Anti-D-dimer monoclonal antibody and its application

Abstract

The present invention discloses an anti-D-dimer monoclonal antibody and its application, and relates to the technical field of detection of D-dimer. The sequence of CDR1-3 of the heavy chain variable region of the anti-D-dimer monoclonal antibody is as shown in SEQ ID NO.1-3 or has at least 95% homology with the sequence shown in SEQ ID NO.1-3; the amino acid sequence of CDR1 and 3 of the light chain variable region is as shown in SEQ ID NO.4 and 5 or has at least 95% homology with the sequence shown in SEQ ID NO.4 and 5; the CDR2 sequence of the light chain variable region is DTS. The above-mentioned monoclonal antibody has very high titer and specificity for D-dimer, and its sensitivity is comparable to that of products of well-known imported manufacturers. It can also significantly prolong the plateau period of the curve of the change during the antigen-antibody reaction, greatly reducing the probability of false negatives due to excessive DD content in the specimen during clinical use.

Description

Technical Field

[0001] The present invention relates to the technical field of D-dimer detection, in particular to anti-D-dimer monoclonal antibodies and applications thereof. Background Art

[0002] D-dimer is a specific molecular marker produced by plasmin acting on cross-linked fibrin. It contains two identical subunits, each of which is composed of three polypeptide chains: α, β, and γ. The so-called "D-dimer" refers to a mixture of D-dimer structures with different fragment sizes (such as DD, DY, XD, XY, DXD, YXD, DXXD, etc.), with a molecular weight of 190kD-720kD. The half-life of D-dimer in the body is about 6-8h, which is significantly longer than other biomarkers, and is mainly cleared from the circulation through the kidneys and reticuloendothelial system.

[0003] D-dimer is a highly significant biomarker that is not only elevated in thrombotic diseases but also associated with reduced overall survival in patients with various cancers and associated with sepsis, myocardial infarction, preeclampsia, acute upper gastrointestinal bleeding, and acute ischemic stroke. However, D-dimer has certain limitations in actual clinical application. In order to fully develop its potential, accurate and reliable D-dimer test results are essential.

[0004] At present, clinical detection of D-dimer is based on immunological methods, including immunochromatography (colloidal gold) method, two-way flow immunoassay, chemiluminescent enzyme-linked immunosorbent assay and immunoturbidimetric assay (including immunoscattering turbidimetric assay and immunoprojection turbidimetric assay, each of which is specifically divided into two types: latex agglutination enhancement and ordinary). These methods all require high-quality D-dimer-specific antibodies. Therefore, it is extremely important to develop a high-titer, high-specificity monoclonal antibody. Summary of the invention

[0005] The present invention provides an anti-D-dimer monoclonal antibody and its application, which has very high D-dimer detection specificity, sensitivity and accuracy. Compared with the existing TCL system or Sekisui reagent system, the detection result of the detection reagent prepared by the anti-D-dimer monoclonal antibody of the present invention has more obvious advantages. It is specifically achieved by the following technology.

[0006] An anti-D-dimer monoclonal antibody, wherein in the heavy chain variable region of the anti-D-dimer monoclonal antibody, the amino acid sequence of the complementary determining region CDR1 is as shown in SEQ ID NO.1 or has at least 95% homology to the sequence shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 is as shown in SEQ ID NO.2 or has at least 95% homology to the sequence shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 is as shown in SEQ ID NO.3 or has at least 95% homology to the sequence shown in SEQ ID NO.3;

[0007] In the light chain variable region of the monoclonal antibody, the amino acid sequence of the complementary determining region CDR1 is as shown in SEQ ID NO.4 or has at least 95% homology with the sequence shown in SEQ ID NO.4, the amino acid sequence of the complementary determining region CDR2 is DTS, and the amino acid sequence of the complementary determining region CDR3 is as shown in SEQ ID NO.5 or has at least 95% homology with the sequence shown in SEQ ID NO.5.

[0008] Further, in the heavy chain variable region of the anti-D-dimer monoclonal antibody, the amino acid sequence of the framework region FR1 is as shown in SEQ ID NO.6 or has at least 95% identity with the sequence shown in SEQ ID NO.6, the amino acid sequence of the framework region FR2 is as shown in SEQ ID NO.7 or has at least 95% identity with the sequence shown in SEQ ID NO.7, the amino acid sequence of the framework region FR3 is as shown in SEQ ID NO.8 or has at least 95% identity with the sequence shown in SEQ ID NO.8, and the amino acid sequence of the framework region FR4 is as shown in SEQ ID NO.9 or has at least 95% identity with the sequence shown in SEQ ID NO.9;

[0009] In the light chain variable region, the amino acid sequence of the framework region FR1 is as shown in SEQ ID NO.10 or has at least 95% identity with the sequence shown in SEQ ID NO.10, the amino acid sequence of the framework region FR2 is as shown in SEQ ID NO.11 or has at least 95% identity with the sequence shown in SEQ ID NO.11, the amino acid sequence of the framework region FR3 is as shown in SEQ ID NO.12 or has at least 95% identity with the sequence shown in SEQ ID NO.12, and the amino acid sequence of the framework region FR4 is as shown in SEQ ID NO.13 or has at least 95% identity with the sequence shown in SEQ ID NO.13.

[0010] Furthermore, the nucleotide sequence of the heavy chain of the anti-D-dimer monoclonal antibody is as shown in SEQ ID NO.14 or has at least 95% identity with the sequence shown in SEQ ID NO.14, and the nucleotide sequence of the light chain is as shown in SEQ ID NO.15 or has at least 95% identity with the sequence shown in SEQ ID NO.15.

[0011] Furthermore, the gene sequence encoding the heavy chain variable region of the anti-D-dimer monoclonal antibody is shown in SEQ ID NO.16, and the gene sequence encoding the light chain variable region of the anti-D-dimer monoclonal antibody is shown in SEQ ID NO.17.

[0012] Furthermore, the gene sequence encoding the heavy chain of the anti-D-dimer monoclonal antibody is shown in SEQ ID NO.18, and the gene sequence encoding the light chain of the anti-D-dimer monoclonal antibody is shown in SEQ ID NO.19.

[0013] The present invention also provides a biomaterial comprising any one of the following:

[0014] (1) A nucleic acid molecule encoding the anti-D-dimer monoclonal antibody described in any one of the above items;

[0015] (2) an expression vector / vector set containing the nucleic acid molecule;

[0016] (3) A host cell containing the nucleic acid molecule or containing an expression vector / vector set.

[0017] The present invention also provides an anti-D-dimer composition, comprising any one of the anti-D-dimer monoclonal antibodies described above.

[0018] Furthermore, in the anti-D-dimer composition, the anti-D-dimer monoclonal antibody is also provided with a covalently or non-covalently linked conjugate, and the conjugate is at least one of an enzyme, a fluorescent protein, a fluorophore, biotin, and streptavidin.

[0019] The present invention also provides an application of any of the above-mentioned anti-D-dimer monoclonal antibodies or the above-mentioned anti-D-dimer composition, characterized in that it is used to prepare a product for detecting D-dimer, or to detect D-dimer for purposes other than disease diagnosis and treatment.

[0020] Furthermore, the product is a detection kit, a detection test strip or a detection chip.

[0021] Furthermore, the detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatography kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit or a radioimmunoassay kit; the detection test strip is a colloidal gold test strip or an ELISA test strip.

[0022] Compared with the prior art, the advantages of the present invention are as follows: the present invention provides an anti-D-dimer monoclonal antibody with very high D-dimer detection specificity, sensitivity and accuracy, and has a better ability to resist background interference and has more advantages than the detection results of the existing gold standard TCL system or Sekisui reagent system; it can be widely used in ELISA and other detections of D-dimer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The heavy chain and light chain SDS-PAGE electrophoresis results of the monoclonal antibody D-36 (2C11) in Example 2 are shown.

[0024] Figure 2 These are the test results of the turbidimetric reagent prepared using the monoclonal antibody D-36 (2C11) and the Siemens control reagent in Example 3.

[0025] Figure 3 These are the test results of the turbidimetric reagent prepared using the monoclonal antibody D-36 (2C11) and the water-logging control reagent in Example 3.

[0026] Figure 4 The change trend of the detection signal of the high concentration sample in the detection results of the turbidimetric reagent prepared by using the monoclonal antibody D-36 (2C11) and the water control reagent in Example 3. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0029] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0030] Generally speaking, the terms used in this specification have the following meanings:

[0031] In this specification, the term "monoclonal antibody" means an antibody derived from a colony of substantially homologous antibodies, that is, each antibody constituting the colony is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody colony, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using all or part of transgenic animals comprising human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0032] In the specification of the present application, the terms "heavy chain" ("CH"), "light chain" ("CL"), "heavy chain constant region", "light chain constant region", "light chain variable region" ("VL"), "heavy chain variable region" ("VH"), "framework region" ("Framework Region, FR"), and "complementarity determining region" ("CDR") are components of antibodies, and different types of antibodies can be formed through different combinations. For example, a conventional IgG antibody is a tetramer composed of two light chains and two heavy chains. Among them, VL and VH are both composed of 3 complementary determining regions (CDR) and 4 framework regions (FR). Among them, CDR contains residues that contact the antigen and determine the antigen specificity of VL and VH, and FR is used to maintain the variable region structure and determine the position of the CDR loop. Usually, the heavy chain variable region and the heavy chain constant region constitute a complete heavy chain, while the light chain variable region and the light chain constant region constitute a complete light chain.

[0033] In the specification of the present application, a conjugate refers to a molecule that, after binding to an antibody, can indicate the location or amount of the antibody by color, chemical reaction, excitation light, mass spectrometry, etc.; including but not limited to alkaline phosphatase, peroxidase, luciferase, fluorescein, fluorescent protein, biotin, streptomycin or isotope, etc.

[0034] In the specification of the present application, the terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably, including but not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (micro RNA), genomic DNA, synthetic DNA, synthetic RNA and / or tRNA.

[0035] In the specification of the present application, the term "vector" (expression vector or vector group) refers to a vector through which a polynucleotide sequence (such as a foreign gene) can be introduced into a host cell to transform the host and promote the expression (such as transcription and translation) of the introduced sequence. Vectors include plasmids, phage vectors, viral vectors, etc. Among them, a "viral vector" is a vector transformed from a viral genome, which introduces foreign genes into a host cell through viral infection. "Plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeasts and actinomycetes, which exists in the cytoplasm or nucleus and has the ability to replicate autonomously, so that it can also maintain a constant copy number in daughter cells and express the genetic information it carries.

[0036] In the specification of the present application, "enzyme-linked immunosorbent assay (ELISA)" refers to a detection method that utilizes the characteristic that antibody molecules can specifically bind to antigen molecules, combines free impurities with the target protein bound to a solid phase carrier, and uses special markers to conduct qualitative or quantitative analysis thereof. The principle of ELISA is: antigens or antibodies can be physically adsorbed on the solid surface and maintain their immune activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immune activity or enzyme activity; after the enzyme conjugates bind to the corresponding antigens or antibodies, the occurrence of the immune reaction can be determined by the color reaction of the added substrate, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen. Various types of detection methods can be designed according to the substance to be detected and the conditions for detection. The double antibody sandwich method is the most commonly used method for detecting antigens.

[0037] Example 1: Natural extraction of D-dimer protein

[0038] 1. Fibrinogen extraction

[0039] (1) Human plasma processing

[0040] Take 100 mL of dissolved human plasma, centrifuge at 10,000 rpm and 4°C for 10 min, and filter through a 0.45 μm filter membrane.

[0041] (2) Ammonium sulfate precipitation

[0042] The treated plasma was added with solid ammonium sulfate at a mass volume ratio of 20%, shaken at 4°C for 30 min, centrifuged at 10,000 rpm for 10 min, and the obtained solid was dialyzed into 0.01 M Tris-HCl pH 7.4 and run on a gel.

[0043] (3) Anion exchange chromatography

[0044] Take the ammonium sulfate precipitate fraction and load it onto the Q column. The loading buffer is 0.01M Tris-HCl pH=7.4, and the elution buffer is 0.01M Tris-HCl, 1M Nacl, pH=7.4. 100mM, 200mM, and 300mM elution fractions are collected respectively.

[0045] 2. Fibrinogen collagenase and enzymatic hydrolysis

[0046] (1) Fibrinogenase

[0047] Combine the 100mM sodium chloride and 200mM sodium chloride components of the above ion exchange chromatography, concentrate and then perform enzyme connection. The protein concentration is 1.67mg / mL, the volume is 37mL, add 92.5mg calcium chloride, 250μL FXIII (final concentration 6μg / mL), 250μL thrombin (final concentration 1.5U / L), mix well, and bathe at 37℃ for 16h to obtain a white gel-like fibrin mass, which is washed 3 times with TBS, 30min each time, and dialyzed into TBS.

[0048] (2) Enzymatic hydrolysis of fibrinogen

[0049] Place the fibrin in 5 mL of the above-mentioned TBS buffer solution, add 25 μL of plasmin (final concentration 5 U / L) to the solution, and react with stirring at 37°C. After 48 hours, add another 25 μL of plasmin and continue enzymolysis until there is no solid matter. During the enzymolysis, samples are taken at regular intervals and aprotinin is added to terminate the reaction.

[0050] 3. Enzymatic hydrolysis reactants pass through molecular sieve

[0051] The product of the enzymatic hydrolysis in the previous step was filtered through a dextran gel filtration medium G-200 (16 / 60), the mobile phase was TBS at pH 7.4, the flow rate was 0.5 mL / min, and the fraction was collected, 2 mL per tube, and the peak 2 obtained by separation was the target protein. The target protein was dialyzed into 20 mM phosphate buffer (Na2HPO4 / NaH2PO4), 0.15 M NaCl, pH = 7.4 buffer; the dialyzed sample was determined by BCA Protein Quantification Kit to have a protein concentration of 3 mg / mL, a total of 28 mg, and 1% NaN3 was added and stored at -20°C.

[0052] Example 2: Preparation of D-dimer monoclonal antibody

[0053] 1. Animal immunization

[0054] Five female Balb / c mice aged 6-8 weeks were selected for immunization. The antigen was purified D-dimer protein mixed with Freund's complete adjuvant, Freund's incomplete adjuvant, and PBS. After complete emulsification, 200 μL was injected into each mouse at multiple points on the back, ventral groove, and axilla. Immunization steps: the first immunization, 50 μg of antigen / mouse, Freund's complete adjuvant was added for subcutaneous injection and multiple points on the back; the second immunization was carried out two weeks later, the dosage route was the same as the first, and Freund's incomplete adjuvant was added; the third immunization was carried out 15 days later, the dosage was the same as above, and Freund's incomplete adjuvant was added, and the intraperitoneal injection was performed; the last immunization was one month apart from the previous immunization, and D-dimer protein 50 μg / mouse was used for muscle booster immunization (without adjuvant), and tail blood was collected 3 days later, and the serum antibody titer was detected by ELISA.

[0055] 2. Construction of B cells

[0056] (1) Cultivation and preparation of myeloma cell lines

[0057] The present invention adopts SP / 20 myeloma cell strain, which has good growth and fusion efficiency and a doubling time of 10-12h. When fusion, cells with good logarithmic growth phase, cell morphology and activity are selected. Myeloma cells should be adaptively cultured before fusion to make the cells grow to the optimal state (i.e., logarithmic growth phase).

[0058] On the day of fusion, use an elbow pipette to gently blow the myeloma cells off the bottle wall, collect them in a 50mL centrifuge tube or fusion tube, centrifuge at 1000r / min for 5-10min, discard the supernatant, add 30mL of culture medium to the precipitate, wash once by centrifugation, centrifuge for 5-10min, discard the supernatant, and mix the precipitate with 20mL of culture medium for later use.

[0059] (2) Preparation of splenocytes

[0060] Take BALB / c mice that have completed animal immunization, remove the eyeballs to collect blood, and use a centrifuge to separate the serum as the positive control serum for antibody detection.

[0061] Mice were killed by cervical dislocation and soaked in 75% alcohol for 5 min. The spleen was removed aseptically in a clean bench and placed in a plate containing 10 mL of culture medium. The spleen was gently washed and the surrounding connective tissue was removed. The spleen was pierced with a sterile syringe until it turned white. The spleen cells in the plate were collected in a 50 mL centrifuge tube and centrifuged at 1000 r / min for 10 min. The supernatant was discarded, 10 mL of culture medium was added, and a small amount of 10 was taken for dilution counting.

[0062] (3) Cell fusion

[0063] Three days before fusion, positive mice were boosted with immunization, mouse spleen cells were mixed with myeloma cells sp2 / 0 at a ratio of 10:1, PEG fused, and then cultured in HAT selection medium. Ten days later, hybridoma cell supernatants were screened by ELISA, and the screened positive hybridoma cells were cloned by limiting dilution. After five screenings, 8 cells were finally determined to be positive cells.

[0064] (4) Screening of hybridoma cells

[0065] Indirect ELISA method: D-dimer protein was coated on a 96-well plate at 10 μg / mL, 50 μL / well, and adsorbed at 37°C for 1 hour. The liquid in the well was discarded, and the well was washed with washing solution for 3 times, patted dry, and 100 μL of blocking solution was added to each well for blocking at 37°C for 1 hour, washed twice, and patted dry. 100 μL of the culture supernatant of the 8 hybridoma cells to be tested was added to each well, and positive, negative and blank control standards were set up at the same time. The plates were incubated at 37°C for 0.5 hours, washed 4 times, and patted dry. Horseradish enzyme-labeled goat anti-mouse IgG 1:10000 was taken, 100 μL per well was added to the ELISA plate, incubated at 37°C for 30 minutes, washed 4 times, and patted dry.

[0066] Finally, add 100μL / well of TMB substrate colorimetric solution, and terminate the reaction with 50μL / well of 2mol / L dilute hydrochloric acid after 15min of color development. As shown in Table 1 below, the OD value of each well was measured at dual wavelengths of 450nm and 630nm. The blank well value was below 0.02, and the negative well value was below 0.1. The distinction between positive and negative was obvious, and it can be judged that the anti-D-dimer hybridoma cells secreted antibodies that can specifically recognize D-dimer protein. The same method was used to measure the titer of mouse eyeball blood, which reached 1:100000, and can be used for cell fusion.

[0067] Table 1

[0068]

[0069]

[0070] 3. Ascites Preparation

[0071] After cell fusion and ELISA assay, 24 hybridoma cell wells were selected and screened repeatedly using subcloning and culture medium and feeder cells until a single hybridoma cell line with high stability and efficacy was screened. A total of 8 hybridoma cell lines that met the above conditions were screened in the experiment, numbered 2C11, 7G9, 4H10, 6G12, 15A1, 6E5, 3D12, and 7F4.

[0072] Before the screened positive hybridomas are injected into the mouse peritoneum, the mice are first sensitized with paraffin to activate the immune system in the mice. One week later, the single stable and high-efficiency positive hybridoma cells obtained by the fusion of the above cells are injected into the mouse peritoneum. The mice are observed for 7-10 days. The abdominal cavity of the mice with abnormally bulging bellies is filled with ascites. After the ascites is removed, it is purified by affinity purification to obtain the antibodies corresponding to the positive hybridoma cell lines.

[0073] 4. Production and purification of monoclonal antibodies

[0074] (1) Preparation of buffer solution: Use high-purity water to prepare the following buffer solution. Filter the buffer solution through a 0.45 μm filter membrane before use.

[0075] Equilibration buffer: 20 mM Na2HPO4, 0.15 M NaCl, pH 7.0.

[0076] Elution buffer: 0.1 M glycine, pH 3.0.

[0077] Neutralization buffer: 1M Tris-HCl, pH 8.5.

[0078] (2) Sample preparation: To ensure that the ionic strength and pH value are favorable for antibody binding, it is necessary to dilute the serum sample. The ascites fluid is diluted 1:1 with the equilibrium buffer and set aside.

[0079] (3) Column loading operation

[0080] Shake well to resuspend the resin, pipette 1 ml of the slurry into a new chromatography column, and pre-add 1 ml of equilibration buffer into the column. Allow the resin to settle naturally and drain the equilibration buffer. Add 5 ml of equilibration buffer to the column to equilibrate the resin, and drain the equilibration buffer at a flow rate of about 1 ml / min.

[0081] (4) Chromatographic purification

[0082] Load the sample into the column at a flow rate of about 1 ml / min, collect the effluent, and use it for subsequent determination of the binding capacity of the resin, such as SDS-PAGE. Wash the resin with 30 ml of equilibration buffer at a flow rate of about 2 ml / min, or until the A280 absorbance of the effluent reaches a stable value.

[0083] Elute the antibody with 10-15 ml of elution buffer, maintaining a flow rate of about 1 ml / min, collect the eluate containing the target immunoglobulin, and immediately add 1 / 10 volume of elution buffer with neutralization buffer to adjust the pH to 7.4.

[0084] The eluted sample was dialyzed into 20 mM phosphate buffer (Na2HPO4 / NaH2PO4), 0.15 M NaCl, pH=7.4 buffer; the antibody concentration of the dialyzed sample was determined by UV spectrophotometer, and 1% NaN3 was added and stored at 4°C.

[0085] 5. Characterization of monoclonal antibodies

[0086] Determination of antibody concentration: The absorbance values ​​of monoclonal antibodies at 280nm and 260nm, A280 and A260, were determined by UV spectrophotometry. The protein content was antibody A (10mg / mL) and antibody B (8mg / mL). The protein content was calculated according to the following formula:

[0087] Protein content (mg / mL) = (A280 × dilution factor) / 1.35.

[0088] Antibody molecular mass identification: SDS-PAGE was used to determine the monoclonal antibody, such as Figure 1 As shown, the heavy chain of the monoclonal antibody is about 46 KD, and the light chain is about 25 KD.

[0089] ELISA titer determination: The purified ascites monoclonal antibodies were determined by indirect ELISA, as shown in Table 2 below. The results showed that the titers after purification were all 1:100,000.

[0090] Table 2

[0091]

[0092] After sequencing, the amino acid sequence of the complementary determining region CDR1 of the heavy chain variable region of the monoclonal antibody D-36 (2C11) is GYTFSSHW, as shown in SEQ ID NO.1; the amino acid sequence of the complementary determining region CDR2 of the heavy chain variable region is ILPGSGNT, as shown in SEQ ID NO.2; the amino acid sequence of the complementary determining region CDR3 of the heavy chain variable region is ARGGNWGYYFDY, as shown in SEQID NO.3.

[0093] The amino acid sequence of the complementary determining region CDR1 of the light chain variable region of monoclonal antibody D-36 (2C11) is SSVNY, as shown in SEQ ID NO.4; the amino acid sequence of the complementary determining region CDR2 of the light chain variable region is DTS; the amino acid sequence of the complementary determining region CDR3 of the light chain variable region is QQWSSYPPYT, as shown in SEQ ID NO.5.

[0094] The amino acid sequence of the framework region FR1 of the heavy chain variable region of monoclonal antibody D-36 (2C11) is LVQLQQSGAELMKPGASVKISCKAT, as shown in SEQ ID NO.6; the amino acid sequence of the framework region FR2 of the heavy chain variable region is IEWVKQRPGHGLEWIGE, as shown in SEQ ID NO.7; the amino acid sequence of the framework region FR3 of the heavy chain variable region is NYNEKFEGKATFTAETSSNTAYMQLSSLTSEDSAVYYC, as shown in SEQ ID NO.8; the amino acid sequence of the framework region FR4 of the heavy chain variable region is WGQGTTLTVSSA, as shown in SEQ ID NO.9.

[0095] The amino acid sequence of the framework region FR1 of the light chain variable region of monoclonal antibody D-36 (2C11) is DIVLTQSPAIMSASPGEKVTMTCSAS, as shown in SEQ ID NO.10; the amino acid sequence of the framework region FR2 of the light chain variable region is MFWYQQKPGSSPRLLIY, as shown in SEQ ID NO.11; the amino acid sequence of the framework region FR3 of the light chain variable region is NLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYC, as shown in SEQ ID NO.12; the amino acid sequence of the framework region FR4 of the light chain variable region is FGGGTKLEIKR, as shown in SEQ ID NO.13.

[0096] The nucleotide sequence of the heavy chain of monoclonal antibody D-36 (2C11) is shown in SEQ ID NO. 14, and the nucleotide sequence of the light chain is shown in SEQ ID NO. 15. The constant regions of the heavy and light chains of monoclonal antibody D-36 (2C11) are both κ chains, and the heavy chain constant region is of IgG1 type.

[0097] The gene sequence encoding the heavy chain variable region of monoclonal antibody D-36 (2C11) is shown in SEQ ID NO.16, and the gene sequence encoding the light chain variable region of monoclonal antibody D-36 (2C11) is shown in SEQ ID NO.17.

[0098] Example 3: Evaluation of specificity and stability of monoclonal antibody D-36 (2C11)

[0099] 1. Specificity evaluation

[0100] The monoclonal antibody D-36 (2C11) obtained in Example 3 was used to detect D-dimer, IL-6 protein, MYO protein, CEA carcinoembryonic antigen, and HE4 protein, respectively. The detection results are shown in Table 3 below.

[0101] Table 3 Specificity evaluation results (indirect method)

[0102]

[0103]

[0104] It can be seen from the above results that the monoclonal antibody D-36 (2C11) provided by the present invention has a very high specificity for D-dimer detection.

[0105] 2. Stability evaluation

[0106] This example studies the stability of the monoclonal antibody D-36 (2C11) under different storage conditions.

[0107] The conjugation steps of monoclonal antibodies are:

[0108] (1) Microsphere activation: 200 μL JSR 219 nm microspheres were added to 1.8 mL pH 6.0 MES 50 mM buffer, 40 μL EDC 10 mg / ml was added and mixed quickly, and activated on a shaker at 37 °C, 200 rpm for 15 min;

[0109] (2) Conjugation with monoclonal antibodies: 400 μg of each monoclonal antibody was dissolved in 1.5 mL of pH 7.5 HEPES 50 mM buffer. The activated microspheres were quickly added to the antibody solution at once and incubated on a shaker at 37°C, 200 rpm for 3 h.

[0110] (3) Blocking: Prepare 6% BSA with 100 mM Gly at pH 8.5, add 600 μL of 6% BSA to the labeled microspheres, shake at 37°C, 200 rpm, and block for 1 h;

[0111] (4) After blocking, the latex was centrifuged at 4°C and 13,000 rpm for 15 min, the supernatant was removed, and 2.5 mL of 50 mM Gly + 12% sucrose solution at pH 8.0 was added to the precipitate for resuspending, and ultrasonicated for 4 min for resuspending for later use.

[0112] Using the newly prepared monoclonal antibody D-36 (2C11) as a control, the above products were frozen at -20°C for 1, 2, 4 and 6 months to test their freezing stability; and thermal stability was tested at 37°C and 45°C for 3-7 days.

[0113] Table 4 - Stability evaluation results of monoclonal antibody D-36 (2C11) stored at 20°C for 1 month

[0114]

[0115] Table 5 - Stability evaluation results of monoclonal antibody D-36 (2C11) stored at 20°C for 2 months

[0116]

[0117] Table 6 - Stability evaluation results of monoclonal antibody D-36 (2C11) stored at 20°C for 4 months

[0118]

[0119] Table 7 - Stability evaluation results of monoclonal antibody D-36 (2C11) stored at 20°C for 6 months

[0120]

[0121] From the evaluation results in Tables 4-7 above, it can be seen that the antibody signal of monoclonal antibody D-36 (2C11) is close to that of the control group when stored at -20°C for 1-6 months, indicating that it has good low-temperature storage stability.

[0122] Table 8 Thermal stability evaluation results of monoclonal antibody D-36 (2C11)

[0123]

[0124]

[0125] From the evaluation results in Table 8 above, it can be seen that the antibody signal of monoclonal antibody D-36 (2C11) was close to that of the control group when stored at 37°C and 45°C for 3-7 days, and both remained within the qualified range, indicating that it has good thermal stability.

[0126] Embodiment 3: Preparation of Latex Enhanced Immuno-Turbidimetric Rapid Detection Reagent for D-Dimer Detection by Using Monoclonal Antibody D-36 (2C11) The components of the D-Dimer determination kit (latex immuno-turbidimetry) include reagent 1 (R1) and reagent 2 (R2), wherein the function of R1 is mainly to dilute the D-Dimer in the sample and balance the reaction environment when the D-Dimer reacts with the antibody; R2 is a latex particle suspension sensitized by mouse anti-human D-Dimer monoclonal antibody (D-36), which can specifically bind to the D-Dimer in R1. The testing principle of the kit is that the D-Dimer in the sample and the latex particles sensitized by mouse anti-human D-Dimer monoclonal antibody (D-36) undergo a specific antigen-antibody reaction, resulting in agglutination and an increase in turbidity. The change in turbidity is measured by a spectrophotometer, and then the content of D-Dimer is obtained.

[0127] 1. Comparison of detection effects between monoclonal antibody D-36 (2C11) and Siemens system

[0128] Preparation process of latex enhanced immunoturbidimetric rapid detection reagent: 300 μL 219 nm microspheres (purchased from JSR, model P0219) were added to 3 mL, pH 6.0, 50 mM MES buffer, EDC was dissolved to 20 mg / mL with MES, 0.03 mL was added, and activated at room temperature for 1 h. 1200 μg of monoclonal antibody D-36 (2C11) was dissolved in 3 mL MES, and the activated microspheres were quickly added to the antibody solution at once, and stirred at room temperature for 1.5 h. The microspheres were centrifuged at 10,000 rpm for 6 min, the supernatant was discarded, 20 mL of buffer (1% BSA, 0.9% NaCl, 2% trehalose, 0.1% P300, 50 mM phosphate buffer, pH 7.4) was added, and ultrasonication was performed on ice for 7 min. The suspension was stirred at room temperature for 2 h and allowed to stand at 37°C overnight to obtain 20 mL of R2, i.e., a latex particle suspension sensitized by mouse anti-human D-dimer monoclonal antibody (D-36). The storage concentration of the microspheres was 1.5 mg / mL.

[0129] R1: 50 mM phosphate buffer, 0.9% NaCl, 3.0% Triton X-100, 0.1% Proclin 300, pH 7.4.

[0130] The reagent of the Siemens system uses the D-dimer assay kit (immunoturbidimetry) produced by Siemens Healthcare Diagnostics, and the detection instrument is the CA7000 fully automatic coagulation analyzer produced by Sysmex Medical Electronics (Shanghai) Co., Ltd. The test results of this test example are compared with the test results of the Siemens system. The reaction parameters are 6μL sample + 150μL R1 + 50μL R2, wavelength 570nm, and reaction time is 10min. The test instrument is a Hitachi 7180 fully automatic analyzer. The test results are shown in Table 9 and Figure 2 shown.

[0131] Table 9

[0132]

[0133]

[0134] After comparing with the test results of the Siemens system, the P value of the comparison between the two groups of data was 0.84>0.05 through t-test, indicating that there was no significant difference between the two groups of data.

[0135] 2. Comparison of detection effects between monoclonal antibody D-36 (2C11) and Sekisui reagent system

[0136] Preparation process of latex enhanced immunoturbidimetric rapid detection reagent: 800 μL, 113 nm microspheres (purchased from JSR, model P0116) were added to 8 mL pH 6.0 50 mM MES buffer, EDC was dissolved to 20 mg / mL with MES, 0.08 mL was added, and the mixture was stirred at room temperature for 1 h for activation. 2400 μg of antibody was dissolved in 4 mL MES, and the activated microspheres were quickly added to the antibody solution at one time and stirred at room temperature for 1.5 h. The microspheres were centrifuged at 10000 rpm for 6 min, the supernatant was discarded, 20 mL of buffer (1% BSA, 0.9% NaCl, 2% trehalose, 0.1% P300, 50 mM phosphate buffer, pH 7.4) was added, ice bath ultrasonication was performed for 7 min, stirring was performed at room temperature for 2 h, and 20 mL of R2, i.e., mouse anti-human D-dimer monoclonal antibody 36 sensitized latex particle suspension, was obtained after standing at 37°C overnight, and the microsphere storage concentration was 2 mg / mL.

[0137] R1: 50 mM phosphate buffer, 0.9% NaCl, 3.0% Triton X-100, 0.1% Proclin 300, pH 7.4.

[0138] The reagent of the Sekisui reagent system adopts the D-dimer detection kit (latex immunoturbidimetry) produced by Sekisui Medical Co., Ltd., and the detection instrument is the 7180 type fully automatic biochemical analyzer produced by Hitachi. The test results of this test example are compared with the test results of the Sekisui reagent system. The reaction parameters are 12μL sample + 100μL R1 + 100μL R2, the main wavelength is 570nm, the secondary wavelength is 800nm, and the reaction time is 10min. The detection instrument is the Hitachi 7180 type fully automatic analyzer. The test results are shown in Tables 10 and 10 below. Figure 3 shown.

[0139] Table 10

[0140]

[0141]

[0142] After comparing with the test results of the Sekisui reagent system, the P value of the comparison between the two groups of data was 0.57>0.05 through t-test, indicating that there was no significant difference between the two groups of data.

[0143] 3. Comparison of the anti-background interference ability of the turbidimetric reagent prepared by monoclonal antibody D-36 (2C11) and Siemens control reagent

[0144] The same ultrapure water without D-dimer was tested 20 times, and the average value of the 20 test results plus 2 times the standard deviation was the minimum detection limit. By comparing the minimum detection limit, the anti-background interference ability of the two reagents can be judged. The specific values ​​are shown in Table 11 below.

[0145] Table 11

[0146]

[0147] It can be seen that the minimum detection limit of the Siemens control reagent detection system is 0.03 mg / L, and the minimum detection limit of the monoclonal antibody D-36 (2C11) turbidimetric reagent provided by the present invention is 0.02 mg / L, which is significantly better than the minimum detection limit of the Siemens reagent detection system. This shows that the monoclonal antibody D-36 (2C11) provided by the present invention has a better ability to resist background interference.

[0148] 4. Comparison of Hook effect concentration points between turbidimetric reagent prepared with monoclonal antibody D-36 (2C11) and water control reagent

[0149] The D-dimer antigen was diluted to high concentration samples with concentrations of 50, 100, 150, 200, 250, 300, 350 and 400 mg / L, respectively, and the change trends of the detection signals of the monoclonal antibody D-36 (2C11) turbidimetric reagent provided by the present invention and the water control reagent for the above series of high concentration samples were compared. The results are as follows: Figure 4 As shown, the size of the Hook effect concentration point is judged, and the signal value is shown in Table 12.

[0150] Table 12

[0151]

[0152] From the trend of signal change, it can be seen that the Hook effect concentration point of the monoclonal antibody D-36 (2C11) turbidimetric reagent provided by the present invention is 250 mg / L, which is higher than 200 mg / L of the water control reagent. This shows that the detection upper limit of the monoclonal antibody D-36 (2C11) turbidimetric reagent provided by the present invention is wider and the detection adaptability is wider.

[0153] The test results of the above test examples show that in the actual application of monoclonal antibody D-36 (2C11), good detection effects can be obtained under different detection systems, and it has excellent application prospects.

[0154] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. An anti-D-dimer monoclonal antibody, characterized in that In the heavy chain variable region of the anti-D-dimer monoclonal antibody, the amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO.3; In the light chain variable region of the monoclonal antibody, the amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the complementary determining region CDR2 is DTS, and the amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO.

5.

2. The anti-D-dimer monoclonal antibody according to claim 1, characterized in that In the heavy chain variable region of the anti-D-dimer monoclonal antibody, the amino acid sequence of the framework region FR1 is shown in SEQ ID NO.6, the amino acid sequence of the framework region FR2 is shown in SEQ ID NO.7, the amino acid sequence of the framework region FR3 is shown in SEQ ID NO.8, and the amino acid sequence of the framework region FR4 is shown in SEQ ID NO.9; In the light chain variable region, the amino acid sequence of the framework region FR1 is shown in SEQ ID NO.10, the amino acid sequence of the framework region FR2 is shown in SEQ ID NO.11, the amino acid sequence of the framework region FR3 is shown in SEQ ID NO.12, and the amino acid sequence of the framework region FR4 is shown in SEQ ID NO.

13.

3. The anti-D-dimer monoclonal antibody according to claim 2, characterized in that The nucleotide sequence of the heavy chain of the anti-D-dimer monoclonal antibody is shown in SEQ ID NO.14, and the nucleotide sequence of the light chain is shown in SEQ ID NO.

15.

4. The anti-D-dimer monoclonal antibody according to claim 3, characterized in that The gene sequence encoding the heavy chain variable region of the anti-D-dimer monoclonal antibody is shown in SEQ ID NO.16, and the gene sequence encoding the light chain variable region of the anti-D-dimer monoclonal antibody is shown in SEQ ID NO.

17.

5. A biomaterial, characterized in that: The biological material includes any one of the following: (1) A nucleic acid molecule encoding the anti-D-dimer monoclonal antibody according to any one of claims 1 to 4; (2) an expression vector / vector set containing the nucleic acid molecule; (3) A host cell containing the nucleic acid molecule or an expression vector / vector set.

6. An anti-D-dimer composition, characterized in that: The anti-D-dimer monoclonal antibody according to any one of claims 1 to 4 is further provided with a covalently or non-covalently linked conjugate, wherein the conjugate is at least one of an enzyme, a fluorescent protein, a fluorophore, biotin, and streptavidin.

7. Use of the anti-D-dimer monoclonal antibody according to any one of claims 1 to 4, or the anti-D-dimer composition according to claim 6, characterized in that: Used to prepare products for detecting D-dimer, or used to detect D-dimer for purposes other than disease diagnosis and treatment.

8. The use according to claim 7, characterized in that: The product is a detection kit, a detection test strip or a detection chip.

9. The use according to claim 8, characterized in that: The detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatography kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit or a radioimmunoassay kit; The test strip is a colloidal gold test strip or an ELISA test strip.

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