Plasmin degradation product d-dimer antibodies and uses thereof

Abstract

The application relates to biotechnology, and particularly discloses a plasmin degradation product D-dimer 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 an antibody for detecting a fibrinolysis system in blood coagulation in vitro diagnosis, a mammalian cell expression system (293F) is adopted, three strains of D-dimer monoclonal antibodies (antibody numbers DD-1, DD-2 and DD-3) with biological activity are successfully expressed and obtained. Compared with a commercially available Roche D-dimer antibody, the three strains of antibodies have consistent affinity to DD antigens, can recognize and detect D-dimer in plasma with high specificity and high sensitivity, have no cross reaction with fibrinogen, primary fibrinolysis (X, Y and D) and soluble fibrin (SF), and can be used for qualitative or quantitative detection of D-dimer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, more particularly, it relates to a fibrinolysin degradation product D-dimer 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, while the cross-strand fibrins are arranged in a half-staggered overlap manner, as shown in the combination of Figure 2 a chain and b chain. 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. Fibrinolysin degrades fibrinogen to produce X, Y, D and E fragments (see Figure 1 ). When fibrinolysin 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 (fibrinogen) degradation products (FDP).

[0004] Plasma D-dimer is one of the molecular markers reflecting the activation of the coagulation and fibrinolysis system. When intravascular thrombosis occurs, a large amount of cross-linked fibrin is produced, and the activity of fibrinolysin is secondarily enhanced, resulting in an increase in the content of plasma D-dimer. Therefore, detecting the content of plasma D-dimer is of great significance for the diagnosis of thrombotic diseases and monitoring of thrombolytic therapy, etc.

[0005] At present, the detection methods of plasma D-dimer mainly include latex agglutination method, enzyme-linked immunosorbent assay, colloidal gold immunopercusivity test and immunoturbidimetry. Enzyme-linked fluorescence analysis method such as VIDAS D-dimer detection method has high sensitivity and negative predictive value, and the bedside rapid diagnosis method is not mature at present. Most of the commercially available D-dimer is mouse monoclonal antibody prepared by traditional hybridoma technology. Hybridoma technology has the disadvantages of limited development species (the monoclonal antibody produced by hybridoma technology is 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 antibody of fibrinolytic system, D-dimer is obtained from fibrinogen by different enzyme catalysis, which has great similarity with fibrinogen structure, which causes great difficulty in screening of hybridoma mouse monoclonal antibody.

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

[0007] In order to solve the above technical problems, the present application provides a fibrinolysin degradation product D-dimer antibody and application thereof.

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

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

[0010] The complementarity determining region CDR of the heavy chain H-DD-1 of the first antibody DD-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-DD-1 of the first antibody DD-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-DD-2 of the second antibody DD-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-DD-2 of the second antibody DD-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-DD-3 of the third antibody DD-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-DD-3 of the third antibody DD-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 D-dimer antibody, which is a first antibody DD-1, the amino acid sequence of the heavy chain variable region of the first antibody DD-1 is shown in SEQ ID NO. 04, and the amino acid sequence of the light chain variable region of the first antibody DD-1 is shown in SEQ ID NO. 09.

[0014] In a third aspect, the present application provides a plasmin degradation product D-dimer antibody, which is a second antibody DD-2, the amino acid sequence of the heavy chain variable region of the second antibody DD-2 is shown in SEQ ID NO. 14, and the amino acid sequence of the light chain variable region of the second antibody DD-2 is shown in SEQ ID NO. 19.

[0015] In a fourth aspect, the present application provides a plasmin degradation product D-dimer antibody, which is a third antibody DD-3, the amino acid sequence of the heavy chain variable region of the third antibody DD-3 is shown in SEQ ID NO. 24, and the amino acid sequence of the light chain variable region of the third antibody DD-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 D-dimer antibody, which is any one of the following:

[0017] (a) a nucleic acid molecule encoding the above-mentioned D-dimer 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 D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit for detecting D-dimer, which contains the above-mentioned plasmin degradation product D-dimer antibody. In a sixth aspect, the present application provides a kit

[0022] In a seventh aspect, the present application provides a conjugate comprising the D-dimer 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 D-dimer antibody as described above, and / or the conjugate as described above, to a solid medium or a semi-solid medium.

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

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

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

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

[0028] In summary, the present application has the following beneficial effects:

[0029] 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 detection antibodies for fibrinolytic system, achieving precise and high-throughput screening.

[0030] 2. The present application isolates 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 small amount of required cells.

[0031] 3. The present application obtains three D-dimer antibodies with strong specificity and high affinity through one-time immunization and screening, which can realize high affinity, high accuracy and high precision determination of D-dimer.

[0032] 4. The monoclonal antibodies screened by the application are expressed in mammalian cells (293F) and have biological activity, and are independent of animals. The mammalian expression system can perform post-translational modifications such as phosphorylation and glycosylation, which enables the expressed recombinant protein to have the correct conformation and high-order structure, close to the natural protein. This system also allows the exogenous recombinant protein to be directly secreted into the culture medium, facilitating 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

[0033] Figure 1 is a schematic diagram of fibrinogen and its degradation products;

[0034] Figure 2 is a schematic diagram of the formation and degradation of fibrin;

[0035] Figure 3 is an immunoelectrophoresis diagram provided by Example 1 of the application;

[0036] Figure 4 is a screening primary electrophoresis diagram provided by Example 2 of the application;

[0037] Figure 5 is the affinity of the ELISA platform antibody to the DD antigen provided by Example 7 of the application;

[0038] Figure 6 is the reactivity of the immune turbidity platform DD antibody to the DD antigen provided by Example 8 of the application;

[0039] Figure 7 is the antibody SDS-PAGE result analysis provided by Example 9 of the application. DETAILED DESCRIPTION

[0040] The embodiments of the application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the application and should not be regarded as limiting the scope of the application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer, and the reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0041] The technical solution of the application is:

[0042] In this embodiment, the mammal is used as an immunogen, and three specific D-dimer antibodies are screened by a single immunization. The specific sequences of the three antibodies are shown in Tables 1, 2 and 3:

[0043] The term "complementarity determining region," "CDR," refers to the highly variable regions of immunoglobulin heavy and light chains, which contain one or more, or even all, of the amino acid residues that play a major role in the binding affinity of an antibody for its recognized antigen or epitope.

[0044] Table 1. Heavy and light chain sequences of the first antibody DD-1

[0045]

[0046]

[0047] Table 2. Heavy and light chain sequences of the second antibody DD-2

[0048]

[0049]

[0050]

[0051] Table 3. Heavy and light chain sequences of the third antibody DD-3

[0052]

[0053]

[0054]

[0055]

[0056] The mammal as an immunogen is preferably a rabbit. The reason for choosing a rabbit as an immunogen is that it is superior to a mouse in many aspects, especially in the development of monoclonal antibody technology and antibody drugs, with the following advantages:

[0057] (1) Immune response diversity: The rabbit's spleen is large, and the antibody spectrum is rich, which can produce a 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.

[0058] (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.

[0059] (3) Antibody diversity: The B cell and antibody repertoire of rabbits exhibit more diversity during ontogeny, providing more options for developing specific antibodies against a variety of diseases.

[0060] (4) Antibody properties: Rabbit IgG antibodies are structurally simpler than mouse IgG, with fewer amino acids and additional disulfide bonds, which makes rabbit mAbs more advantageous in stability, which is crucial for long-term stability and effectiveness of antibody drugs.

[0061] (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.

[0062] The present embodiment also provides a nucleic acid molecule, an expression cassette, a recombinant vector, and a recombinant cell:

[0063] In the process of preparing or obtaining the first antibody DD-1, the second antibody DD-2, or the third antibody DD-3, nucleic acid molecules expressing these antibodies can be used, linked with different vectors, and then expressed in different cells to obtain the corresponding antibodies.

[0064] 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 it actually includes any one of the complementary double strands, or both. 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.

[0065] Preferably, the nucleic acid molecule encoding the variable region of the first antibody DD-1, the second antibody DD-2, or the third antibody DD-3 is as shown in Table 4:

[0066] Table 4. Base sequence encoding antibody variable region

[0067]

[0068]

[0069]

[0070] Preferably, the vector in the recombinant vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0071] Preferably, the recombinant cell is obtained by introducing the above-mentioned vector into the 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.

[0072] Based on the amino acid sequences of the antibodies of the present disclosure, it is easy for those skilled in the art to conceive that the antibodies are prepared by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, isolated and purified from the culture products of recombinant cells capable of recombinantly expressing the antibodies according to any one of the above, which is easy for those skilled in the art to achieve. Therefore, no matter what technique is used to prepare the antibodies of the present disclosure, it falls within the protection scope of the present disclosure.

[0073] The present embodiment also provides a conjugate, a conjugate, a kit:

[0074] The D-dimer antibody in the present application can be used in combination with any detection reagent or therapeutic preparation, for example, combined with diagnostic nuclides, nanomaterials, etc. The target site is detected by the radioactivity of the nuclide, and then the information of the target site is obtained. It can also be combined with therapeutic nuclides, and the target cells, tissues, etc. are specifically killed by the radioactivity of the nuclides.

[0075] The conjugate in the present application is a D-dimer antibody covalently linked to 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.

[0076] The conjugate in the present application includes a solid medium or a semi-solid medium combined with the D-dimer antibody, including a substance capable of being suspended or dispersed in a liquid phase (for example, a particle, a magnetic bead, etc. solid phase carrier), or a solid phase capable of accommodating or carrying a liquid phase (for example, 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 part includes at least one selected from the group consisting of magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

[0077] The kit in the present application includes the aforementioned dimer antibody, conjugate or conjugate. Such a kit can effectively qualitatively or quantitatively detect D-dimer. As described above, the antibodies in some specific embodiments or examples of the present application have higher binding activity to D-dimer, and therefore the reagent or kit containing the antibodies has higher detection sensitivity or specificity.

[0078] 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.

[0079] Embodiment

[0080] Example 1 in vitro degradation, purification of immunogen

[0081] 1) In a 7 mL silicon bottle, add 2 mL of reconstituted quality control plasma and 2 mL of 25 mM CaCl2buffer, mix well.

[0082] 2) Add 2 μL of 120 IU / mL thrombin (CAS No.: 9002-04-4, manufacturer: Hubei Belo Biological Technology Co., Ltd., product code: EP1204-1000KU), mix well, and let the blood clot at 37°C without stirring for 1 hour. Put in a magnetic stirrer, add 2 μL of 150 mU / mL fibrinolysin (CAS No.: 9001-91-6, manufacturer: SIGMA, product code: P1867), stir at 800 rpm for 72 hours of degradation.

[0083] 3) The degradation product is separated and purified by polyacrylamide gel electrophoresis (manufacturer: Solarbio, product code: P1200) and protein gel recovery method (manufacturer: Solarbio, product code: G7200) to obtain D-dimer fragments with a purity of >90% (as shown in Figure 3 ).

[0084] Example 2 screening in vitro degradation, purification

[0085] Fibrinogen degradation fragments include X, Y and D, and the preparation method comprises:

[0086] 1. Degradation of fibrinogen

[0087] Fibrinogen is a commercially available product (CAS No.: 9001-32-5, manufacturer: sigma; product code: F3879). In a 1 mL solution of fibrinogen with a concentration of 1 mg / mL, add 20 μL of 150 μg / mL fibrinolysin solution, and place it at 37°C for degradation for 30 min. Add 10 μL of aprotinin solution with a concentration of 1 mg / mL to terminate the fibrinolysis reaction, and obtain mixed FDP.

[0088] 1. Preparation of X fragment, Y fragment and D fragment

[0089] The degraded mixed FDP is subjected to SDS-PAGE, and electrophoresis is performed using 8% separation gel and 5% concentration gel. The 5% concentration gel is subjected to electrophoresis using a voltage of 80V, and the 8% separation gel is subjected to electrophoresis using a voltage of 120V. After electrophoresis for 1 h, staining is performed for 30 min, and the results are observed after decolorization.

[0090] As shown in the SDS-PAGE results, Figure 4 the molecular weights of fragments X, Y and D are 250 kd, 150 kd and 100 kd, respectively.

[0091] 3) SF preparation method

[0092] In 1 mL of fibrinogen solution with a concentration of 1 mg / mL, 2 μL of 120 IU / mL thrombin was added, mixed evenly, and the blood clot was allowed to stand at 37°C without stirring for 1 hour.

[0093] Example 3 animal immunization

[0094] Eight rabbits were immunized, and the pre-immune sera of the eight rabbits were used to detect the immunogen, and four rabbits with low background were selected for immunization.

[0095] During the immunization process, the immunogen (D-dimer fragment prepared in Example 1) was used for serum ELISA detection, and Table 5 shows the ELISA detection results of the 3rd, 4th and 5th immunization sera of the selected rabbit (No. E14143). After the standard 63-day immunization program was completed, the rabbit was killed, and the spleen was collected for monoclonal antibody screening.

[0096] Table 5. Rabbit serum ELISA detection

[0097]

[0098] Example 4 flow cytometry sorting and positive clone amplification

[0099] In this embodiment, during the B cell sorting stage, a fluorescence-labeled multi-parameter flow cytometry sorting method is used to sort individual B cells, achieving precise and high-throughput screening. The fluorescence-labeled multi-parameter flow cytometry sorting method sorts the target antigen-specific antibodies by adding fluorescence-labeled antibodies for labeling B cell surface markers and fluorescence-labeled target antigens (for binding to B cell surface BCR), achieving rapid, accurate and high-throughput separation of B cells and multi-parameter simultaneous analysis.

[0100] The specific sorting process is as follows:

[0101] B lymphocytes in the spleen were separated, and the original B lymphocytes were screened with FITC-labeled D-dimer for fluorescence labeling. The cells were enriched with a flow cytometer, and then the cells were sorted into 96-well plates, with one cell per well, and a total of 2000 single cells were separated. The single B cells were cultured in the 96-well plates, and the cultured B cells secreted a small amount of antibody supernatant. After 10-14 days, the cell supernatant was taken for ELISA verification and cross-antibody screening.

[0102] The antibody genes of the selected cell strains were extracted, and an expression module pcDNA 3.4 was constructed, and 293F cells were transfected for positive clone amplification and expression.

[0103] Example 5 293F cell transfection

[0104] The present embodiment realizes expression of the above-mentioned screened monoclonal antibodies 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, glycosylation and the like, this makes the expressed recombinant protein have 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.

[0105] The specific process is as follows:

[0106] 1. Preparation before transfection

[0107] The 293F cells with a density of 0.3-0.35*10 6 cells / mL are subcultured in 20 mL SMM 293-TII culture medium (manufacturer: SinoBiogical, item number: M293TII). Culturing is carried out 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 with the culture medium SMM 293-TII, the cell liquid volume is 20 mL per bottle, then the bottle mouth is tightly screwed and placed in the shaker for continuous culturing. After 2-4 hours, transfection can be carried out.

[0108] 2) Preparation of transfection solution

[0109] 10 μg of plasmid is diluted with 0.15 M NaCl, mixed and then placed at room temperature for 5 min. 50 μL of Sinofection (manufacturer: SinoBiogical, item number: STF02) is added to the plasmid dilution solution, the total volume of the final transfection solution is 1 mL, and after mixing, it is placed for 10 min.

[0110] 3) Transfection

[0111] The prepared transfection solution is added dropwise to the cell culture solution, mixed, the bottle mouth is tightly screwed and placed in the shaker, and culturing is carried out at 37°C, 120 rpm and 5% CO2.

[0112] 4) Observation and process test after transfection

[0113] After 20-24 h of transfection, the SMS293-SUPI culture medium feeding solution (0.7 mL / 20 mL) (manufacturer: SinoBiogical, item number: M293-SUPI) is added, and the feeding culture is carried out every other day thereafter, and the state and quantity of the cells are observed and recorded. On the third day after transfection, 0.5 mL of sample is taken for ELISA process identification and detection, and the cell culture supernatant is collected after 4-8 days of continued shaker culturing according to the cell viability.

[0114] Example 6 Antibody Purification

[0115] 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.

[0116] 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.

[0117] 3) Sample loading: Load the pretreated cell supernatant at a flow rate of 10 rpm and collect the flow-through.

[0118] 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.

[0119] 5) Sample elution and collection: Add 10% of the expected sample volume of Tris at pH 9.0 to the collection tube, and use Elution Buffer (pH 3.0 glycine) to elute the column at a flow rate of 10 rpm to collect the sample.

[0120] 6) Concentration and dialysis: After overnight dialysis with PBS, concentrate the protein to a concentration >0.5 mg / mL.

[0121] Example 7: ELISA platform for screening antibodies

[0122] Screened pro-D-dimer (DD), fibrinogen (FIB), 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.

[0123] 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: DD-1, DD-2, DD-3) to the coated ELISA plate and incubate at 37°C for 1 hour. After washing the ELISA plate 3-5 times with washing buffer, drain as much water as possible.

[0124] 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.

[0125] 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.

[0126] The ELISA reader was set to a wavelength of 450 nm, and the OD values ​​were read. The results are shown in Table 6. The affinity between the antibody and DD antigen on the ELISA platform is as follows: Figure 5 As shown.

[0127] Table 6. ELISA Platform Detection Results

[0128]

[0129] From Table 6 and Figure 5 As can be seen, on the ELISA platform, under the same antibody and antigen concentrations, the three D-dimer recombinant rabbit monoclonal antibodies of this invention (antibody numbers DD-1, DD-2, and DD-3) have consistent affinity for DD antigens compared to commercially available Roche D-dimer antibodies. They can identify and detect D-dimers in plasma with high specificity and high sensitivity. At the same time, they have no cross-reactivity with fibrinogen, primary fibrinolytics (Table 6, X, Y, and D), and SF. They can be applied to the fields of DD indirect ELISA and Western blotting detection and screening.

[0130] Example 8: Immunoturbidimetric platform coating verification of antibody-antigen reactivity

[0131] Activate the latex with 50 mM MES (pH 7.0) buffer at room temperature, add 62.5 μg / mL EDC and NHS, and mix well at room temperature in the dark for 20 min. After activation, add a certain amount of antibody (DD-1, DD-2, DD-3, and commercially available Roche D-dimer antibody) and couple at room temperature for 2 h. Add 2% BSA and block at room temperature for 2 h. Centrifuge, transfer to 50 mM glycine (pH 7.0) for storage, and sonicate (power: 45% Φ6, 2s 2s, 4 min).

[0132] The intensity of transmitted or scattered light was measured using a turbidimeter to assess the reactivity of DD antibody with DD antigen. The results are as follows: Figure 6as shown.

[0133] By Figure 6 It can be seen that, in the immunoturbidimetry platform, under the same antibody and antigen concentration: 2 of the 3 D-dimer recombinant rabbit monoclonal antibodies (antibody numbers DD-1, DD-2, and DD-3) of the present application can be applied to the immunoturbidimetry platform (antibody numbers DD-1 and DD-3, see the experimental results in detail in Figure 6 ), compared with the commercially available Roche D-dimer antibody, have consistent affinity to DD antigen, can recognize and detect D-dimer in plasma with high specificity and high sensitivity, and at the same time, in the immunoturbidimetry platform, have no cross-reaction with fibrinogen, primary fibrinolysis, and SF.

[0134] Example 9 SDS-PAGE identification

[0135] 1) Gel preparation: load the glass plate into the gel maker, prepare 10 ml of 10% separation gel in a beaker, and sequentially add 2.7 ml of ultrapure water, 3.3 ml of 30% acrylamide (Acr) and bisacrylamide (Bis) mixture (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 persulfate (AP), and 4 μl of tetramethyl ethylenediamine (TEMED). After mixing well, slowly add the prepared 5 ml separation gel solution between the gel maker slots along one corner of the glass plate, add 400 μl or more of distilled water to seal, and place at room temperature for 30 min. After the separation gel solidifies, discard the distilled water, prepare 3 ml of 5% concentrated gel in a beaker, and sequentially add 2.1 ml of ultrapure water, 0.5 ml of 30% Acr / Bis (29:1, v / v), 0.38 ml of 1M Tris-HCl (pH 6.8), 0.03 ml of 10% SDS, 0.03 ml of 10% AP, and 0.003 ml of TEMED. After mixing well, slowly add between the gel maker slots along one corner of the glass plate until the liquid surface reaches about 0.3 cm from the concave plate, and quickly insert the comb. Place at room temperature for 30 min until the concentrated gel solidifies.

[0136] 2) Electrophoresis: the denatured protein sample was sequentially spotted according to a certain order, 4 μl of protein Marker (manufacturer: Solarbio, item number: PR1920) was added into the excess hole, and a certain volume of 1x SDS loading buffer was added to fill up. Finally, the electrophoresis tank was connected to the electrophoresis instrument, and electrophoresis was performed at 80V for about 30 min, and after the sample entered the separation gel, electrophoresis was performed at 120V for 2-3h.

[0137] Reduction electrophoresis, the sample needs to be boiled with loading buffer containing β-mercaptoethanol for 5 minutes, so that the disulfide bond between the proteins can be opened under the action of the reducing agent, and the obtained is the primary structure of the protein.

[0138] The results are shown in Figure 7 After 293F expression, the heavy chains of the three strains of antibodies obtained finally were about 50kDa, and the light chains were about 22kDa.

[0139] In summary, the single B cell antibody preparation technology and flow cytometry sorting method are applied to the detection of the fibrinolytic system in the coagulation in vitro diagnosis for the first time. The D-dimer monoclonal antibodies of three strains (antibody numbers DD-1, DD-2, and DD-3) with biological activity are successfully expressed and obtained by using a mammalian cell expression system (293F). The variable regions of the heavy chains and the light chains 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:

[0140] Table 7. Information of antibodies

[0141]

[0142] The present application finally screens three strains of D-dimer antibodies with high specificity and high affinity by one-time immunization. The core raw material import substitution is realized, and the application is applied to the development of the D-dimer detection kit. The diversification selection of the D-dimer antibody is realized.

[0143] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A plasmin degradation product D-dimer antibody, characterized in that, The D-dimer antibody comprises any one of a first antibody DD-1, a second antibody DD-2 or a third antibody DD-3; The complementary determining regions CDR of the heavy chain H-DD-1 of the first antibody DD-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-DD-1 of the first antibody DD-1 are as follows: CDR1 shown in SEQ ID NO. 06, CDR2 shown in SEQ ID NO. 07, CDR3 shown in SEQ ID NO. 08; The complementary determining regions CDR of the heavy chain H-DD-2 of the second antibody DD-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-DD-2 of the second antibody DD-2 are as follows: CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 17, CDR3 shown in SEQ ID NO. 18; The complementary determining regions CDR of the heavy chain H-DD-3 of the third antibody DD-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-DD-3 of the third antibody DD-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.

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

09.

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

19.

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

29.

5. A biomaterial associated with the antibody to the degradation product of plasmin D-dimer 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 D-dimer antibody according to any one of claims 1-4; (b) an expression cassette comprising the nucleic acid molecule of (a); (c) a recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b); (d) a recombinant cell containing the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).

6. A kit for detecting D-dimer, characterized by, The kit contains the plasmin degradation product D-dimer antibody according to any one of claims 1 to 4.

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

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

9. Use of the D-dimer antibody according to any one of claims 1 to 4, the conjugate according to claim 7, or the conjugate according to claim 8, for the manufacture of a product for the detection of D-dimer.

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