Anti-t-paic monoclonal antibody, coding sequence and application thereof
By designing anti-t-PAIC monoclonal antibodies with specific CDR and backbone regions, the problems of insufficient affinity and specificity for t-PAIC detection in existing technologies have been solved, achieving highly sensitive t-PAIC detection and meeting clinical testing needs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN YAREWELL HONGYUAN BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-06-25
- Publication Date
- 2026-07-31
AI Technical Summary
Current technologies for detecting t-PAIC antibodies suffer from insufficient affinity and specificity, making it difficult to meet the requirements for high sensitivity and specificity, especially in the diagnosis and monitoring of hematological diseases such as atherosclerosis.
A monoclonal antibody against t-PAIC was developed. By designing specific CDR region amino acid sequences and backbone regions, the antibody can be ensured to bind to t-PAIC with high affinity and high specificity, and can be applied to chemiluminescence reagent detection and immunological detection.
It achieves high-sensitivity detection of t-PAIC, with a detection limit of 1 ng/mL and a correlation coefficient R2 ≥ 0.950, meeting clinical testing requirements and suitable for quantitative and immunological detection of t-PAIC.
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Figure CN122483206A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedicine or biopharmaceutical technology, and in particular to an anti-t-PAIC monoclonal antibody, its encoding sequence, and its applications. Background Technology
[0002] Under normal circumstances, hemostatic thrombi formed after tissue injury will gradually dissolve after fulfilling their hemostatic function, thus ensuring the patency of blood vessels. The dissolution of hemostatic thrombi mainly depends on the fibrinolytic system, including plasminogen activator (PA), plasminase, plasminogen activator (PA), and fibrinolysis inhibitor. Specifically, plasminogen activates plasminase under the action of PA, and plasminase dissolves fibrin or fibrinogen to form fibrin degradation products. During this process, if the fibrinolytic system is inactive, fibrin / fibrinogen degradation decreases, exacerbating thromboembolism.
[0003] The tissue-type plasminogen activator-plasminogen activator inhibitor complex (t-PAIC) is a complex formed by the binding of tissue-type plasminogen activator (t-PA) released from the vascular endothelium into the bloodstream and the physiological inhibitory factor plasminogen activator inhibitor-1 (PAI-1) in a 1:1 ratio. The main function of t-PA is to convert plasminogen in the blood into plasmin, while PAI-1 inhibits fibrin degradation, promotes fibrin deposition in the vascular wall, and stimulates smooth muscle cell proliferation.
[0004] The concentration of PAI-1 in the blood is five times that of t-PA, therefore it can be assumed that almost all t-PA released into the blood forms a complex with PAI-1. Thus, a high plasma t-PAIC value indicates an increased t-PA concentration. During vascular endothelial injury, both PAI-1 and t-PA are released into the blood, leading to an increased t-PAIC concentration. Elevated t-PAIC concentration is both a marker of vascular endothelial injury and a marker of fibrinolytic system activation.
[0005] t-PAIC can reveal the functional status of the fibrinolytic system and determine the balance between thrombus formation and dissipation. This indicator is a molecular marker of fibrinolytic system activation and is indicative of disseminated intravascular coagulation (DIC) and arterial and venous thrombosis. Simultaneously, it is also a risk indicator for venous thromboembolism (VTE) and myocardial infarction, and has high clinical value in the diagnosis of DIC.
[0006] Currently, most methods for detecting t-PAIC use immunoassays such as ELISA and CLIA. CLIA technology has advantages over ELISA, including simpler operation, higher sensitivity, more stable reagents, and lower cost. Therefore, developing antibodies with high affinity and high specificity to recognize t-PAIC is of great significance for the development of t-PAIC detection reagents and for the diagnosis and monitoring of hematological diseases such as atherosclerosis. Summary of the Invention
[0007] To address one or more problems existing in the prior art, this invention provides an anti-t-PAIC monoclonal antibody. The technical solution adopted by this invention to solve the above problems is that the antibody or its antigen-binding fragment can specifically bind to t-PAIC, and the antibody or its antigen-binding fragment is any one of the following three: The heavy chain variable region of the first antibody or its antigen-binding fragment comprises: CDR-H1 with an amino acid sequence of SEQ ID NO:1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:3 or a variant thereof; The light chain variable region of the first antibody or its antigen-binding fragment comprises: CDR-L1 with an amino acid sequence of SEQ ID NO:4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:6 or a variant thereof; The heavy chain variable region of the second type of antibody or its antigen-binding fragment comprises: CDR-H1 with an amino acid sequence of SEQ ID NO:7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:9 or a variant thereof; The second type of antibody or its antigen-binding fragment has a light chain variable region comprising: CDR-L1 with an amino acid sequence of SEQ ID NO:10 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:11 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:12 or a variant thereof. The heavy chain variable region of the third type of antibody or its antigen-binding fragment comprises: CDR-H1 with an amino acid sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:15 or a variant thereof. The light chain variable region of the third type of antibody or its antigen-binding fragment comprises: CDR-L1 with an amino acid sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:18 or a variant thereof.
[0008] In some embodiments, the variant of the amino acid sequence contains an amino acid mutation compared to its source amino acid sequence, the amino acid mutation being any one or more of the substitution, deletion, or addition of one or more amino acids.
[0009] Furthermore, the substitutions in the amino acid mutations are conservative substitutions.
[0010] Furthermore, the identity of the variant of the amino acid sequence to its derived amino acid sequence is any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%.
[0011] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region and a light chain constant region, wherein the constant region is a heavy chain constant region and a light chain constant region of any of the following species: mouse, rat, or human.
[0012] Furthermore, the heavy chain constant region is the IgG heavy chain constant region, and the light chain constant region is the κ or λ light chain constant region.
[0013] In some embodiments, the antibody or its antigen-binding fragment is any one of IgG, IgM, IgE, IgD, and IgA antibodies.
[0014] In some embodiments, the antigen-binding fragment is selected from any one of scFv, Fab, (Fab')2, Fd, Fv, CDR fragment, nanobody, disulfide-linked Fv(dsFv), biantibody, bispecific antibody, and multispecific antibody; and / or, the antibody is any one of murine antibody, chimeric antibody, and humanized antibody.
[0015] In some embodiments, the first antibody or its antigen-binding fragment, the second antibody or its antigen-binding fragment, and the third antibody or its antigen-binding fragment further include a backbone region of a heavy chain variable region and a backbone region of a light chain variable region; The backbone region of the heavy chain variable region of the first antibody or its antigen-binding fragment includes: FR1-VH1, FR2-VH1, FR3-VH1 and FR4-VH1; The backbone region of the light chain variable region of the first antibody or its antigen-binding fragment includes: FR1-VL1, FR2-VL1, FR3-VL1 and FR4-VL1; The backbone region of the heavy chain variable region of the second type of antibody or its antigen-binding fragment includes: FR1-VH2, FR2-VH2, FR3-VH2 and FR4-VH2; The backbone region of the light chain variable region of the second type of antibody or its antigen-binding fragment includes: FR1-VL2, FR2-VL2, FR3-VL2 and FR4 VL2; The backbone region of the heavy chain variable region of the third type of antibody or its antigen-binding fragment includes: FR1-VH3, FR2-VH3, FR3-VH3 and FR4-VH3; The backbone region of the light chain variable region of the third type of antibody or its antigen-binding fragment includes: FR1-VL3, FR2-VL3, FR3-VL3 and FR4 VL3.
[0016] Application of an anti-t-PAIC monoclonal antibody, wherein the antibody or its antigen-binding fragment described above is applied to any one or more of chemiluminescent reagent detection, quantitative detection of t-PAIC, and immunological detection.
[0017] A nucleic acid sequence for encoding the antibody or its antigen-binding fragment described above, wherein the nucleic acid sequence SEQ ID NO:43-60 sequentially encodes amino acid sequences SEQ ID NO:1-18.
[0018] The technical advantages achieved by this invention are: the above-mentioned antibody or its antigen-binding fragment can specifically bind to t-PAIC, which can be used to prepare chemiluminescent reagents and for the quantitative and immunological detection of t-PAIC, with a detection sensitivity of up to 1 ng / mL and a correlation coefficient R. 2 ≥ 0.950, which shows a high correlation with the measured value of clinical plasma samples and meets the requirements of clinical testing. Attached Figure Description
[0019] Figure 1 This is a schematic diagram illustrating the correlation between the measured values of clinical samples from the combination t-PA-03-2A7 / PAI-05-2A5 in the example-reagent correlation test. Figure 2 This is a schematic diagram illustrating the correlation between clinical sample measurements of the combination t-PA-03-2A7 / PAI-05-2E6 in the example-reagent correlation test. Detailed Implementation
[0020] To make the above-mentioned objects, features, and advantages of the present invention more readily understood, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0021] This invention discloses an anti-t-PAIC monoclonal antibody, wherein the antibody or its antigen-binding fragment can specifically bind to t-PAIC, and the antibody or its antigen-binding fragment is any one of the following three (binding the amino acid and nucleic acid sequence of the CDR region of the t-PAIC antibody in Table 1). The heavy chain variable region of the first antibody or its antigen-binding fragment comprises: CDR-H1 with an amino acid sequence of SEQ ID NO:1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:3 or a variant thereof; The light chain variable region of the first antibody or its antigen-binding fragment comprises: CDR-L1 with an amino acid sequence of SEQ ID NO:4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:6 or a variant thereof; The heavy chain variable region of the second type of antibody or its antigen-binding fragment comprises: CDR-H1 with an amino acid sequence of SEQ ID NO:7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:9 or a variant thereof; The second type of antibody or its antigen-binding fragment has a light chain variable region comprising: CDR-L1 with an amino acid sequence of SEQ ID NO:10 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:11 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:12 or a variant thereof. The heavy chain variable region of the third type of antibody or its antigen-binding fragment comprises: CDR-H1 with an amino acid sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:15 or a variant thereof. The light chain variable region of the third type of antibody or its antigen-binding fragment comprises: CDR-L1 with the amino acid sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the amino acid sequence of SEQ ID NO:18 or a variant thereof.
[0022] Each of the above includes a heavy chain variable region and a light chain variable region, wherein both the heavy chain variable region and the light chain variable region contain three CDRs and are defined by the Kabat numbering system.
[0023] Specifically, the first type of antibody or its antigen-binding fragment, the second type of antibody or its antigen-binding fragment, and the third type of antibody or its antigen-binding fragment further include the backbone region of the heavy chain variable region and the backbone region of the light chain variable region (as shown in Table 1). The backbone region of the heavy chain variable region of the first antibody or its antigen-binding fragment includes: FR1-VH1, FR2-VH1, FR3-VH1 and FR4-VH1; The backbone region of the light chain variable region of the first antibody or its antigen-binding fragment includes: FR1-VL1, FR2-VL1, FR3-VL1 and FR4-VL1; The backbone region of the heavy chain variable region of the second type of antibody or its antigen-binding fragment includes: FR1-VH2, FR2-VH2, FR3-VH2 and FR4-VH2; The backbone region of the light chain variable region of the second type of antibody or its antigen-binding fragment includes: FR1-VL2, FR2-VL2, FR3-VL2 and FR4 VL2; The backbone region of the heavy chain variable region of the third type of antibody or its antigen-binding fragment includes: FR1-VH3, FR2-VH3, FR3-VH3 and FR4-VH3; The backbone region of the light chain variable region of the third type of antibody or its antigen-binding fragment includes: FR1-VL3, FR2-VL3, FR3-VL3 and FR4 VL3.
[0024] The amino acid sequence of FR1-VH1 is SEQ ID NO:19; The amino acid sequence of FR2-VH1 is SEQ ID NO:20; The amino acid sequence of FR3-VH1 is SEQ ID NO:21; The amino acid sequence of FR4-VH1 is SEQ ID NO:22; The amino acid sequence of FR1-VL1 is SEQ ID NO:23; The amino acid sequence of FR2-VL1 is SEQ ID NO:24; The amino acid sequence of FR3-VL1 is SEQ ID NO:25; The amino acid sequence of FR4-VL1 is SEQ ID NO:26; The amino acid sequence of FR1-VH2 is SEQ ID NO:27; The amino acid sequence of FR2-VH2 is SEQ ID NO:28; The amino acid sequence of FR3-VH2 is SEQ ID NO:29; The amino acid sequence of FR4-VH2 is SEQ ID NO:30; The amino acid sequence of FR1-VL2 is SEQ ID NO:31; The amino acid sequence of FR2-VL2 is SEQ ID NO:32; The amino acid sequence of FR3-VL2 is SEQ ID NO:33; The amino acid sequence of FR4-VL2 is SEQ ID NO:34; The amino acid sequence of FR1-VH3 is SEQ ID NO:35; The amino acid sequence of FR2-VH3 is SEQ ID NO:36; The amino acid sequence of FR3-VH3 is SEQ ID NO:37; The amino acid sequence of FR4-VH3 is SEQ ID NO:38; The amino acid sequence of FR1-VL3 is SEQ ID NO:39; The amino acid sequence of FR2-VL3 is SEQ ID NO:40; The amino acid sequence of FR3-VL3 is SEQ ID NO:41; The amino acid sequence of FR4-VL3 is SEQ ID NO:42.
[0025] SEQ ID NO:19: QVQLQQSGAELVKPGASVKLSCKAS; SEQ ID NO:20: INWVRQRPEQGLEWIGW; SEQ ID NO:21: KYNEKFKGKATLTTDKSSSTAYMQLSRLTSEDSAVYFC; SEQ ID NO:22: WGAGTTVTVSS; SEQ ID NO:23: QIVLTQSPAIMSASPGEKVTMTCSAS; SEQ ID NO:24: MHWYQQKSGTSPKRWIY; SEQ ID NO:25: KLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC; SEQ ID NO:26: FGAG TKLELK; SEQ ID NO:27: QVQLQQSGAELARPGASVKMSCKAS; SEQ ID NO:28: MWVKQRPGQGLEWIGY; SEQ ID NO:29: NYIQRFKDKATLTADKSSSTAYMQVSSLTSEDSAVYYC; SEQ ID NO:30: WGQGTSVTVSS; SEQ ID NO:31: NIVMTQSPKSMSMSVGERVTLTCKAS; SEQ ID NO:32: VSWYQQKPEQSPKLLIY; SEQ ID NO:33: NRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHC; SEQ ID NO:34: FGGGTKLEIK; SEQ ID NO:35: QVQLQQSGAELVRPGTSVKVSCKAS; SEQ ID NO:36: IEWVKQRPGQGLEWIGV; SEQ ID NO:37: NYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFC; SEQ ID NO:38: WGQGTLVTVSA; SEQ ID NO:39: QIVLTQSPAIMSASPGEKVTITCSAS; SEQ ID NO:40: MNWFQQKPGTSPKLWIY; SEQ ID NO:41: NLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYC; SEQ ID NO:42: FGGGTKLEIK。
[0026] Specifically, the antibody or its antigen-binding fragment has any one of the following light chain variable regions and heavy chain variable regions (refer to Table 1): 1. The heavy chain variable region VH shown in SEQ ID NO:61 and the light chain variable region VL shown in SEQ ID NO:62; 2. The heavy chain variable region VH shown in SEQ ID NO:63 and the light chain variable region VL shown in SEQ ID NO:64; 3. The heavy chain variable region VH shown in SEQ ID NO:65 and the light chain variable region VL shown in SEQ ID NO:66.
[0027] A nucleic acid sequence encoding the antibody described above or its antigen-binding fragment, as shown in Table 1 for the amino acid and nucleic acid sequences of the CDR region of the t-PAIC antibody, wherein the nucleic acid sequence SEQ ID NO:43-60 sequentially encodes the amino acid sequences SEQ ID NO:1-18.
[0028] Application of an anti-t-PAIC monoclonal antibody, wherein the antibody or its antigen-binding fragment is used in any one or more of chemiluminescent reagent detection, quantitative detection of t-PAIC, and immunological detection. "Immunological detection" refers to assays that utilize the specific interaction / binding between antigen and antibody, and are generally used to detect the presence or level of a specific antigen or antibody in a sample. Such immunological assays are well known to those skilled in the art and include, but are not limited to, enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, immunoturbidimetry, and surface plasmon resonance assays.
[0029] It should be noted that the variant of the amino acid sequence contains amino acid mutations compared to its source amino acid sequence. These mutations are any one or more substitutions, deletions, or additions of one or more amino acids (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids). Preferably, the substitutions in the amino acid mutations are conserved substitutions. The identity of the variant of the amino acid sequence to its source amino acid sequence is any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
[0030] Specifically, in some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) and a light chain constant region (CL), wherein the constant region is a heavy chain constant region and a light chain constant region of any species of mouse, rat, or human; wherein the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or for example, the mouse IgG1, IgG2a, IgG2b, or IgG3 constant region; and the light chain constant region is a κ or λ light chain constant region, such as the human λ light chain constant region. In some embodiments, the antibody or its antigen-binding fragment is any one of IgG, IgM, IgE, IgD, or IgA antibodies.
[0031] In some embodiments, the antigen-binding fragment is selected from any one of scFv, Fab, (Fab')2, Fd, Fv, CDR fragment, nanobody, disulfide-linked Fv (dsFv), diabody, bispecific antibody, and multispecific antibody; and / or, the antibody is any one of murine antibody, chimeric antibody, and humanized antibody.
[0032] The term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system. For a given antibody, those skilled in the art will readily identify the CDRs as defined by each numbering system. In this invention, the CDRs contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art.
[0033] As used in this application, the term "framework region" or "FR" residue refers to those amino acid residues in the antibody variable region other than the CDR residues as defined above. The term "antibody" is not limited to any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.
[0034] Table 1. Amino acid and nucleic acid sequences of the CDR region of t-PAIC antibody Example - Preparation of anti-t-PAIC monoclonal antibody: 1. Animal immunization: The gene sequences of t-PA (sequence 1) and PAI-1 (sequence 2) were synthesized and inserted into the expression vector pCAG (with BamHI / EcoRI restriction sites on both sides) to construct and extract the expression vectors pCAG-t-PA and pCAG-PAI-1.
[0035] Babl / c mice were immunized with the extracted expression vector at a dose of 60-100 μg / mouse. A second immunization was performed two weeks later. Three days after the fourth immunization, tail vein blood was collected to test the serum titer. Cell fusion was performed if the titer was greater than 400,000. Three days before cell fusion, intraperitoneal pulse immunization was performed with t-PAIC complex antigen (catalog number YA0182, Shenzhen Yawei Century Technology Co., Ltd.) at a dose of 60-100 μg / mouse.
[0036] Sequence 1 (t-PA): MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEFCSTPACSEGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRP。
[0037] Sequence 2 (PAI-1): MQMSPALTCLVLGLALVFGEGSAVHHPPSYVAHLASDFGVRVFQQVAQASKDRNVVFSPYGVASVLAMLQLTTGGETQQQIQAAMGFKIDDKGMAPALRH LYKELMGPWNKDEISTTDAIFVQRDLKLVQGFMPHFFRLFRSTVKQVDFSEVERARFIINDWVKTHTKGMISNLLGKGAVDQLTRLVLVNALYFNGQWKTP FPDSSTHRRLFHKSDGSTVSVPMMAQTNKFNYTEFTTPDGHYYDILELPYHGDTLSMFIAAPYEKEVPLSALTNILSAQLISHWKGNMTRLLPRLLVLPKFS LETEVDLRKPLENLGMTDMFRQFQADFTSLSDQEPLHVAQALQKVKIEVNESGTVASSSTAVIVSARMAPEEIIMDRPFLFVVRHNPTGTVLFMGQVMEP.
[0038] 2. SP2 / 0 cell treatment: Mouse myeloma cells (SP2 / 0 cells) were cultured for one week, then digested with trypsin, collected, centrifuged and the supernatant was discarded. The SP2 / 0 cells were washed three times with 30 mL of DMEM basal medium, and finally resuspended in 20 mL of DMEM basal medium.
[0039] 3. Spleen cell isolation and shock immunization: The spleen of the above-mentioned experimental mice was taken, ground into a homogenate, and 30 ml of 1XPBS (buffered saline) was added. Red blood cells were lysed using methods known in the art, and then resuspended in culture medium to obtain isolated spleen cells.
[0040] 4. Cell fusion: Mix spleen cells and SP2 / 0 cells at a ratio of 2:1, centrifuge to remove the supernatant, and slowly add PEG to mix; add 14 mL of DMEM basal medium and incubate at 37°C; add 20 mL of DMEM basal medium, centrifuge to remove the supernatant, and plate the cells with DMEM medium containing HAT. Culture the cells for 6-10 days. In this example, the DMEM basal medium was purchased from Hyclone.
[0041] 5. Fusion cells were screened and plated with t-PAIC complex antigen (catalog number YA0182, Shenzhen Yawei Century Technology Co., Ltd.). Positive clones were screened using indirect ELISA. Single clones were prepared using limiting dilution and ELISA methods. Cell lines t-PA-03-2A7 (which binds specifically to both the t-PAIC complex and t-PA) and PAI-05-2A5 and PAI-05-2E6 (which bind specifically to both the t-PAIC complex and PAI) were obtained.
[0042] 6. Antibody preparation: Paraffin was injected into the peritoneal cavity of mice, and then the hybridoma cells obtained above were injected into the peritoneal cavity of mice. After the mouse abdomen swelled, the ascites fluid was collected. The mouse ascites fluid was centrifuged at 4°C, the supernatant was collected, filtered through a 0.45µm filter membrane, and air bubbles in the solution were removed by sonication. The purified antibody was obtained by elution with 50mM glycine solution using the Protein G pre-packed column purification method.
[0043] 7. Sequence acquisition: Total RNA was extracted from the monoclonal hybridoma cells obtained in this example using a commercially available RNA Isolater Total RNA Extraction Reagent kit. The total RNA was reverse transcribed, amplified by PCR, and the amplified products were sequenced to obtain the amino acid sequence of the antibody produced by the hybridoma cells and the corresponding DNA coding sequence.
[0044] Example - Preparation of chemiluminescent reagents: 1. Preparation of magnetic bead reagent: Purified PAI-05-2A5 and PAI-05-2E6 monoclonal antibodies were conjugated with tosylate magnetic beads (manufacturer: JSR or Thermo). The conjugation reaction buffer was CBS, pH 9.6, and the conjugation catalyst was a conjugation buffer solution containing 3M ammonium sulfate. The magnetic bead conjugation procedure was as follows: 20 μg antibody / mg magnetic bead, magnetic bead reaction volume was 10 mg / ml, reaction buffer was 2 / 3 conjugation reaction buffer and 1 / 3 conjugation catalyst, reaction was carried out at 37℃ for 16-24 h, excess unconjugated magnetic beads were washed with PBST, and excess unconjugated magnetic beads were blocked with PBS containing 5% casein and 5% BSA, pH 7.4, for 12-24 h. After the blocking process is complete, add 50 mM TBS, 5% BSA, 0.02% Tween 20, and 0.05% Proclin 300 to dilute the magnetic beads to a concentration of 0.15-0.3 mg / ml, thus obtaining the working solution of reagent 1 magnetic beads.
[0045] 2. Preparation of luminescent labeling reagent: Take 1 mg of t-PA-03-2A7 antibody and add 30 μg of NHS-SA-acridine. Incubate at room temperature for 2 h. After purification by molecular sieve, the labeled antibody solution is obtained. Dilute with acridine diluent to 0.1-0.4 μg / ml according to the antibody concentration. The acridine diluent is 50 mM MES, 0.15 M NaCl, 0.5% BSA, 0.05% Tween 20, and 0.5% casein to obtain the acridine working solution.
[0046] 3. Luminescent substrate reagents: Luminescent reagent 1: 0.1 mol / L hydrochloric acid + 0.3% hydrogen peroxide; Luminescent reagent 2: 0.15 mol / L sodium hydroxide + 0.05% Triton 100.
[0047] 4. Reagent kit reaction procedure: 50 μL sample, 50 μL magnetic bead working solution, 50 μL acridine working solution, incubate for 20 min, magnetically separate and wash 3 times, add 1-100 μL luminescent reagent, read the luminescence signal value after adding 2-100 μL luminescent reagent; the entire reaction system can be completed in 25 min.
[0048] Example - Reagent Sensitivity Test: Test method: Five samples with concentrations close to the detection limit of 1 ng / mL were tested, and each sample was tested five times.
[0049] Testing standard: The number of test results below the blank limit of 0.5 ng / mL should be less than or equal to 3.
[0050] The test results of the reagents prepared by the combination t-PA-03-2A7 / PAI-05-2A5 are shown in Table 2, and the test results of the reagents prepared by the combination t-PA-03-2A7 / PAI-05-2E6 are shown in Table 3. It can be seen that the detection sensitivity of both antibody combinations reaches 1 ng / mL.
[0051] Table 2: Sensitivity Test Results of Combination t-PA-03-2A7 / PAI-05-2A5 Table 3: Sensitivity Test Results of Combination t-PA-03-2A7 / PAI-05-2E6 Example - Reagent Correlation Test: Forty to fifty clinical plasma samples were selected and tested using a self-prepared luminescent reagent. Correlation analysis was performed on the test results, and the results are shown in Table 4.
[0052] Table 4: Antibody-pair correlation detection Correlation analysis between the reagent preparation of the combination t-PA-03-2A7 / PAI-05-2A5 and clinical measurements (detection method: Sysmex t-PAIC chemiluminescence reagent kit) is shown in [link to relevant data]. Figure 1 Correlation coefficient R 2 = 0.9847; Correlation analysis between the reagents prepared by combination t-PA-03-2A7 / PAI-05-2A5 and clinical measurements is shown in [link to relevant data]. Figure 2 Correlation coefficient R 2 = 0.961. The reagents prepared from both antibody combinations showed good correlation with clinical measurements.
[0053] In summary, the antibody or its antigen-binding fragment of this application can specifically bind to t-PAIC, and can be used to prepare chemiluminescent reagents for the quantitative and immunological detection of t-PAIC. The detection sensitivity can reach 1 ng / mL, and the correlation coefficient R is [value missing]. 2 ≥ 0.950, which shows a high correlation with the measured value of clinical plasma samples and meets the requirements of clinical testing.
[0054] The embodiments described above are merely illustrative of one or more implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.
Claims
1. An anti-t-PA IC monoclonal antibody, characterized in that, The antibody or its antigen-binding fragment can specifically bind to t-PAIC, and the antibody or its antigen-binding fragment is any one of the following three: The heavy chain variable region of the first antibody or its antigen-binding fragment comprises: CDR-H1 with an amino acid sequence of SEQ ID NO:1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:3 or a variant thereof; The light chain variable region of the first antibody or its antigen-binding fragment comprises: CDR-L1 with an amino acid sequence of SEQ ID NO:4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:6 or a variant thereof; The heavy chain variable region of the second type of antibody or its antigen-binding fragment comprises: CDR-H1 with an amino acid sequence of SEQ ID NO:7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:9 or a variant thereof; The light chain variable region of the second type of antibody or its antigen-binding fragment comprises: CDR-L1 with an amino acid sequence of SEQ ID NO:10 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:11 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:12 or a variant thereof. The heavy chain variable region of the third type of antibody or its antigen-binding fragment comprises: CDR-H1 with an amino acid sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:15 or a variant thereof. The light chain variable region of the third type of antibody or its antigen-binding fragment comprises: CDR-L1 with the amino acid sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the amino acid sequence of SEQ ID NO:18 or a variant thereof. Among them, SEQ ID NO:1: GFTFTSYY, SEQ ID NO:2: IYPGDGST, SEQ ID NO:3: AREGGLRFFDY, SEQ ID NO:4: QDINSY, SEQ ID NO:5: RAN, SEQ ID NO:6: LQNDELYT, SEQ ID NO:7: GDFFSRYW, SEQ ID NO:8: INPGSNTI, SEQ ID NO:9: ARLDGYYGYAMDY, SEQ ID NO:10: ESVDSHDNSF, SEQ ID NO:11: LAS, SEQ ID NO:12: QQNNEDPWT, SEQ ID NO:13: GFNIKDTY, SEQ ID NO:14: IDPANGNS, SEQ ID NO:15: ADGYYEGTMDY, SEQ ID NO:16: QDIKSY, SEQ ID NO:17: YAT, SEQ ID NO:18: LQHGESPFT.
2. The anti-t-PA IC monoclonal antibody according to claim 1, characterized by The variant of the amino acid sequence contains an amino acid mutation compared to its source amino acid sequence, wherein the amino acid mutation is any one or more of the substitution, deletion, or addition of one or more amino acids.
3. The anti-t-PA IC monoclonal antibody according to claim 2, characterized by The identity of the variant of the amino acid sequence to the amino acid sequence from which it originates is any one of the following values: at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100%.
4. The anti-t-PA IC monoclonal antibody according to claim 1, characterized by The antibody or its antigen-binding fragment comprises a heavy chain constant region and a light chain constant region, wherein the constant region is the heavy chain constant region and light chain constant region of any of the following genera: mouse, rat, or human.
5. The anti-t-PA IC monoclonal antibody according to claim 4, characterized in that, The heavy chain constant region is the IgG heavy chain constant region, and the light chain constant region is the κ or λ light chain constant region.
6. The anti-t-PAIC monoclonal antibody according to claim 1, characterized in that, The antibody or its antigen-binding fragment is any one of IgG, IgM, IgE, IgD, or IgA antibodies.
7. The anti-t-PAIC monoclonal antibody according to claim 1, characterized in that, The antigen-binding fragment is selected from any one of scFv, Fab, (Fab')2, Fd, Fv, CDR fragment, nanobody, disulfide-linked Fv (dsFv), biantibody, bispecific antibody, and multispecific antibody; And / or, the antibody is any one of mouse antibody, chimeric antibody, or humanized antibody.
8. The anti-t-PAIC monoclonal antibody according to any one of claims 1-7, characterized in that, The first type of antibody or its antigen-binding fragment, the second type of antibody or its antigen-binding fragment, and the third type of antibody or its antigen-binding fragment further include the backbone region of the heavy chain variable region and the backbone region of the light chain variable region; The backbone region of the heavy chain variable region of the first antibody or its antigen-binding fragment includes: FR1-VH1, FR2-VH1, FR3-VH1 and FR4-VH1; The backbone region of the light chain variable region of the first antibody or its antigen-binding fragment includes: FR1-VL1, FR2-VL1, FR3-VL1 and FR4-VL1; The backbone region of the heavy chain variable region of the second type of antibody or its antigen-binding fragment includes: FR1-VH2, FR2-VH2, FR3-VH2 and FR4-VH2; The backbone region of the light chain variable region of the second type of antibody or its antigen-binding fragment includes: FR1-VL2, FR2-VL2, FR3-VL2 and FR4 VL2; The backbone region of the heavy chain variable region of the third type of antibody or its antigen-binding fragment includes: FR1-VH3, FR2-VH3, FR3-VH3 and FR4-VH3; The backbone region of the light chain variable region of the third type of antibody or its antigen-binding fragment includes: FR1-VL3, FR2-VL3, FR3-VL3 and FR4 VL3; The amino acid sequence of FR1-VH1 is SEQ ID NO:19; The amino acid sequence of FR2-VH1 is SEQ ID NO:20; The amino acid sequence of FR3-VH1 is SEQ ID NO:21; The amino acid sequence of FR4-VH1 is SEQ ID NO:22; The amino acid sequence of FR1-VL1 is SEQ ID NO:23; The amino acid sequence of FR2-VL1 is SEQ ID NO:24; The amino acid sequence of FR3-VL1 is SEQ ID NO:25; The amino acid sequence of FR4-VL1 is SEQ ID NO:26; The amino acid sequence of FR1-VH2 is SEQ ID NO:27; The amino acid sequence of FR2-VH2 is SEQ ID NO:28; The amino acid sequence of FR3-VH2 is SEQ ID NO:29; The amino acid sequence of FR4-VH2 is SEQ ID NO:30; The amino acid sequence of FR1-VL2 is SEQ ID NO:31; The amino acid sequence of FR2-VL2 is SEQ ID NO:32; The amino acid sequence of FR3-VL2 is SEQ ID NO:33; The amino acid sequence of FR4-VL2 is SEQ ID NO:34; The amino acid sequence of FR1-VH3 is SEQ ID NO:35; The amino acid sequence of FR2-VH3 is SEQ ID NO:36; The amino acid sequence of FR3-VH3 is SEQ ID NO:37; The amino acid sequence of FR4-VH3 is SEQ ID NO:38; The amino acid sequence of FR1-VL3 is SEQ ID NO:39; The amino acid sequence of FR2-VL3 is SEQ ID NO:40; The amino acid sequence of FR3-VL3 is SEQ ID NO:41; The amino acid sequence of FR4-VL3 is SEQ ID NO:42; SEQ ID NO:19: QVQLQQSGAELVKPGASVKLSCKAS; SEQ ID NO:20: INWVRQRPEQGLEWIGW; SEQ ID NO:21: KYNEKFKGKATLTTDKSSSTAYMQLSRLTSEDSAVYFC; SEQ ID NO:22: WGAGTTVTVSS; SEQ ID NO:23: QIVLTQSPAIMSASPGEKVTMTCSAS; SEQ ID NO:24: MHWYQQKSGTSPKRWIY; SEQ ID NO:25: KLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC; SEQ ID NO:26: FGAG TKLELK; SEQ ID NO:27: QVQLQQSGAELARPGASVKMSCKAS; SEQ ID NO:28: MWVKQRPGQGLEWIGY; SEQ ID NO:29: NYIQRFKDKATLTADKSSSTAYMQVSSLTSEDSAVYYC; SEQ ID NO:30: WGQGTSVTVSS; SEQ ID NO:31: NIVMTQSPKSMSMSVGERVTLTCKAS; SEQ ID NO:32: VSWYQQKPEQSPKLLIY; SEQ ID NO:33: NRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHC; SEQ ID NO:34: FGGGTKLEIK; SEQ ID NO:35: QVQLQQSGAELVRPGTSVKVSCKAS; SEQ ID NO:36: IEWVKQRPGQGLEWIGV; SEQ ID NO:37: NYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFC; SEQ ID NO:38: WGQGTLVTVSA; SEQ ID NO:39: QIVLTQSPAIMSASPGEKVTITCSAS; SEQ ID NO:40: MNWFQQKPGTSPKLWIY; SEQ ID NO:41: NLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYC; SEQ ID NO:42: FGGGTKLEIK.
9. The application of an anti-t-PAIC monoclonal antibody, wherein the antibody or its antigen-binding fragment as described in any one of claims 1-8 is applied to any one or more of chemiluminescent reagent detection, quantitative detection of t-PAIC, and immunological detection.
10. A nucleic acid sequence, characterized in that, The nucleic acid sequence is used to encode the antibody or its antigen-binding fragment according to any one of claims 1-8, wherein the nucleic acid sequence SEQ ID NO:43-60 sequentially encodes the amino acid sequence SEQ ID NO:1-18; Among them, SEQ ID NO:43: GGCTTCACCTTCACAAGCTACTAT; SEQ ID NO: 44: ATTTATCCTGGAGATGGTAGTACT; SEQ ID NO:45: GCAAGGGAAGGAGGATTACGATTCTTTGACTAC; SEQ ID NO:46: CAGGACATTAATAGCTAT; SEQ ID NO:47:CGTGCAAAC; SEQ ID NO: 48: CTACAGAATGATGAGTTGTACACG; SEQ ID NO:49: GGATTCGATTTTAGTAGATACTGG; SEQ ID NO: 50: ATTAATCCAGGAAGCAATACGATA; SEQ ID NO:51:GCAAGACTCGATGGTTACTACGGCTATGCTATGGACTAC; SEQ ID NO:52:GAAAGTGTTGATAGTCATGACAATAGTTTT; SEQ ID NO:53:CTTGCATCC; SEQ ID NO:54:CAGCAAAATAATGAGGATCCGTGGACG; SEQ ID NO:55:GGCTTCAACATTAAAGACACCTAT; SEQ ID NO:56:ATTGATCCTGCGAATGGTAATAGT; SEQ ID NO:57:GCCGATGGTTACTACGAGGGGACTATGGACTAT; SEQ ID NO:58:CAGGACATTAAAAGTTAT; SEQ ID NO:59:TATGCAACA; SEQ ID NO:60:CTACAACATGGTGAGAGCCCATTCACG。