An anti-PIC monoclonal antibody, its encoding sequence and applications

Abstract

This invention discloses an anti-PIC monoclonal antibody, its encoding sequence, and its applications. The antibody or its antigen-binding fragment can specifically bind to the complex of α2-plasmin inhibitor and plasmin. The antibody or its antigen-binding fragment includes heavy chain variable regions CDR-H1, CDR-H2, and CDR-H3, and light chain variable regions CDR-L1, CDR-L2, and CDR-L3. This antibody or its antigen-binding fragment can specifically bind to the complex of α2-plasmin inhibitor and plasmin, and can be used to prepare chemiluminescent reagents for the quantitative detection of PIC. The detection sensitivity can reach 0.05 μg / mL, and it has a high correlation with the measured values ​​in clinical plasma samples, with a correlation coefficient R0. 2 ≥ 0.950, which meets the requirements for clinical testing.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine or biopharmaceutical technology, and in particular to an anti-PIC monoclonal antibody, its encoding sequence, and its applications. Background Technology

[0002] PIC is a product of the interaction between α2-plasmin inhibitor (α2-PI) and plasmin in the body. Under physiological conditions, plasminogen activators such as tissue-plasminogen activator (tPA) and urokinase-plasminogen activator (uPA) activate PLG into plasmin, which rapidly acts on fibrin to degrade it, a process known as fibrinolysis. Meanwhile, α2-PI in the body forms a plasmin-α2-PI complex (PIC) with free plasmin in a 1:1 ratio, thus inactivating it.

[0003] Plasmin is a key factor in the fibrinolytic system, its main function being to dissolve fibrin(ogen) and thus dissolve thrombi. Excessive plasmin production can lead to hyperfibrinolysis, causing uncontrollable bleeding. Similar to thrombin, plasmin has a short half-life of only a few seconds, making it difficult to measure. PIC is a complex formed by the 1:1 binding of plasmin and α2 antiplasmin after plasmin production, with a half-life of approximately 6 hours. PIC level detection overcomes the limitations of plasmin's short half-life and difficulty in detection, directly reflecting plasmin production and indicating hyperfibrinolysis. PIC is a marker of the activation of the fibrinolytic system, reflecting the degree of plasmin activation, predicting thrombus formation, and guiding antifibrinolytic therapy.

[0004] Generally, a TAT / PIC ratio of approximately 5:1 indicates a relatively balanced state between coagulation and fibrinolysis. A ratio greater than 5:1 suggests that the coagulation system is dominant, making the body more prone to thrombosis, while a lower ratio indicates a greater likelihood of bleeding. Elevated PIC indicates thrombosis has occurred and reflects the production of plasmin by the fibrinolytic system, helping to assess the degree of fibrinolytic activation. Elevated PIC is seen in early disseminated intravascular coagulation (DIC) and during antifibrinolytic therapy, and is a common specific diagnostic indicator for venous thrombotic diseases (VTE) in clinical practice. Excessive PIC elevation in pregnant women can predict the risk of preeclampsia and preeclampsia. PIC levels are also elevated in rheumatic diseases and SLE, and are correlated with disease activity.

[0005] PIC, as one of the new thrombosis detection items (TAT, PIC, TM, tPAI-C), provides a basis for the diagnosis, medication guidance, and prediction of many diseases, such as thrombosis, cancer, and cardiovascular and cerebrovascular diseases. Traditional thrombosis detection indicators in my country include prothrombin time, fibrinogen (FIB), D-dimer, and triglycerides (TT). These traditional items are mainly used for preoperative screening, and the results are relatively delayed, limiting their ability to diagnose early disease development. Currently, PIC detection mostly uses immunoassays such as ELISA and CLIA. CLIA technology has advantages over ELISA, including simpler operation, higher sensitivity, more stable reagents, and lower cost. Immunoassays rely on highly specific and sensitive monoclonal antibodies (pairs). Therefore, the preparation of highly specific and sensitive monoclonal antibodies has significant market and social value for developing high-quality PIC detection methods and reducing medical costs. Summary of the Invention

[0006] To address one or more problems existing in the prior art, this invention provides an anti-PIC monoclonal antibody, its encoding sequence, and its applications. The technical solution adopted by this invention to solve the above problems is: an anti-PIC monoclonal antibody, wherein the antibody or its antigen-binding fragment can specifically bind to a complex of α2-plasmin inhibitor and plasmin, 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.

[0007] 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.

[0008] Furthermore, the substitutions in the amino acid mutations are conservative substitutions.

[0009] 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%.

[0010] 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.

[0011] 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.

[0012] In some embodiments, the antibody or its antigen-binding fragment is any one of IgG, IgM, IgE, IgD, and IgA antibodies.

[0013] 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.

[0014] 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.

[0015] Application of an anti-PIC monoclonal antibody, wherein the antibody described above or its antigen-binding fragment is used in chemiluminescent reagent detection and / or immunological detection.

[0016] 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.

[0017] The technical effects achieved by this invention are as follows: the above-mentioned antibody or its antigen-binding fragment can specifically bind to the complex of α2-plasmin inhibitor and plasmin, and can be used to prepare chemiluminescent reagents for quantitative detection in PIC, with a detection sensitivity of 0.05 μg / mL, and a high correlation with the measured values ​​of clinical plasma samples, with a correlation coefficient R. 2 ≥ 0.950, which meets the requirements for clinical testing. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the correlation between the measured values ​​of clinical samples from the combination PLG-12-5B10 / 2PI-11-1A6 in the example-reagent correlation test. Figure 2This is a schematic diagram illustrating the correlation between clinical sample measurements of the combination PLG-12-5A10 / 2PI-11-1A6 in the example-reagent correlation test. Detailed Implementation

[0019] 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.

[0020] This invention discloses an anti-PIC monoclonal antibody, wherein the antibody or its antigen-binding fragment can specifically bind to the complex of α2-plasmin inhibitor and plasmin, 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 PIC 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 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. 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.

[0021] 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.

[0022] 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.

[0023] SEQ ID NO:19: QVQLQQSGAELVRPGALVKLSCKAS; SEQ ID NO:20: IHWVKQRPKQGLEWIGW; SEQ ID NO:21: FFDPSFQGKASLATDTSSNTAFLQLSSLTSEDSVVYYC; SEQ ID NO:22: WGAGTTVTVSS; SEQ ID NO:23: DIELTQSPASLSASVGETVTITCQAS; SEQ ID NO:24: LAWHQQKQGKSPQLLVY; SEQ ID NO:25: TLAGGVSSRFSGSGSGTHFSLKIKSLQPEDFGIYYC; SEQ ID NO:26: FGGGTKLEI; SEQ ID NO:27: QVKLQESGAELARPGASVKLSCKAS; SEQ ID NO:28: MQWVKQR PGQGLDWIGA; SEQ ID NO:29: RYTHKFKGKATLTADKSSSTAYMQLSSLASEDSGVYYC; SEQ ID NO:30: WG QGTTVTVSS; SEQ ID NO:31: DIMMTQSPATLSVTPGDRVSLSCRAS; SEQ ID NO:32: LHWYQQKSHESPRLLIK; SEQ ID NO:33: QSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC; SEQ ID NO:34: FGAGTKLELK; SEQ ID NO:35: QVQLQQSDAELVKPGASVKISCKVS; SEQ ID NO:36: FHWMKQRPEQGLEWIGY; SEQ ID NO:37: DYNEKFKGKATLTADRSSSTVYMQLNSLTSEDSAVYFC; SEQ ID NO:38: WGQGTLVTVSA; SEQ ID NO:39: ENVLTQSPAIMSATLGERVTMTCRAS; SEQ ID NO:40: MYWFQQKSGTSPKLWIY; SEQ ID NO:41: DLASGVPPRFSGSGSGTSYSLTISSVEAEDAATYYC; SEQ ID NO:42: FGTGTKLEI。

[0024] 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.

[0025] 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 PIC antibody CDR region, wherein the nucleic acid sequence SEQ ID NO:43-60 sequentially encodes the amino acid sequences SEQ ID NO:1-18.

[0026] An application of an anti-PIC monoclonal antibody, wherein the antibody or its antigen-binding fragment described above is used in chemiluminescent reagent detection and / or immunological detection. "Immunological detection" refers to assays performed using the specific interaction / binding between antigen and antibody, which 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.

[0027] 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%.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Table 1: Amino acid and nucleic acid sequences of the CDR region of PIC antibody Example - Preparation of anti-PIC monoclonal antibody: 1. Animal immunization: The gene sequences of Plasmin (sequence 1) and α2-PI (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-Plasmin and pCAG-α2-PI. 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 shock immunization was performed with PIC complex antigen (catalog number YA0203, Shenzhen Yawei Century Technology Co., Ltd.) at a dose of 60-100 μg / mouse.

[0033] Sequence 1 (Plasmin): MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN。

[0034] Sequence 2 (α2-PI): MALLWGLLVLSWSCLQGPCSVFSPVSAMEPLGRQLTSGPNQEQVSPLTLLKLGNQEPGGQTALKSPPGVCSRDPTPEQTHRLARAMMAFTADLFSLVAQTSTCPNLILSPLSVALALSHLALG AQNHTLQRLQQVLHAGSGPCLPHLLSRLCQDLGPGAFRLAARMYLQKGFPIKEDFLEQSEQLFGAKPVSLTGKQEDDLANINQWVKEATEGKIQEFLSGLPEDTVLLLLNAIHFQGFWRNKFD PSLTQRDSFHLDEQFTVPVEMMQARTYPLRWFLLEQPEIQVAHFPFKNNMSFVVLVPTHFEWNVSQVLANLSWDTLHPPLVWERPTKVRLPKLYLKHQMDLVATLSQLGLQELFQAPDLRGIS EQSLVVSGVQHQSTLELSEVGVEAAAATSIAMSRMSLSSFSVNRPFLFFIFEDTTGLPLFVGSVRNPNPSAPRELKEQQDSPGNKDFLQSLKGFPRGDKLFGPDLKLVPPMEEDYPQFGSPK.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 5. Screening of fusion cells: Plated with PIC complex antigen (catalog number YA0182, Shenzhen Yawei Century Technology Co., Ltd.), positive clones were screened by indirect ELISA, and single clones were prepared by limiting dilution method and ELISA. Cell lines PLG-12-5B10 and PLG-12-5A10, which can bind specifically to both the PIC complex and Plasmin, and cell line 2PI-11-1A6, which can bind specifically to both the PIC complex and α2-PI, were obtained.

[0039] 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.

[0040] 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.

[0041] Example - Preparation of chemiluminescent reagents: 1. Preparation of magnetic bead reagent: Purified PLG-12-5B10 and PLG-12-5A10 monoclonal antibodies were conjugated to toluenesulfonyl 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 blocking, 50 mM ammonium sulfate was added. The magnetic bead working solution of reagent 1 is obtained by diluting TBS, 5% BSA, 0.02% Tween 20, and 0.05% Proclin 300 to a magnetic bead concentration of 0.15-0.3 mg / ml.

[0042] 2. Preparation of luminescent labeling reagent: Take 1 mg of 2PI-11-1A6 antibody and add 30 μg of NHS-SA-acridine. Incubate at room temperature for 2 h. After purification by molecular sieve, obtain the labeled antibody solution. 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.

[0043] 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.

[0044] 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.

[0045] Example - Reagent Sensitivity Test: Test method: Five samples with a concentration close to the detection limit of 0.05 μg / mL were tested, and each sample was tested five times.

[0046] Test standard: The number of test results below the blank limit of 0.025 μg / mL should be less than or equal to 3.

[0047] The test results of the reagents prepared by the combination PLG-12-5B10 / 2PI-11-1A6 are shown in Table 2, and the test results of the reagents prepared by the combination PLG-12-5A10 / 2PI-11-1A6 are shown in Table 3. It can be seen that the detection sensitivity of both antibody combinations reached 0.05 μg / mL.

[0048] Table 2: Sensitivity Test Results of PLG-12-5B10 / 2PI-11-1A6 Combination Table 3: Sensitivity Test Results of PLG-12-5A10 / 2PI-11-1A6 Combination 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.

[0049] Table 4: Antibody-pair correlation detection Correlation analysis between the reagent preparation of the PLG-12-5B10 / 2PI-11-1A6 combination and clinical measurements (detection method: Sysmex PIC chemiluminescence reagent kit) is shown in [link to relevant documentation]. Figure 1 Correlation coefficient R 2 = 0.9841; Correlation analysis between the reagents prepared by the combination PLG-12-5A10 / 2PI-11-1A6 and clinical measurements is shown in [link to relevant data]. Figure 2 Correlation coefficient R 2 =0.9875. The reagents prepared from both antibody combinations showed good correlation with clinical measurements.

[0050] In summary, the antibody or its antigen-binding fragment of this application can specifically bind to the complex of α2-plasmin inhibitor and plasmin, and can be used to prepare chemiluminescent reagents for quantitative detection in PIC. The detection sensitivity can reach 0.05 μg / mL, and it has a high correlation with the measured values ​​in clinical plasma samples, with a correlation coefficient R. 2 ≥ 0.950, which meets the requirements for clinical testing.

[0051] 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-PIC monoclonal antibody, characterized in that, The antibody or its antigen-binding fragment can specifically bind to the complex of α2-plasmin inhibitor and plasmin, 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: GYSFTDYT, SEQ ID NO:2: INPYNGGT, SEQ ID NO:3: ARRGDRYDGGYYAMDY, SEQ ID NO:4: SSVSF, SEQ ID NO:5: ATS, SEQ ID NO:6: QQWSSNTPT, SEQ ID NO:7: GYSFTDYT, SEQ ID NO:8: INPYNGGT, SEQ ID NO: 9: ARRGDRYDGGYYAMDY, SEQ ID NO: 10: SSVSF, SEQ ID NO: 11: ATS, SEQ ID NO: 12: QQWSSNTPT, SEQ ID NO: 13: GYSITSGYY, SEQ ID NO: 14: INYDGYS, SEQ ID NO: 15: ARDEDSYFDY, SEQ ID NO: 16: QDVRTA, SEQ ID NO:17: WAS, SEQ ID NO:18: QQHYSTPFT.

2. The anti-PIC monoclonal antibody according to claim 1, characterized in that, 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-PIC monoclonal antibody according to claim 2, characterized in that, 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-PIC monoclonal antibody according to claim 1, characterized in that, 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-PIC 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-PIC 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-PIC 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-PIC 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: QVQLQQSGAELVRPGALVKLSCKAS; SEQ ID NO:20: IHWVKQRPKQGLEWIGW; SEQ ID NO:21: FFDPSFQGKASLATDTSSNTAFLQLSSLTSEDSVVYYC; SEQ ID NO:22: WGAGTTVTVSS; SEQ ID NO:23: DIELTQSPASLSASVGETVTITCQAS; SEQ ID NO:24: LAWHQQKQGKSPQLLVY; SEQ ID NO:25: TLAGGVSSRFSGSGSGTHFSLKIKSLQPEDFGIYYC; SEQ ID NO:26: FGGGTKLEI; SEQ ID NO:27: QVKLQESGAELARPGASVKLSCKAS; SEQ ID NO:28: MQWVKQRPGQGLDWIGA; SEQ ID NO:29: RYTHKFKGKATLTADKSSSTAYMQLSSLASEDSGVYYC; SEQ ID NO:30: WGQGTTVTVSS; SEQ ID NO:31: DIMMTQSPATLSVTPGDRVSLSCRAS; SEQ ID NO:32: LHWYQQKSHESPRLLIK; SEQ ID NO:33: QSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC; SEQ ID NO:34: FGAGTKLELK; SEQ ID NO:35: QVQLQQSDAELVKPGASVKISCKVS; SEQ ID NO:36: FHWMKQRPEQGLEWIGY; SEQ ID NO:37: DYNEKFKGKATLTADRSSSTVYMQLNSLTSEDSAVYFC; SEQ ID NO:38: WGQGTLVTVSA; SEQ ID NO:39: ENVLTQSPAIMSATLGERVTMTCRAS; SEQ ID NO:40: MYWFQQKSGTSPKLWIY; SEQ ID NO:41: DLASGVPPRFSGSGSGTSYSLTISSVEAEDAATYYC; SEQ ID NO:42: FGTGTKLEI.

9. The application of an anti-PIC monoclonal antibody, characterized in that, The antibody or its antigen-binding fragment as described in any one of claims 1-8 may be used in chemiluminescent reagent detection and / or 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: GGTTACTCATTCACTGACTACACC; SEQ ID NO:44: ATTAATCCTTACAATGGTGGTACT; SEQ ID NO:45: GCAAGAAGGGGGGGATAGGTACGACGGGGGTTACTATGCTATGGACTAC; SEQ ID NO:46: TCAAGTGTAAGTTTC; SEQ ID NO:47: GCCACATCC; SEQ ID NO: 48: CAGCAGTGGAGTAGTAATACACCCACG; SEQ ID NO:49: GGTTACTCATTCACTGACTACACC; SEQ ID NO:50: ATTAATCCTTACAATGGTGGTACT; SEQ ID NO:51: GCAAGAAGGGGGGATAGGTACGACGGGGGTTACTATGCTATGGACTAC; SEQ ID NO:52:TCAAGTGTAAGTTTC; SEQ ID NO:53:GCCACATCC; SEQ ID NO:54:CAGCAGTGGAGTAGTAATACACCCACG; SEQ ID NO:55:GGCTACTCCATCACCAGTGGTTATTAC; SEQ ID NO:56:ATAAATTACGACGGTTACAGT; SEQ ID NO:57:GCAAGAGATGAGGACTCCTACTTTGACTAC; SEQ ID NO:58:CAGGATGTGAGAACTGCT; SEQ ID NO:59:TGGGCATCC; SEQ ID NO:60:CAACAACATTATAGCACTCCATTCACG。

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