Plasmin degradation product FDP antibody and application thereof

Abstract

The invention relates to a biotechnology, and particularly discloses a plasmin degradation product FDP antibody and application thereof. According to the present invention, the single B cell antibody preparation technology and the flow cytometry sorting method are firstly applied to the blood coagulation in vitro diagnosis fibrinolysis system detection antibody development, and the mammalian cell expression system is adopted to successfully express and obtain the three FDP monoclonal antibody strains (the numbers are FDP-1, FDP-2 and FDP-3) with the biological activity; on an ELISA (enzyme-linked immuno sorbent assay) platform, the three FDP recombinant rabbit monoclonal antibodies have specific reaction with primary fibrinolytic substances (X and Y) and secondary fibrinolytic substances (DD); on an immunoturbidimetric platform, the FDP-1 has specific reaction with the primary fibrinolytic substance and the secondary fibrinolytic substance, and the FDP-2 only has specific reaction with the primary fibrinolytic substance on the immunoturbidimetric platform. The three FDP antibodies provided by the invention can make up the vacancy of domestic FDP antibodies on the market, ensure the joint monitoring suitability of D-dimer and FDP, and realize the detection integrity of fibrinolytic system.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and more specifically, to an antibody against a plasmin degradation product FDP and its application. Background Art

[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. Its structure is as follows Figure 1 As shown. Activation of the coagulation system leads to the generation of thrombin, which converts fibrinogen into fibrin by catalyzing proteolytic removal of fibrinopeptide A and fibrinopeptide B. These cleavages expose two polymerization sites in the E region of fibrinogen to form soluble fibrin (SF), to which the D region from the fibrinogen molecule can bind. Soluble fibrin spontaneously polymerizes into double-stranded fibrils, with the fibrin monomers in each chain arranged end-to-end, while the cross-chain fibrins are arranged in a semi-staggered overlapping manner, as shown in Figure 2 The combination of chain a and chain b is shown in Figure 1. Factor XIIIa catalyzes the covalent cross-linking of adjacent D regions through isopeptide bonds to form cross-linked and stable fibrin.

[0003] The basic process of 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 and stable fibrin, it produces diverse fragments due to different cleavage sites (see Figure 2 ), as shown in the figure DD / E, DY / YD, DXY / YXD and DXD / YY, where DD / E is the smallest degradation fragment (D-dimer). All the above fragments are collectively referred to as fibrin (original) degradation products (Fibrin (Fibrinogen) degradation products, FDP).

[0004] At present, there are many studies and related kits on D-dimer, but there are fewer types of kits for detecting FDP, which are mainly based on immunoturbidimetry and rely on imports. The development of FDP antibodies required for the development of the kit is the rate-limiting step in the development of the kit. According to research, the domestically produced FDP antibodies on the market have poor reactivity with primary fibrinolytic substances (fragments X, Y, D and E) and cannot meet clinical needs.

[0005] In addition, most of the existing fibrinolytic degradation products are mouse monoclonal antibodies prepared using traditional hybridoma technology. Hybridoma technology has the disadvantages of limited development species (monoclonal antibodies produced using hybridoma technology are mostly mouse-derived), long preparation cycle, cumbersome operation steps, easy to produce human anti-mouse antibody (HAMA) reaction, and quickly cleared in the human circulatory system. For the detection of monoclonal antibodies related to the fibrinolytic system, D-dimer, FDP and SF are all obtained from fibrinogen by different enzyme catalysis, and have great similarity with fibrinogen structure, which makes it very difficult to screen hybridoma mouse monoclonal antibodies.

[0006] In view of this, there is an urgent need to develop an antibody that can effectively bind to the plasmin degradation product FDP and is easy to screen and obtain. Summary of the invention

[0007] In order to solve the above technical problems, the present application provides antibodies against the plasmin degradation product FDP and applications thereof.

[0008] This application adopts the following technical solutions: In a first aspect, the present application provides an antibody against a plasmin degradation product FDP, wherein the FDP antibody comprises any one of a first antibody FDP-1, a second antibody FDP-2 or a third antibody FDP-3; The complementary determining region CDR of the heavy chain H-FDP-1 of the first antibody FDP-1 is 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 complementary determining region CDR of the light chain L-FDP-1 of the first antibody FDP-1 is as follows: CDR1 shown in SEQ ID NO.06, CDR2 shown in SEQ ID NO.07, and CDR3 shown in SEQ ID NO.08; The complementary determining region CDR of the heavy chain H-FDP-2 of the second antibody FDP-2 is 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 complementary determining region CDR of the heavy chain L-FDP-2 of the second antibody FDP-2 is as follows: CDR1 shown in SEQ ID NO.16, CDR2 shown in SEQ ID NO.17, and CDR3 shown in SEQ ID NO.18; The complementary determining region CDR of the heavy chain H-FDP-3 of the third antibody FDP-3 is 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 complementary determining region CDR of the heavy chain L-FDP-3 of the third antibody FDP-3 is as follows: CDR1 shown in SEQ ID NO.26, CDR2 shown in SEQ ID NO.27, and CDR3 shown in SEQ ID NO.28.

[0009] In a second aspect, the present application provides an antibody against a plasmin degradation product FDP, wherein the FDP antibody is a first antibody FDP-1, wherein the amino acid sequence of the heavy chain variable region of the first antibody FDP-1 is shown in SEQ ID NO.04, and the amino acid sequence of the light chain variable region of the first antibody FDP-1 is shown in SEQ ID NO.09.

[0010] In a third aspect, the present application provides an antibody against a plasmin degradation product FDP, wherein the FDP antibody is a second antibody FDP-2, wherein the amino acid sequence of the heavy chain variable region of the second antibody FDP-2 is shown in SEQ ID NO.14, and the amino acid sequence of the light chain variable region of the second antibody FDP-2 is shown in SEQ ID NO.19.

[0011] In a fourth aspect, the present application provides an antibody against a plasmin degradation product FDP, wherein the FDP antibody is a third antibody FDP-3, wherein the amino acid sequence of the heavy chain variable region of the third antibody FDP-3 is shown in SEQ ID NO.24, and the amino acid sequence of the light chain variable region of the third antibody FDP-3 is shown in SEQ ID NO.29.

[0012] In a fifth aspect, the present application provides a biomaterial related to the above-mentioned plasmin degradation product FDP antibody, and the biomaterial is any one of the following: (a) a nucleic acid molecule encoding the above-mentioned FDP antibody; (b) an expression cassette comprising the nucleic acid molecule described in (a); (c) a recombinant vector comprising the nucleic acid molecule in (a) or the expression cassette in (b); (d) A recombinant cell comprising the nucleic acid molecule in (a), the expression cassette in (b) or the recombinant vector in (c).

[0013] In a sixth aspect, the present application provides a kit for detecting FDP, wherein the kit contains the above-mentioned antibody against the plasmin degradation product FDP.

[0014] In a seventh aspect, the present application provides a conjugate comprising the FDP antibody as described above covalently linked to a chemical marker or a biological marker.

[0015] In an eighth aspect, the present application provides a conjugate, which is formed by coupling the above-mentioned FDP antibody, and / or the above-mentioned binding substance with a solid medium or a semi-solid medium.

[0016] In a ninth aspect, the present application provides a use of the above-mentioned FDP antibody, conjugate or coupling in the preparation of a product for detecting FDP expression.

[0017] In a tenth aspect, the present application provides a method for detecting FDP, comprising: The above-mentioned FDP antibody, kit, conjugate or coupling substance is contacted with the sample to be detected to form an immune complex; Based on the signal of the immune complex, it is determined whether the sample to be tested contains FDP or the content of FDP.

[0018] In an eleventh aspect, the present application provides a method for preparing the above-mentioned plasmin degradation product FDP antibody, which comprises: (1) Using D-dimer as an immunogen to immunize rabbits, and collecting lymphocytes from the spleen of the immunized rabbits; (2) Using fluorescently labeled D-dimer as a screening agent to fluorescently label B lymphocytes, and sorting them using flow cytometry to obtain positive clone cell lines; (3) extracting the antibody gene of the positive clone cell line, constructing an expression vector, and transfecting it into 293F cells to amplify and express the positive clone; (4) Collecting the cell culture fluid, purifying the antibodies, and detecting the antibodies using an ELISA platform coated with fibrinogen degradation fragments.

[0019] Furthermore, the degradation fragments of fibrinogen include primary fibrinolytic products (X, Y, and D), secondary fibrinolytic products (D-dimer), and soluble fibrin SF.

[0020] In summary, this application has the following beneficial effects: 1. Compared with the traditional mouse hybridoma cell antibody screening technology, the present invention optimizes the immune species (rabbit source), screening scheme and antibody expression in multiple directions. For the first time, single B cell antibody preparation technology and flow cytometry sorting method are applied to the development of fibrinolytic system detection antibodies to achieve accurate and high-throughput screening.

[0021] 2. The present application separates antigen-specific B cells from immune animal tissues or peripheral blood, amplifies IgG heavy chain and light chain variable region genes from single antibody-secreting B cells by single-cell PCR technology, and then expresses them in mammalian cells to obtain monoclonal antibodies with biological activity. This technology retains the natural pairing of light and heavy chain variable regions, and has the advantages of good gene diversity, high efficiency, and a small number of cells required.

[0022] 3. This application screened and obtained three strains of FDP antibodies with strong specificity and high affinity through a single immunization, which can achieve high affinity, high accuracy and high precision determination of FDP.

[0023] 4. The monoclonal clone obtained by screening in this application is expressed in mammalian cells (293F) and obtains a monoclonal antibody FDP with biological activity, which is independent of animals. The mammalian expression system can perform post-translational modifications, such as phosphorylation, glycosylation, etc., which makes the expressed recombinant protein have the correct configuration and higher-order structure, close to the natural protein. This system can also make the exogenous recombinant protein directly secreted into the culture medium, which is convenient for purification and application. In addition, the mammalian expression system has the advantages of high genetic stability and good repeatability, making it widely used in biomedical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the structure of fibrinogen and its degradation products; Figure 2 Schematic diagram of the formation and degradation of fibrin; Figure 3 is the immunogen electrophoresis diagram provided in Example 1 of the present application; Figure 4 It is the original electrophoresis diagram of the screening provided in Example 2 of the present application; Figure 5 It is the affinity between the ELISA platform antibody and the DD antigen provided in Example 7 of the present application; Figure 6 It is the reactivity of FDP antibody and secondary fibrinolysis in the immunoturbidimetric platform provided in Example 8 of the present application; Figure 7 It is the reactivity of FDP antibody and primary fibrinolysis material on the immunoturbidimetric platform provided in Example 8 of the present application; Figure 8 This is the antibody SDS-PAGE result analysis provided in Example 9 of the present application. DETAILED DESCRIPTION

[0025] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0026] The technical solution of the present invention is: In this embodiment, mammals are used as immunogens, and the specific sequences of three specific FDP antibodies obtained by screening through one immunization are shown in Table 1, Table 2 and Table 3: The terms "complementarity determining region" and "CDR" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to the regions containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody to the antigen or epitope it recognizes.

[0027] Table 1. Heavy chain and light chain sequences of the primary antibody FDP-1 Table 2. Heavy and light chain sequences of the second antibody FDP-2 Table 3. Heavy and light chain sequences of the third antibody FDP-3 The mammal used as the immunogen is preferably a rabbit. The rabbit is selected as the immunogen because it is superior to mice in many aspects, especially in monoclonal antibody technology and antibody drug development, and has the following advantages: (1) Diversity of immune response: Rabbits have large spleens and rich antibody spectrum, and can produce stronger immune responses than humans and mice. They can recognize some antigens that mouse antibodies cannot recognize, thereby producing antibodies that recognize more unique epitopes.

[0028] (2) Immune response characteristics: Rabbits will produce obvious immune responses to small molecules and haptens, while rodents will not produce obvious immune responses, which provides a better model for studying the immune response to small molecules and drugs.

[0029] (3) Antibody diversity: The rabbit's B cell repertoire and antibody repertoire show more diversity during individual development, which provides more options for developing specific antibodies against a variety of diseases.

[0030] (4) Antibody properties: Rabbit IgG antibodies are simpler in structure than mouse IgG, with fewer amino acids and additional disulfide bonds, which makes rabbit monoclonal antibodies more advantageous in stability, which is crucial for the long-term stability and effectiveness of antibody drugs.

[0031] (5) Application advantages: Since rabbits are larger, more B cells can be obtained from a single rabbit, which is very beneficial for large-scale monoclonal antibody production.

[0032] This embodiment also provides a nucleic acid molecule, an expression cassette, a recombinant vector and a recombinant cell: In the process of preparing or obtaining the first antibody FDP-1, the second antibody FDP-2 or the third antibody FDP-3, nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0033] Wherein, the nucleic acid molecules that can encode the above-mentioned antibodies include DNA and RNA. It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art should understand that they actually include any one of the complementary double strands, or two. For convenience, in this article, although only one strand is given in most cases, another strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0034] Preferably, the nucleic acid molecules encoding the variable regions of the first antibody FDP-1, the second antibody FDP-2 or the third antibody FDP-3 are shown in Table 4: Table 4. Base sequences encoding antibody variable regions Preferably, the vector in the recombinant vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0035] Preferably, the recombinant cell is obtained by introducing the above-mentioned vector into a cell. The cell is not particularly limited and can be a prokaryotic cell, a eukaryotic cell or a bacteriophage. Preferably, the cell is a mammalian cell.

[0036] Based on the amino acid sequence of the antibody disclosed herein, it is easy for those skilled in the art to conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody, for example, separating and purifying the antibody from the culture product of a recombinant cell that can recombinantly express the antibody as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody disclosed herein, it belongs to the protection scope of the present disclosure.

[0037] This embodiment also provides a conjugate, a conjugate, and a kit: The FDP antibody in the present application can be used in combination with any detection reagent or therapeutic preparation, for example, in combination with diagnostic nuclides, nanomaterials, etc., to detect the target site through the radioactivity of the nuclide, and then obtain information about the target site; it can also be used in combination with therapeutic nuclides to use the radioactivity of the nuclide to specifically kill target cells, tissues, etc.

[0038] The conjugate in the present application is a FDP antibody covalently linked to a chemical label or a biological label, wherein the chemical label includes one or more selected from radioisotopes, fluorophores, rhodamine and its derivatives, luciferase and luciferin; the biological label includes one or more selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase.

[0039] The conjugate in the present application includes a solid medium or semi-solid medium that binds to the FDP antibody, including a substance that can be suspended or dispersed in a liquid phase (for example, solid phase carriers such as particles and magnetic beads), or a solid phase that can accommodate or carry a liquid phase (for example, a support such as a plate, a membrane, a test tube, and a container such as a well plate, a microfluidic channel, a glass capillary, a nanocolumn, a monolithic column, etc.). Preferably, the conjugated part includes at least one selected from magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0040] The kit in the present application includes: the aforementioned FDP antibody, conjugate or conjugate. This kit can effectively detect FDP qualitatively or quantitatively. As mentioned above, the antibodies in some specific embodiments or examples of the present invention have higher binding activity with FDP, so the reagent or kit containing the FDP antibody has higher detection sensitivity or specificity.

[0041] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Example

[0042] Example 1 In vitro degradation and purification of immunogens 1) Add 2 mL of reconstituted quality control plasma and 2 mL of 25 mM CaCl to a 7 mL siliconized bottle. 2 Buffer, mix well.

[0043] 2) Add 2 μL 120 IU / mL thrombin (CAS No.: 9002-04-4, manufacturer: Hubei Beiluo Biotechnology Co., Ltd., item number: EP1204-1000KU), mix well, and coagulate at 37°C without stirring for 1 hour. Add a magnetic stir bar, add 2 μL 150 mU / mL plasmin (CAS No.: 9001-91-6, manufacturer: SIGMA, item number: P1867), and stir at 800 rpm for 72 hours.

[0044] 3) The degradation products were separated and purified by polyacrylamide gel electrophoresis (manufacturer: Solarbio, catalog number: P1200) and protein gel recovery method (manufacturer: Solarbio, catalog number: G7200) to obtain D-dimer fragments with a purity of >90% (such as Figure 3 shown).

[0045] Example 2 Screening of in vitro degradation and purification of protozoa The fibrinogen degradation fragments include X, Y and D, and the preparation method thereof includes: 1. Degradation of Fibrinogen Fibrinogen was purchased from the market (CAS No.: 9001-32-5, manufacturer: Sigma; item number: F3879). 20 μl of 150 μg / ml plasmin solution was added to 1 mL of 1 mg / mL fibrinogen solution, and placed at 37°C for degradation for 30 min. 10 μL of 1 mg / mL aprotinin solution was added to terminate the fibrinolytic reaction to obtain mixed FDP.

[0046] 1. Preparation of fragment X, fragment Y and fragment D The mixed FDP obtained by degradation was subjected to SDS-PAGE, and 8% separation gel and 5% concentrated gel were used for electrophoresis. The 5% concentrated gel was electrophoresed at 80V, and the 8% separation gel was electrophoresed at 120V. After electrophoresis for 1 hour, staining was performed for 30 minutes, and decolorization was performed to observe the results.

[0047] like Figure 4 As shown in the SDS-PAGE results, the molecular weights of fragments X, Y and D are 250 kd, 150 kd and 100 kd, respectively.

[0048] 3) SF preparation method Add 2 μL of 120 IU / mL thrombin to 1 mL of 1 mg / mL fibrinogen solution, mix well, and allow the blood to coagulate at 37°C without stirring for 1 hour.

[0049] Example 3 Animal Immunization Rabbits were used as immunized animals, and pre-immune sera from 8 rabbits were taken for immunogen detection, and 4 rabbits with low background were selected for immunization.

[0050] During the immunization process, the immunogen (D-dimer fragment prepared in Example 1) was used for serum ELISA detection. Table 5 shows the results of serum ELISA detection of the 3rd, 4th and 5th immunizations of the rabbit (No. E14143) finally selected. After the standard 63-day immunization program, the rabbits were killed, and the spleens were collected for screening of monoclonal antibodies.

[0051] Table 5. Rabbit serum ELISA test Example 4 Flow cytometry sorting and positive clone expansion In this embodiment, in the B cell sorting stage, a fluorescent labeling multi-parameter flow cytometry method is used to sort single B cells to achieve accurate and high-throughput screening. The fluorescent labeling multi-parameter flow cytometry method is used to add fluorescent antibodies for marking B cell surface markers and fluorescently labeled target antigens (used to bind to the B cell surface BCR), thereby sorting out target antigen-specific antibodies. Rapid, accurate, high-throughput separation of B cells and simultaneous analysis of multiple parameters are achieved.

[0052] The specific sorting process is: B lymphocytes in the spleen were isolated and fluorescently labeled with FITC-labeled D-dimer screening agents. These cells were enriched using flow cytometry, and then sorted into 96-well plates, with one cell per well, for a total of 2,000 single cells. Single B cells were cultured in 96-well plates, and the cultured B cells secreted a small amount of antibody supernatant. After 10 to 14 days, the cell supernatant was taken for ELISA verification and cross-antibody screening.

[0053] The antibody genes of the selected cell lines were extracted, and the expression module pcDNA3.4 was constructed and transfected into 293F cells for positive clone amplification and expression.

[0054] Example 5 293F cell transfection This embodiment achieves the expression of the above-screened monoclonal in mammalian cells (293F) and obtains monoclonal antibodies with biological activity, without relying on animals. Since the mammalian expression system can perform post-translational modifications, such as phosphorylation, glycosylation, etc., the expressed recombinant protein has the correct configuration and higher-order structure, close to the natural protein; and the mammalian expression system can also directly secrete the exogenous recombinant protein into the culture medium, which is convenient for purification and application.

[0055] The specific process is as follows: 1. Preparation before transfection The density is 0.3~0.35*10 6 293F cells with a concentration of 10 cells / mL were subcultured and inoculated into 20 mL of SMM 293-TII medium (manufacturer: SinoBiogical, catalog number: M293TII). The cells were incubated at 37°C, 120 rpm, and 5% CO 2 After 3 days, the cell density is about 2-3*10 6 cells / mL, dilute the cells to a density of 1*10 cells / mL with medium SMM 293-TII. 6 cells / mL, the volume of each bottle of cell liquid is 20mL, then tighten the bottle mouth and place it in a shaker for continued culture, and transfection can be carried out after 2 to 4 hours.

[0056] 2) Preparation of transfection solution Dilute 10 μg of plasmid with 0.15 M NaCl, mix well and place at room temperature for 5 minutes. Add 50 μL of Sinofection (manufacturer: SinoBiogical, catalog number: STF02) to the plasmid dilution, and the total volume of the final transfection solution is 1 mL. Mix well and place for 10 minutes.

[0057] 3) Transfection Add the prepared transfection solution dropwise into the cell culture medium, shake well, tighten the bottle cap and place it on a shaker at 37°C, 120 rpm, 5% CO 2 cultured under the conditions of .

[0058] 4) Post-transfection observation and process testing 20-24h after transfection, add SMS293-SUPI culture medium feed solution (0.7mL / 20mL) (manufacturer: SinoBiogical, catalog number: M293-SUPI), and feed culture every other day, and observe and record the status and number of cells. On the third day after transfection, sample 0.5mL for ELISA process identification and detection, continue to shake the flask to culture cells for 4-8 days, and collect the cell culture supernatant depending on the cell viability.

[0059] Example 6 Antibody Purification 1) Pre-sampling treatment: The pH of the collected cell supernatant was adjusted to 7.2-7.4 and filtered through 0.22 μm.

[0060] 2) Column pretreatment: After the Protein G column (manufacturer: Navi, catalog number: 16255-17015-090100) was equilibrated to room temperature, 5 to 10 column volumes of equilibration solution (20 mM PB + 0.15 M NaCl, pH 7.0) were used to equilibrate the protein column at a flow rate of 10 rpm.

[0061] 3) Sample loading: The pretreated cell supernatant was loaded at a flow rate of 10 rpm, and the flow-through was collected.

[0062] 4) Impurity removal: Use equilibrium solution (20 mM PB + 0.15 M NaCl, pH 7.0) at a flow rate of 10 rpm to wash 5 to 10 column volumes.

[0063] 5) Sample elution and collection: Add 10% of the expected sample volume of Tris (pH 9.0) to the collection tube, use Elution Buffer (pH 3.0 glycine) to elute the column at a flow rate of 10 rpm, and collect the sample.

[0064] 6) Concentration and fluid replacement: Use PBS for overnight dialysis and then concentrate to a protein concentration of > 0.5 mg / mL.

[0065] Example 7 ELISA platform coating screening antibody The screening original D-dimer (DD), fibrinogen (FIB), soluble fibrin (SF), and fibrinogen degradation fragments X, Y, and D obtained in Example 2 were coated at a coating amount of 100 ng / well and incubated overnight at 4°C. The coating buffer (0.05 M phosphate buffer) was deducted, and the blocking buffer (0.05 M phosphate buffer, 3% BSA) was added to the ELISA plate coated with the antibody at 200 μL / well, and incubated at 37°C for 2 hours for blocking.

[0066] Wash the blocked ELISA plate with washing buffer (PBS + 0.05% Tween-20) for 3-5 times and try to dry the water. Using the commercially available Roche D-dimer antibody as a control, add the antibody to be tested (antibody number: FDP-1, FDP-2, FDP-3) to the coated ELISA plate and incubate at 37°C for 1 hour. Wash the ELISA plate with washing buffer for 3-5 times and try to dry the water.

[0067] Add secondary antibody (manufacturer: Zhongshan Jinqiao, item number: ZB-5301): dilute the secondary antibody with homemade dilution buffer (PBS+0.2% Tween-20+1% BSA) at 1:5000, add 50μL / well to the ELISA plate, and incubate at 37℃ for 45 minutes. Wash 3-5 times with washing buffer and control the water as much as possible. Add 100μL / well of color development solution (manufacturer: Solarbio, item number: PR1210) (after mixing A and B) to the ELISA plate, and color at 37℃ for 10-15 minutes.

[0068] The stop solution (manufacturer Solarbio, catalog number: C1058) was added into the ELISA plate at 50 μL / well to stop the color development reaction.

[0069] The wavelength of the microplate reader was set to 450 nm, and the OD value was read. The results are shown in Table 6. The affinity between the antibody and the antigen on the ELISA platform is as follows Figure 5 shown.

[0070] Table 6. ELISA platform test results From Table 6 and Figure 5 It can be seen that on the ELISA platform, at the same antibody and antigen concentrations: the three FDP recombinant rabbit monoclonal antibodies of the present application (antibody numbers FDP-1, FDP-2, FDP-3) have specific reactions with primary fibrinolytic products (X and Y) and secondary fibrinolytic products (DD), and FDP-3 has no cross-reaction with FIB and SF. FDP-3 can be used in the field of FDP indirect ELISA and Western blotting detection and screening.

[0071] Example 8: Immunoturbidimetric platform coating to verify antibody and antigen reactivity Take 50mM MES (pH7.0) buffer to activate latex at room temperature, add 62.5μg / mL EDC and NHS, stir and mix at room temperature in the dark for 20min. After activation, add a certain amount of antibody (FDP-1, FDP-2, FDP-3 antibody), and couple at room temperature for 2h. Add 2% BSA to block at room temperature for 2h. Centrifuge, transfer to 50mM glycine (pH7.0) for storage, and ultrasonically disperse (power: 45%Φ6, 2s'2s, 4min).

[0072] The intensity of transmitted light or scattered light was measured by turbidimeter to evaluate the reactivity of FDP antibody with primary fibrinolytic products and secondary fibrinolytic products. Figure 6 and Figure 7 shown.

[0073] Depend on Figure 6 and Figure 7 It can be seen that in the immunoturbidimetric platform, at the same antibody and antigen concentrations: 3 strains of FDP recombinant rabbit monoclonal antibodies (antibody numbers FDP-1, FDP-2, FDP-3), 2 strains can be used in the immunoturbidimetric platform (antibody numbers FDP-1 and FDP-2), FDP-1 has specific reactions with both primary and secondary fibrinolytic products, and FDP-2 has specific reactions only with primary fibrinolytic products in the immunoturbidimetric platform. In addition, FDP-1 and FDP-2 have no cross-reaction with FIB and SF in the immunoturbidimetric platform, and can be used as raw materials for the development of FDP reagents in the immunoturbidimetric platform.

[0074] Example 9 SDS-PAGE identification 1) Gel preparation: Place the glass plate into the gel casting apparatus, prepare 10 ml of 10% separation gel in a beaker, and add 2.7 ml of ultrapure water, 3.3 ml of 30% acrylamide (Acr) and bisacrylamide (Bis) mixed solution (Acr / Bis, 29:1, v / v), 3.8 ml of 1.5M Tris-HCl (pH 8.8), 0.1 ml of 10% sodium dodecyl sulfate (SDS), 0.1 ml of 10% ammonium persulphate (AP) and 4 μl of tetramethylethylenediamine (TEMED). After thorough mixing, slowly add 5 ml of the separation gel solution along one corner of the glass plate into the gap between the gel casting plates, add more than 400 μl of distilled water to seal, and leave at room temperature for 30 minutes. After the separation gel solidifies, discard the distilled water, prepare 3ml of 5% concentrated gel in a beaker, and add 2.1ml ultrapure water, 0.5ml 30% Acr / Bis (29:1, v / v), 0.38ml 1M Tris-HCl (pH 6.8), 0.03ml 10% SDS, 0.03ml 10% AP and 0.003ml TEMED in sequence. After fully mixing, slowly add it between the gel plates along one corner of the glass plate until the liquid level reaches about 0.3cm from the concave plate, and quickly insert the comb. Leave it at room temperature for 30 minutes to allow the concentrated gel to solidify.

[0075] 2) Electrophoresis: Denatured protein samples were spotted in a certain order, 4 μl protein marker (manufacturer: Solarbio, item number: PR1920), and a certain volume of 1×SDS loading buffer was added to the excess wells to make up. Finally, the electrophoresis tank was connected to the electrophoresis instrument, and electrophoresis was performed at 80V for about 30 minutes. After the sample entered the separation gel, electrophoresis was performed at 120V for 2 to 3 hours.

[0076] For reducing electrophoresis, the sample needs to be boiled in a loading buffer containing β-mercaptoethanol for 5 minutes. This will open the disulfide bonds between proteins under the action of the reducing agent and obtain the primary structure of the protein.

[0077] The results are as follows Figure 8 As shown, the heavy chains of the three antibodies finally obtained by 293F expression were about 50 kDa, and the light chains were about 22 kDa.

[0078] 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 in vitro diagnosis of coagulation and fibrinolysis system detection. Using a mammalian cell expression system (293F), three strains of FDP monoclonal antibodies with biological activity (antibody numbers FDP-1, FDP-2, and FDP-3) were successfully expressed and obtained. The heavy chain variable regions and light chain variable regions were sequenced respectively. The amino acid sequences are shown in Tables 1-3, the gene sequences are shown in Table 4, and the information of the three antibodies is summarized in Table 7.

[0079] Table 7. Summary of Antibody Information This application screened and obtained three strains of FDP antibodies with strong specificity and high affinity through one immunization. It achieved the substitution of imported core raw materials and was applied to the development of FDP detection kits. It made up for the lack of domestic FDP antibodies on the market, ensured the compatibility of D-dimer and FDP joint monitoring, and realized the integrity of fibrinolytic system detection.

[0080] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A plasmin degradation product FDP antibody, characterized in that: The FDP antibody includes any one of the first antibody FDP-1, the second antibody FDP-2 or the third antibody FDP-3; The complementary determining region CDR of the heavy chain H-FDP-1 of the first antibody FDP-1 is 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 complementary determining region CDR of the light chain L-FDP-1 of the first antibody FDP-1 is as follows: CDR1 shown in SEQ ID NO.06, CDR2 shown in SEQ ID NO.07, and CDR3 shown in SEQ ID NO.08; The complementary determining region CDR of the heavy chain H-FDP-2 of the second antibody FDP-2 is 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 complementary determining region CDR of the light chain L-FDP-2 of the second antibody FDP-2 is as follows: CDR1 shown in SEQ ID NO.16, CDR2 shown in SEQ ID NO.17, and CDR3 shown in SEQ ID NO.18; The complementary determining region CDR of the heavy chain H-FDP-3 of the third antibody FDP-3 is 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 complementary determining region CDR of the light chain L-FDP-3 of the third antibody FDP-3 is as follows: CDR1 shown in SEQ ID NO.26, CDR2 shown in SEQ ID NO.27, and CDR3 shown in SEQ ID NO.

28.

2. A plasmin degradation product FDP antibody, characterized in that: The FDP antibody is the first antibody FDP-1, the amino acid sequence of the heavy chain variable region of the first antibody FDP-1 is shown in SEQ ID NO.04, and the amino acid sequence of the light chain variable region of the first antibody FDP-1 is shown in SEQ ID NO.

09.

3. A plasmin degradation product FDP antibody, characterized in that: The FDP antibody is the second antibody FDP-2, the amino acid sequence of the heavy chain variable region of the second antibody FDP-2 is shown in SEQ ID NO. 14, and the amino acid sequence of the light chain variable region of the second antibody FDP-2 is shown in SEQ ID NO.

19.

4. A plasmin degradation product FDP antibody, characterized in that: The FDP antibody is the third antibody FDP-3, the amino acid sequence of the heavy chain variable region of the third antibody FDP-3 is shown in SEQ ID NO. 24, and the amino acid sequence of the light chain variable region of the third antibody FDP-3 is shown in SEQ ID NO.

29.

5. A biomaterial related to the antibody against the plasmin degradation product FDP according to any one of claims 1 to 4, characterized in that: The biological material is any of the following: (a) a nucleic acid molecule encoding the FDP antibody according to any one of claims 1 to 4; (b) an expression cassette comprising the nucleic acid molecule described in (a); (c) a recombinant vector comprising 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 FDP, characterized in that: The kit contains the antibody against the plasmin degradation product FDP as described in any one of claims 1 to 4.

7. A combination, characterized in that The conjugate comprises the FDP antibody according to any one of claims 1 to 4 covalently linked to a chemical label or a biological label.

8. A conjugate, characterized in that: The conjugate is formed by coupling the FDP antibody according to any one of claims 1 to 4 and / or the conjugate according to claim 7 with a solid medium or a semi-solid medium.

9. Use of the FDP antibody according to any one of claims 1 to 4, the conjugate according to claim 7 or the conjugate according to claim 8 in the preparation of a product for detecting the expression of a plasmin degradation product FDP.

10. A method for detecting FDP, characterized in that: include: The FDP antibody according to any one of claims 1 to 4, the kit according to claim 6, the conjugate according to claim 7 or the conjugate according to claim 8 is contacted with a sample to be detected to form an immune complex; Based on the signal of the immune complex, it is determined whether the sample to be tested contains FDP or the content of the FDP.

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