins to be directly secreted into the culture medium, facilitating purification and application.

[0110] The specific process is as follows:

[0111] 1. Preparation before transfection

[0112] With a density of 0.3 to 0.35 × 10⁻⁶ 6 293F cells per cell / mL were passaged and seeded in 20 mL of SMM 293-TII medium (manufacturer: SinoBiogical, catalog number: M293TII). The cells were cultured at 37°C, 120 rpm, and 5% CO2. After 3 days, the cell density was approximately 2–3 × 10⁻⁶ cells / mL. 6 When the cell density was 1*10 cells / mL, it was diluted with SMM 293-TII culture medium to a density of 1*10. 6 The cell culture volume is 20 mL per bottle. Then tighten the bottle cap and place it in a shaker for further culture. Transfection can be performed after 2-4 hours.

[0113] 2) Preparation of transfection solution

[0114] Dilute 10 μg of plasmid with 0.15 M NaCl, mix well, and incubate at room temperature for 5 min. Add 50 μL of Sinofection (manufacturer: SinoBiogical, catalog number: STF02) to the plasmid dilution buffer, making the final total volume of transfection buffer 1 mL. Mix well and incubate for 10 min.

[0115] 3) Transfection

[0116] Add the prepared transfection solution dropwise to the cell culture medium, shake well, tighten the bottle cap, and place it on a shaker for incubation at 37°C, 120 rpm, and 5% CO2.

[0117] 4) Post-transfection observation and process testing

[0118] 20-24 hours after transfection, add SMS293-SUPI medium (0.7mL / 20mL) (manufacturer: SinoBiogical, catalog number: M293-SUPI). Continue to culture every other day, and observe and record the cell status and number. On the 3rd day after transfection, take 0.5mL of sample for ELISA identification and detection. Continue to culture the cells in shake flasks for 4-8 days, and collect the cell culture supernatant according to the cell viability.

[0119] Example 6 Antibody Purification

[0120] 1) Pretreatment before loading: Adjust the pH of the collected cell supernatant to 7.2-7.4 and filter it through a 0.22μm filter.

[0121] 2) Column pretreatment: After equilibrating the Protein G column (manufacturer: Navi, catalog number: 16255-17015-090100) to room temperature, equilibrate the protein column with 5 to 10 column volumes of equilibration buffer (20mM PB + 0.15M NaCl, pH 7.0) at a flow rate of 10 rpm.

[0122] 3) Sample loading: Load the pretreated cell supernatant at a flow rate of 10 rpm and collect the flow-through.

[0123] 4) Impurity removal: Wash 5 to 10 column volumes with equilibration buffer (20mM PB + 0.15M NaCl, pH 7.0) at a flow rate of 10 rpm.

[0124] 5) Sample elution and collection: Add 10% of the expected sample volume of Tris (pH 9.0) to the collection tube, and elute the column using Elution Buffer (pH 3.0 glycine) at a flow rate of 10 rpm to collect the sample.

[0125] 6) Concentration and dialysis: After overnight dialysis with PBS, concentrate the protein to a concentration >0.5 mg / mL.

[0126] Example 7: ELISA platform containing screening antibodies

[0127] The pre-D-dimer (DD), soluble fibrin (SF), and fibrinogen degradation fragments X, Y, and D obtained in Example 2 were coated at a rate of 100 ng / well and incubated overnight at 4°C. The coating buffer (0.05 M phosphate buffer) was removed, and blocking buffer (0.05 M phosphate buffer, 3% BSA) was added at a rate of 200 μL / well to the antibody-coated ELISA plate. The plate was then incubated at 37°C for 2 hours for blocking.

[0128] After sealing the ELISA plate, wash it 3-5 times with washing buffer (PBS + 0.05% Tween-20) and drain as much water as possible. Using commercially available Roche D-dimer antibody as a control, add the antibodies to be tested (antibody numbers: FDP-1, FDP-2, FDP-3) to the coated ELISA plate and incubate at 37°C for 1 hour. Wash the ELISA plate 3-5 times with washing buffer and drain as much water as possible.

[0129] Add secondary antibody (manufacturer: Zhongshan Jinqiao, catalog number: ZB-5301): Dilute the secondary antibody 1:5000 with homemade dilution buffer (PBS + 0.2% Tween-20 + 1% BSA), and add 50 μL / well to the ELISA plate. Incubate at 37°C for 45 minutes. Wash 3-5 times with washing buffer and drain as much water as possible. Add 100 μL / well of chromogenic solution (manufacturer: Solarbio, catalog number: PR1210) (A and B mixed) to the ELISA plate and incubate at 37°C for 10-15 minutes.

[0130] The stop solution (manufacturer Solarbio, product number: C1058) was added to the ELISA plate at a rate of 50 μL / well to terminate the colorimetric reaction.

[0131] The wavelength of the microplate reader was set to 450 nm, and the OD values ​​were read. The results are shown in Table 6.

[0132] Table 6. ELISA Platform Detection Results

[0133]

[0134] As shown in Table 6, on the ELISA platform, under the same antibody and antigen concentrations, the three SF recombinant rabbit monoclonal antibodies (antibody numbers SF-1, SF-2, and SF-3) of this application all showed specific reactions with SF and no cross-reactivity with primary fibrinolytic products (X, Y, and D). They can be applied to SF indirect ELISA and Western blotting detection and screening.

[0135] In summary, this application is the first to apply single B cell antibody preparation technology and flow cytometry sorting to the development of antibodies for coagulation in vitro diagnostic fibrinolysis system detection. Using a mammalian cell expression system (293F), three biologically active SF monoclonal antibodies (antibody numbers SF-1, SF-2, and SF-3) were successfully expressed and obtained. The heavy chain variable region and light chain variable region of each antibody were sequenced. The amino acid sequences are shown in Tables 1-3, and the gene sequences are shown in Table 4.

[0136] This application yielded three highly specific and high-affinity SF antibodies through a single immunization screening process. This achieves import substitution of core raw materials and is applied to the development of SF detection kits. It fills the gap in the market for domestically produced SF antibodies, ensuring the completeness of fibrinolysis system detection.

[0137] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A soluble fibronectin SF antibody, characterized in that, The SF antibody comprises any one of a first antibody SF-1, a second antibody SF-2 or a third antibody SF-3; The complementary determining regions CDR of the heavy chain H-SF-1 of the first antibody SF-1 are as follows: CDR1 shown in SEQ ID NO. 01, CDR2 shown in SEQ ID NO. 02, CDR3 shown in SEQ ID NO. 03; the complementary determining regions CDR of the light chain L-SF-1 of the first antibody SF-1 are as follows: the sequence of CDR1 is shown in SEQ ID NO. 06, the sequence of CDR2 is KAS, the sequence of CDR3 is shown in SEQ ID NO. 08; The complementary determining regions CDR of the heavy chain H-SF-2 of the second antibody SF-2 are as follows: CDR1 shown in SEQ ID NO. 11, CDR2 shown in SEQ ID NO. 12, CDR3 shown in SEQ ID NO. 13; the complementary determining regions CDR of the light chain L-SF-2 of the second antibody SF-2 are as follows: the sequence of CDR1 is shown in SEQ ID NO. 16, the sequence of CDR2 is SAS, the sequence of CDR3 is shown in SEQ ID NO. 18; The complementary determining regions CDR of the heavy chain H-SF-3 of the third antibody SF-3 are as follows: CDR1 shown in SEQ ID NO. 21, CDR2 shown in SEQ ID NO. 22, CDR3 shown in SEQ ID NO. 23; the complementary determining regions CDR of the light chain L-SF-3 of the third antibody SF-3 are as follows: the sequence of CDR1 is shown in SEQ ID NO. 26, the sequence of CDR2 is QAS, the sequence of CDR3 is shown in SEQ ID NO.

28.

2. A soluble fiber protein (SF) antibody, characterized in that, The SF antibody is the first antibody SF-1, the amino acid sequence of the heavy chain variable region of the first antibody SF-1 is shown in SEQ ID NO. 04, and the amino acid sequence of the light chain variable region of the first antibody SF-1 is shown in SEQ ID NO.

09.

3. A soluble fiber protein (SF) antibody, characterized in that, The SF antibody is the second antibody SF-2, the amino acid sequence of the heavy chain variable region of the second antibody SF-2 is shown in SEQ ID NO. 14, and the amino acid sequence of the light chain variable region of the second antibody SF-2 is shown in SEQ ID NO.

19.

4. A soluble fibronectin SF antibody, characterized in that, The SF antibody is the third antibody SF-3, the amino acid sequence of the heavy chain variable region of the third antibody SF-3 is shown in SEQ ID NO. 24, and the amino acid sequence of the light chain variable region of the third antibody SF-3 is shown in SEQ ID NO.

29.

5. A biomaterial associated with the soluble fibronectin SF antibody according to any one of claims 1 to 4, characterized in that, The biological material is any one of the following: (a) a nucleic acid molecule encoding the SF antibody according to any one of claims 1-4; (b) an expression cassette containing the nucleic acid molecule in (a); (c) a recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b); (d) a recombinant cell containing the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c).

6. A kit for detecting soluble fibrin (SF), characterized by, The kit comprises a soluble fibrin SF antibody according to any one of claims 1 to 4.

7. A composite, characterized in that, The conjugate comprises a soluble fibrin SF antibody according to any one of claims 1 to 4 covalently linked to a chemical or biological label.

8. Use of a soluble fibrin SF antibody according to any one of claims 1 to 4, or of a conjugate according to claim 7, for the manufacture of a product for the detection of soluble fibrin SF.

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