Plasmin degradation product fdp antibodies and uses thereof

Abstract

The application relates to biotechnology and particularly discloses a fibrinolysin degradation product FDP 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 fibrinolysis system detection antibody in a blood coagulation in-vitro diagnosis, a mammalian cell expression system is adopted, three strains (FDP-1, FDP-2 and FDP-3) of FDP monoclonal antibodies with biological activity are successfully expressed and obtained. On an ELISA platform, the three strains of FDP recombinant rabbit monoclonal antibodies have specific reactions with primary fibrinolysis (X and Y) and secondary fibrinolysis (DD); on an immunoturbidimetry platform, FDP-1 has specific reactions with primary fibrinolysis and secondary fibrinolysis, and FDP-2 has a specific reaction with only primary fibrinolysis on the immunoturbidimetry platform. The three strains of FDP antibodies provided by the application can make up for the vacancy of domestic FDP antibodies on the market, guarantee the adaptability of D-dimer and FDP combined monitoring, and realize the integrity of fibrinolysis system detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, more particularly, it relates to a fibrinolysin degradation product FDP 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 fibrin monomers in each strand arranged in an end-to-end manner, and trans-strand fibrins arranged in a half-staggered overlap manner, as shown in the combination of Figure 2 and b chains. 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 of the above fragments are collectively referred to as 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, mainly based on immunoturbidimetry, and relying on imports. The development of FDP antibodies necessary for the formation of kits is the rate-limiting step of kit development. According to research, the domestic FDP antibodies on the market have poor reactivity with primary fibrinolyte (X, Y, D and E fragments), which cannot meet the clinical needs.

[0005] In addition, most of the existing plasmin degradation products are mouse monoclonal antibodies prepared by traditional hybridoma technology. The hybridoma technology has the disadvantages of limited development species (the monoclonal antibodies produced by the hybridoma technology are mostly of mouse origin), long preparation period, complicated operation steps, easy to produce human anti-mouse antibody (HAMA) reaction, and quickly eliminated in the human circulatory system. For the detection of related monoclonal antibodies of the fibrinolytic system, D-dimer, FDP and SF are all obtained from fibrinogen by different enzyme catalysis, which has great similarity with the structure of fibrinogen, which causes great difficulty in screening of hybridoma mouse monoclonal antibodies.

[0006] Therefore, it is urgent to develop an antibody which can effectively bind to the plasmin degradation product FDP and is easy to screen and obtain. SUMMARY

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

[0008] The present application adopts the following technical solutions:

[0009] In a first aspect, the present application provides a plasmin degradation product FDP antibody, wherein the FDP antibody comprises any one of a first antibody FDP-1, a second antibody FDP-2 or a third antibody FDP-3.

[0010] The complementarity 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, CDR3 shown in SEQ ID NO. 03; the complementarity 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, CDR3 shown in SEQ ID NO. 08;

[0011] The complementarity 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, CDR3 shown in SEQ ID NO. 13; the complementarity 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, CDR3 shown in SEQ ID NO. 18;

[0012] The complementarity determining regions CDR of the heavy chain H-FDP-3 of the third antibody FDP-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 complementarity determining regions CDR of the heavy chain L-FDP-3 of the third antibody FDP-3 are as follows: CDR1 shown in SEQ ID NO. 26, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 28.

[0013] In a second aspect, the present application provides a plasmin degradation product FDP antibody, which is a 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.

[0014] In a third aspect, the present application provides a plasmin degradation product FDP antibody, which is a 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.

[0015] In a fourth aspect, the present application provides a plasmin degradation product FDP antibody, which is a 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.

[0016] In a fifth aspect, the present application provides a biological material related to the above-mentioned plasmin degradation product FDP antibody, which is any one of the following:

[0017] (a) a nucleic acid molecule encoding the above-mentioned FDP antibody;

[0018] (b) an expression cassette containing the nucleic acid molecule in (a);

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

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

[0021] In a sixth aspect, the present application provides a kit for detecting FDP, which contains the above-mentioned plasmin degradation product FDP antibody.

[0022] In a seventh aspect, the present application provides a conjugate comprising the FDP antibody as described above covalently linked to a chemical label or a biological label.

[0023] In an eighth aspect, the present application provides a conjugate formed by coupling the FDP antibody as described above, and / or the conjugate as described above, with a solid medium or a semi-solid medium.

[0024] In a ninth aspect, the present application provides use of the FDP antibody, the conjugate or the conjugate as described above in the preparation of a product for detecting expression of FDP.

[0025] In a tenth aspect, the present application provides a method for detecting FDP, comprising:

[0026] contacting the FDP antibody, the kit, the conjugate or the conjugate as described above with a sample to be detected to form an immune complex;

[0027] determining whether the sample to be detected contains FDP or the content of FDP based on the signal of the immune complex.

[0028] In an eleventh aspect, the present application provides a method for preparing the FDP antibody of the plasmin degradation product, comprising:

[0029] (1) immunizing a rabbit with D-dimer as an immunogen, and collecting lymphocytes in the spleen of the rabbit after immunization;

[0030] (2) using fluorescent group-labeled D-dimer as a screening agent to fluorescently label B lymphocytes, and using a flow cytometer to sort to obtain a positive clone cell strain;

[0031] (3) extracting an antibody gene of the positive clone cell strain, constructing an expression vector, and transfecting into 293F cells for amplification and expression of the positive clone;

[0032] (4) collecting cell culture solution, purifying the antibody, and detecting the antibody through an ELISA platform coated with fibrinogen degradation fragments.

[0033] Further, the fibrinogen degradation fragments include primary fibrinolysis (X, Y and D), secondary fibrinolysis (D-dimer), and soluble fibrin SF.

[0034] In summary, the present application has the following beneficial effects:

[0035] 1. Compared with the traditional mouse hybridoma cell antibody screening technology, the present application optimizes the immunization species (rabbit source), the screening scheme and the antibody expression in multiple directions, and for the first time applies single B cell antibody preparation technology and flow cytometry sorting method to the development of fibrinolysis system detection antibodies, and realizes precise and high-throughput screening.

[0036] 2. The application separates antigen-specific B cells from the immune animal tissue 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 and obtains biologically active monoclonal antibodies in mammalian cells. This technology retains the natural pairing of light and heavy chain variable regions, has the advantages of good genetic diversity, high efficiency and less required cells.

[0037] 3. The application obtains three FDP antibodies with strong specificity and high affinity through one immunization and screening, and can realize high-affinity, high-accuracy and high-precision determination of FDP.

[0038] 4. The monoclonal antibody FDP obtained by screening in the application is expressed in mammalian cells (293F) and has biological activity, and does not depend on animals. The mammalian expression system can perform post-translational modifications such as phosphorylation and glycosylation, which makes the expressed recombinant protein have correct conformation and high-order structure, close to natural protein. This system can also make foreign recombinant proteins directly secret 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, and is widely used in biomedical research. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a schematic diagram of fibrinogen and its degradation products;

[0040] Figure 2 is a schematic diagram of fibrin formation and degradation;

[0041] Figure 3 is an immunogen electrophoresis diagram provided in Example 1 of the application;

[0042] Figure 4 is a screening original electrophoresis diagram provided in Example 2 of the application;

[0043] Figure 5 is the affinity of ELISA platform antibody to DD antigen provided in Example 7 of the application;

[0044] Figure 6 is the reactivity of FDP antibody to secondary fibrinolysate in the immunoturbidimetry platform provided in Example 8 of the application;

[0045] Figure 7 is the reactivity of FDP antibody to primary fibrinolysate in the immunoturbidimetry platform provided in Example 8 of the application;

[0046] Figure 8 is the antibody SDS-PAGE result analysis provided in Example 9 of the application. DETAILED DESCRIPTION

[0047] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0048] The technical solution of the present application is:

[0049] The specific sequences of the three specific FDP antibodies screened by one immunization with the mammal as the immunogen are shown in Table 1, Table 2 and Table 3.

[0050] The term "complementarity determining region", "CDR" refers to the highly variable region of the heavy and light chains of immunoglobulins, which refers to the region containing one or more or even all of the main amino acid residues that affect the binding affinity of the antibody to the antigen or epitope it recognizes.

[0051] Table 1. Heavy and light chain sequences of the first antibody FDP-1

[0052]

[0053]

[0054]

[0055]

[0056] Table 2. Heavy and light chain sequences of the second antibody FDP-2

[0057]

[0058]

[0059]

[0060]

[0061] Table 3. Heavy and light chain sequences of the third antibody FDP-3

[0062]

[0063]

[0064]

[0065]

[0066] The mammal as an immunogen is preferably a rabbit. The reason for choosing a rabbit as an immunogen is that it has advantages over mice in many aspects, especially in the development of monoclonal antibodies and antibody drugs, which are as follows:

[0067] (1) Immune response diversity: Rabbits have large spleens and rich antibody spectra, can produce stronger immune response than humans and mice, and can recognize some antigens that mouse antibodies cannot recognize, thereby producing antibodies that recognize more unique epitopes.

[0068] (2) Immune response characteristics: Rabbits produce a significant immune response to small molecules and haptens, while rodents do not produce a significant immune response, which provides a better model for studying the immune response of small molecules and drugs.

[0069] (3) Antibody diversity: The B cell library and antibody library of rabbits show more diversity during individual development, which provides more options for developing specific antibodies for various diseases.

[0070] (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.

[0071] (5) Application advantages: Due to the larger size of rabbits, more B cells can be obtained from a single rabbit, which is very advantageous for large-scale production of monoclonal antibodies.

[0072] The present embodiment also provides a nucleic acid molecule, an expression cassette, a recombinant vector, and a recombinant cell:

[0073] 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 used, which are connected with different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0074] Among them, the nucleic acid molecule can encode the above-mentioned antibodies, including DNA and RNA. It should be noted that for the nucleic acid molecules mentioned herein, those skilled in the art should understand that any one of the complementary double strands or both are actually included. For convenience, in this text, although only one strand is given in most cases, the other complementary strand is also disclosed. In addition, the sequence of molecules in the present application includes DNA or RNA form, and the disclosure of one means the disclosure of the other.

[0075] Preferably, the nucleic acid molecule encoding the variable region of the first antibody FDP-1, the second antibody FDP-2 or the third antibody FDP-3 is as shown in Table 4:

[0076] Table 4. Base sequence encoding antibody variable region

[0077]

[0078]

[0079]

[0080] Preferably, the vector in the recombinant vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0081] 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.

[0082] Based on the amino acid sequence of the antibody of the present disclosure, it is easy for those skilled in the art to conceive that the antibody is prepared by using genetic engineering technology or other technologies (chemical synthesis, recombinant expression), for example, the antibody is isolated and purified from the culture product of a recombinant cell capable of recombinantly expressing the antibody as described in any one of the above, which is easy for those skilled in the art to achieve. Therefore, no matter what technology is used to prepare the antibody of the present disclosure, it belongs to the protection scope of the present disclosure.

[0083] The present embodiment also provides a conjugate, a coupling agent and a kit:

[0084] The FDP antibody in the present application can be used in combination with any detection reagent or therapeutic preparation, for example, in combination with a diagnostic nuclide, a nanomaterial, etc. The target site is detected by the radioactivity of the nuclide, and then information of the target site is obtained. The FDP antibody can also be used in combination with a therapeutic nuclide, which specifically kills target cells, tissues, etc. by using the radioactivity of the nuclide.

[0085] The conjugate in the present application is a FDP antibody covalently linked with a chemical label or a biological label. The chemical label includes one or more selected from the group consisting of a radioisotope, a fluorophore, rhodamine and its derivatives, luciferase and fluorescein; and the biological label includes one or more selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase.

[0086] The conjugate in the present application includes a solid medium or semi-solid medium combined with the FDP antibody, including a substance capable of being suspended or dispersed in a liquid phase (e.g., a particle, a magnetic bead, etc. solid phase carrier), or a solid phase capable of accommodating or carrying a liquid phase (e.g., a plate, a membrane, a test tube, etc. support, and a container such as a well plate, a microchannel, a glass capillary, a nanocolumn, a monolithic column, etc.). Preferably, the conjugate portion includes at least one selected from a magnetic microsphere, a plastic microsphere, a plastic microparticle, a microwell plate, glass, a capillary, nylon, and a nitrocellulose membrane.

[0087] The kit in the present application includes the aforementioned FDP antibody, conjugate or conjugate. Such a kit can effectively qualitatively or quantitatively detect FDP. As described above, the antibody in some specific embodiments or examples of the present application has higher binding activity with FDP, and therefore the reagent or kit containing the FDP antibody has higher detection sensitivity or specificity.

[0088] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0089] EMBODIMENT

[0090] Example 1 Immunogen degradation in vitro, purification

[0091] 1) Add 2 mL of reconstituted quality control plasma and 2 mL of 25 mM CaCl2buffer to a 7 mL siliconized bottle, and mix well.

[0092] 2) Add 2 μL of 120 IU / mL thrombin (CAS No.: 9002-04-4, manufacturer: Hubei Belo Biological Technology Co., Ltd., product No.: EP1204-1000KU), mix well, and incubate the coagulation at 37°C without stirring for 1 hour. Add 2 μL of 150 mU / mL fibrinolysin (CAS No.: 9001-91-6, manufacturer: SIGMA, product No.: P1867) with a magnetic stirrer, and stir at 800 rpm for 72 hours of degradation.

[0093] 3) The degradation product is separated and purified by polyacrylamide gel electrophoresis (manufacturer: Solarbio, product No.: P1200) and protein gel recovery method (manufacturer: Solarbio, product No.: G7200) to obtain D-dimer fragments with a purity of > 90% (as shown in Figure 3 ).

[0094] Example 2 Screening of original in vitro degradation and purification

[0095] The fibrinogen degradation fragment includes X, Y and D, and the preparation method includes:

[0096] 1. Degradation of fibrinogen

[0097] Fibrinogen was purchased commercially (CAS number: 9001-32-5, manufacturer: sigma; item number: F3879). 150 μg / ml of plasmin solution was added to 1 mL of fibrinogen solution with a concentration of 1 mg / mL, and the degradation was carried out at 37°C for 30 min. The fibrinolysis reaction was terminated by adding 10 μL of aprotinin solution with a concentration of 1 mg / mL, and mixed FDP was obtained.

[0098] 1. Preparation of X fragment, Y fragment and D fragment

[0099] The degraded mixed FDP was subjected to SDS-PAGE, and electrophoresis was carried out using 8% separation gel and 5% concentration gel. The 5% concentration gel was subjected to electrophoresis using a voltage of 80V, and the 8% separation gel was subjected to electrophoresis using a voltage of 120V. After electrophoresis for 1 h, staining was carried out for 30 min, and the results were observed after decolorization.

[0100] As shown in the SDS-PAGE results Figure 4 , the molecular weights of fragments X, Y and D were 250 kd, 150 kd and 100 kd, respectively.

[0101] 3) SF preparation method

[0102] In 1 mL of fibrinogen solution with a concentration of 1 mg / mL, 2 μL of thrombin with a concentration of 120 IU / mL was added, and the mixture was uniformly mixed. Coagulation was carried out at 37°C without stirring for 1 h.

[0103] Example 3 Animal immunization

[0104] Rabbits were used as immunized animals, and the pre-immune sera of 8 rabbits were subjected to immunogen detection, and 4 rabbits with low background were selected for immunization.

[0105] During the immunization process, the immunogen (D-dimer fragment prepared in Example 1) was used for serum ELISA detection, and Table 5 shows the serum ELISA detection results of the 3rd, 4th and 5th immunization sera of the finally selected rabbit (number E14143). After the standard 63-day immunization program was completed, the rabbit was killed, and the spleen was collected for monoclonal antibody screening.

[0106] Table 5. Rabbit serum ELISA detection

[0107]

[0108]

[0109] Example 4 Flow cytometry sorting and positive clone amplification

[0110] The embodiment is in the B cell sorting stage, and a fluorescence labeling multi-parameter flow cytometry sorting method is selected for single B cell sorting, so as to realize precise and high-throughput screening. The fluorescence labeling multi-parameter flow cytometry sorting method sorts the target antigen specific antibody by adding fluorescence antibodies for labeling B cell surface markers and fluorescence labeled target antigens (for binding to B cell surface BCR), so as to realize rapid, accurate and high-throughput separation of B cells and multi-parameter simultaneous analysis.

[0111] The specific sorting process is as follows:

[0112] B lymphocytes in the spleen are separated, and the B lymphocytes are screened by FITC labeled D-dimer. The cells are enriched by a flow cytometer, and then the cells are sorted into 96-well plates, one cell per well, and a total of 2000 single cells are separated. The single B cells are cultured in the 96-well plates, and the cultured B cells will secrete a small amount of antibody supernatant. After 10-14 days, the cell supernatant is taken for ELISA verification and cross antibody screening.

[0113] The antibody gene of the selected cell strain is extracted, an expression module pcDNA3.4 is constructed, 293F cells are transfected, and positive clone amplification and expression are performed.

[0114] Example 5: 293F cell transfection

[0115] The embodiment realizes expression of the monoclonal antibodies screened above in mammalian cells (293F) and obtains the monoclonal antibodies with biological activity, which is independent of animals. Since the mammalian expression system can perform post-translational modifications such as phosphorylation and glycosylation, the expressed recombinant protein has a correct configuration and high-order structure, which is close to the natural protein; and the mammalian expression system can also make the exogenous recombinant protein directly secreted into the culture medium, which is convenient for purification and application.

[0116] The specific process is as follows:

[0117] 1. Preparation before transfection

[0118] The 293F cells with a density of 0.3-0.35*10 6 cells / mL are subcultured in 20 mL SMM 293-TII medium (manufacturer: SinoBiological, product number: M293TII). The cells are cultured at 37°C, 120 rpm and 5% CO2. After 3 days, when the cell density is about 2-3*10 6 cells / mL, the cell density is diluted to 1*10 6 cells / mL by using the culture medium SMM 293-TII, and the cell liquid volume is 20 mL per bottle. Then, the bottle mouth is tightly screwed and placed in the shaker for continuous culture. After 2-4 hours, transfection can be performed.

[0119] 2) Preparation of transfection solution

[0120] 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.

[0121] 3) Transfection

[0122] 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.

[0123] 4) Post-transfection observation and process testing

[0124] 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.

[0125] Example 6 Antibody Purification

[0126] 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.

[0127] 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.

[0128] 3) Sample loading: Load the pretreated cell supernatant at a flow rate of 10 rpm and collect the flow-through.

[0129] 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.

[0130] 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.

[0131] 6) Concentration exchange: After overnight dialysis using PBS, concentration to protein concentration > 0.5 mg / mL.

[0132] Example 7 ELISA platform coated screening antibody

[0133] Screening original D-dimer (DD), fibrinogen (FIB), soluble fibrin (SF), fibrinogen degradation fragments X, Y and D obtained in Example 2 were coated at a coating amount of 100 ng / well, 4°C overnight incubation. After deducting the coating buffer (0.05M phosphate buffer), blocking buffer (0.05M phosphate buffer, 3% BSA) was added to the coated ELISA plate at 200 μL / well, 37°C incubation for 2 hours blocking.

[0134] After washing the blocked ELISA plate with washing buffer (PBS + 0.05% Tween-20) for 3-5 times, try to dry the water. As a control, commercially available Roche D-dimer antibody was added to the coated ELISA plate, and the antibody to be tested (antibody number: FDP-1, FDP-2, FDP-3) was added to the coated ELISA plate, 37°C incubation for 1 hour. After washing the ELISA plate with washing buffer for 3-5 times, try to dry the water.

[0135] Add secondary antibody (manufacturer: Zhongshanjinqiao, product number: ZB-5301): dilute the secondary antibody with self-made dilution buffer (PBS + 0.2% Tween-20 + 1% BSA) at 1:5000, add 50 μL / well to the ELISA plate, 37°C incubation for 45 minutes. After washing with washing buffer for 3-5 times, try to dry the water. Add color developing solution (manufacturer: Solarbio, product number: PR1210) (mixed after A, B) to the ELISA plate at 100 μL / well, 37°C color development for 10-15 minutes.

[0136] Add stop solution (manufacturer: Solarbio, product number: C1058) to the ELISA plate at 50 μL / well to terminate the color development reaction.

[0137] Set the wavelength of the enzyme label instrument to 450 nm, read the OD value, and the results are shown in Table 6, and the affinity of the ELISA platform antibody to the antigen is shown in Table 6. Figure 5

[0138] Table 6. ELISA platform detection results

[0139]

[0140]

[0141] From Table 6 and Figure 5 ​As can be seen, on the ELISA platform, under the same antibody and antigen concentrations: the three FDP recombinant rabbit monoclonal antibodies (antibody numbers FDP-1, FDP-2, and FDP-3) of the present application have specific reactions with primary fibrin (X and Y) and secondary fibrin (DD), and FDP-3 has no cross-reaction with FIB and SF. FDP-3 can be applied to the field of FDP indirect ELISA and Western blotting detection and screening.

[0142] Example 8 Coating verification of antibody and antigen reactivity on the immunoturbidimetry platform

[0143] Take 50 mM MES (pH 7.0) buffer to activate the latex at room temperature, add 62.5 μg / mL EDC and NHS, stir evenly in the dark at room temperature for 20 min. After activation, add a certain amount of antibody (FDP-1, FDP-2, FDP-3 antibody), couple at room temperature for 2 h. Add 2% BSA and block at room temperature for 2 h. Centrifuge, replace with 50 mM glycine (pH 7.0) for storage, and ultrasonic dispersion (power: 45% Φ6, 2s'2s, 4 min).

[0144] The intensity of transmitted light or scattered light was measured by turbidimetry to evaluate the reactivity of FDP antibodies with primary fibrin and secondary fibrin, and the results are shown in Figure 6 and Figure 7 As can be seen, on the immunoturbidimetry platform, under the same antibody and antigen concentrations: the three FDP recombinant rabbit monoclonal antibodies (antibody numbers FDP-1, FDP-2, and FDP-3) of the present application have specific reactions with primary fibrin (X and Y) and secondary fibrin (DD), and FDP-3 has no cross-reaction with FIB and SF. FDP-3 can be applied to the field of FDP indirect ELISA and Western blotting detection and screening.

[0145] As can be seen from Figure 6 and Figure 7 As can be seen, on the immunoturbidimetry platform, under the same antibody and antigen concentrations: the three FDP recombinant rabbit monoclonal antibodies (antibody numbers FDP-1, FDP-2, and FDP-3) of the present application have specific reactions with primary fibrin (X and Y) and secondary fibrin (DD), and FDP-3 has no cross-reaction with FIB and SF. FDP-3 can be applied to the field of FDP indirect ELISA and Western blotting detection and screening.

[0146] Example 9 SDS-PAGE identification

[0147] 1) Gel preparation: Put the glass plate into the gel maker, prepare 10 ml 10% separating gel in a beaker, add 2.7 ml ultrapure water, 3.3 ml 30% acrylamide (Acr) and bisacrylamide (Bis) mixed solution (Acr / Bis, 29:1, v / v), 3.8 ml 1.5 M Tris-HCl (pH 8.8), 0.1 ml 10% sodium dodecyl sulfate (SDS), 0.1 ml 10% ammonium persulphate (AP) and 4 μl N,N,N,N-tetramethylethylenediamine (TEMED) in turn. After mixing well, add the prepared 5 ml separating gel solution between the gel maker gap along one corner of the glass plate, add more than 400 μl distilled water to seal, and place at room temperature for 30 min. After the separating gel solidifies, discard the distilled water, prepare 3 ml 5% concentrated gel in a beaker, add 2.1 ml ultrapure water, 0.5 ml 30% Acr / Bis (29:1, v / v), 0.38 ml 1 M Tris-HCl (pH 6.8), 0.03 ml 10% SDS, 0.03 ml 10% AP and 0.003 ml TEMED in turn. After mixing well, add between the gel maker along one corner of the glass plate until the liquid surface reaches about 0.3 cm away from the concave plate, and quickly insert the comb. Place at room temperature for 30 min until the concentrated gel solidifies.

[0148] 2) Electrophoresis: The denatured protein sample was added in a certain order, 4 μl protein Marker (manufacturer: Solarbio, product number: PR1920), and the rest of the wells were filled with a certain volume of 1x SDS loading buffer. Finally, connect the electrophoresis tank with the electrophoresis instrument, 80 V electrophoresis for about 30 min, and then 120 V electrophoresis for 2-3 h after the sample enters the separating gel.

[0149] Reducing electrophoresis, the sample needs to be boiled with loading buffer containing β-mercaptoethanol for 5 minutes, so that the disulfide bonds between proteins are opened under the action of reducing agent, and the primary structure of the protein is obtained.

[0150] The results are shown in Figure 8 The final 3 strains of antibody heavy chains obtained by 293F expression are about 50 kDa, and the light chains are about 22 kDa.

[0151] In summary, the application first applies single B cell antibody preparation technology and flow cytometry sorting method to the development of antibodies for detecting fibrinolysis system in coagulation in vitro diagnosis. Three strains of FDP monoclonal antibodies (antibody numbers FDP-1, FDP-2, and FDP-3) with biological activity are successfully expressed and obtained by using a mammalian cell expression system (293F). The heavy chain variable region and the light chain variable region of each of the three strains are sequenced, and the amino acid sequences are shown in Tables 1-3, the gene sequences are shown in Table 4, and the information of the three strains of antibodies is shown in Table 7.

[0152] Table 7. Information of antibodies

[0153]

[0154] The application screens three strains of FDP antibodies with strong specificity and high affinity through one-time immunization. The application realizes import substitution of core raw materials and is applied to the development of FDP detection kits. The application fills the gap of domestic FDP antibodies on the market and ensures the adaptability of D-dimer and FDP joint monitoring and the integrity of fibrinolysis system detection.

[0155] The specific embodiments are only an explanation of the application, and are not a limitation of the application. Those skilled in the art can make modifications to the embodiments according to the needs after reading the specification, and the modifications do not contribute to the creativity as long as the modifications are within the scope of the claims of the application.

Claims

1. An antibody against FDP, a plasmin degradation product, 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 complementarity-determining regions (CDRs) of the heavy chain H-FDP-1 of the first antibody FDP-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-FDP-1 of the first antibody FDP-1 are as follows: the sequence of CDR1 is shown in SEQ ID NO.06, the sequence of CDR2 is AAS, and the sequence of CDR3 is shown in SEQ ID NO.

08. The complementarity-determining regions (CDRs) of the heavy chain H-FDP-2 of the second antibody FDP-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 light chain L-FDP-2 of the second antibody FDP-2 are as follows: the sequence of CDR1 is shown in SEQ ID NO.16, the sequence of CDR2 is STS, and the sequence of CDR3 is shown in SEQ ID NO.

18. The complementarity-determining regions (CDRs) of the heavy chain H-FDP-3 of the third antibody FDP-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 light chain L-FDP-3 of the third antibody FDP-3 are as follows: the sequence of CDR1 is shown in SEQ ID NO.26, the sequence of CDR2 is AAS, and the sequence of CDR3 is shown in SEQ ID NO.

28.

2. An antibody against FDP, a plasmin degradation product, characterized in that, The FDP antibody is a 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. An antibody against FDP, a plasmin degradation product, characterized in that, The FDP antibody is a 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. An antibody against FDP, a plasmin degradation product, characterized in that, The FDP antibody is the third antibody FDP-3, and 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 plasmin degradation product FDP antibody according to any one of claims 1-4, characterized in that, The biomaterial is any one of the following: (a) A nucleic acid molecule encoding the FDP antibody as described in any one of claims 1-4; (b) Expression cassette containing the nucleic acid molecules from (a); (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b); (d) Recombinant cells containing the nucleic acid molecules 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 an antibody against the plasmin degradation product FDP as described in any one of claims 1-4.

7. A composite, characterized in that, The conjugate comprises an FDP antibody as described in any one of claims 1-4, covalently linked to a chemical or biological marker.

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

9. The use of an FDP antibody as described in any one of claims 1-4, a conjugate as described in claim 7, or a conjugate as described in claim 8 in the preparation of a product for detecting plasmin degradation product FDP.

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