Antibody for detecting plasmin-alpha 2 plasmin inhibitor complex and application thereof

Abstract

The invention relates to the field of antibodies, and discloses an anti-plasmin antibody, an anti-alpha2 plasmin inhibitor antibody and application of the anti-alpha2 plasmin inhibitor antibody. The antibody comprises a heavy chain complementarity determining region and a light chain complementarity determining region, and can provide an important raw material for detection of a plasmin-alpha 2 plasmin inhibitor. The antibody provided by the invention has good affinity and activity, the pairing of the two antibodies has an obvious gradient in the conventional linear range of PIC, and the antibody can be effectively applied to the related detection field.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, in particular to an antibody against an anti-plasmin and an antibody against an α2 plasmin inhibitor and applications thereof. Background Art

[0002] Plasminogen (Pg) is a single-chain glycoprotein composed of 287 amino acids with a molecular weight of approximately 72 kDa. It is synthesized in the liver and transported throughout the body via the blood. Under physiological conditions, plasminogen exists in an inactive form but can be activated into active plasmin under specific conditions. Plasmin activation is primarily catalyzed by t-PA (tissue plasminogen activator) and u-PA (urokinase plasminogen activator). These activators recognize the N-terminal domain of plasminogen and cleave it into two fragments: a light chain and a heavy chain, forming active plasmin. α2-Plasmin inhibitor (α2PI) is a serine protease inhibitor belonging to the Kunitz inhibitor family. It consists of 206 amino acids and has a molecular weight of approximately 22 kDa. α2PI binds to the active site of plasmin via its N-terminal Kunitz domain, forming a 1:1 complex and inhibiting plasmin activity. This inhibitory effect is reversible, but under physiological conditions, the binding of α2PI to plasmin is very stable. PIC (Plasmin-α2-Plasmin Inhibitor Complex) is a complex formed by plasmin and α2PI. The formation of this complex involves a specific interaction between the active site of plasmin and the Kunitz domain of α2PI. The formation of PIC not only inhibits the activity of plasmin, but also changes the conformation of plasmin, making it unable to be further activated by t-PA. Structurally, PIC is composed of the heavy chain of plasmin and the N-terminal part of α2PI. The heavy chain of plasmin contains multiple functional domains, including a fibrin-binding domain (Fibronectin-like domain), a carboxyl-terminal domain (C-terminal domain), and an active site. The Kunitz domain of α2PI binds to the active site of plasmin through its conserved Arg-Ile-Gly sequence to form a stable complex.

[0003] In the human body, the dynamic equilibrium of PIC formation and dissociation regulates the activity of the fibrinolytic system, thereby maintaining the balance between hemostasis and fibrinolysis. Under normal physiological conditions, α2PI binds to plasmin, preventing its overactivation and thus excessive fibrin degradation. However, under pathological conditions, such as thrombotic and hemorrhagic diseases, the dynamic equilibrium of PIC formation and dissociation may be disrupted, leading to abnormal activation or inhibition of the fibrinolytic system.

[0004] In clinical practice, the detection of PIC has important diagnostic and therapeutic value. First, the level of PIC can be used as an important indicator to evaluate the activity of the fibrinolytic system. In thrombotic diseases, such as deep vein thrombosis and pulmonary embolism, the level of PIC may be elevated, reflecting the inhibitory state of the fibrinolytic system. In hemorrhagic diseases, such as urokinase-type plasminogen activator deficiency, the level of PIC may be decreased, reflecting the overactivation of the fibrinolytic system. In addition, the detection of PIC can also be used to evaluate the efficacy of antifibrinolytic therapy. For example, in thrombotic diseases, antifibrinolytic drugs (such as tranexamic acid) inhibit the activity of α2PI and increase the activity of plasmin, thereby promoting the degradation of fibrin. The detection of PIC can be used to evaluate the efficacy of these drugs and guide the formulation of individualized treatment plans.

[0005] Currently, the main methods for determining PIC include enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and Western blotting. ELISA is a commonly used quantitative detection method with high sensitivity and specificity. This method uses specific anti-PIC antibodies to detect PIC levels in plasma or serum. Immunoprecipitation uses anti-PIC antibodies to purify PIC complexes from plasma, followed by analysis by SDS-PAGE and Western blotting. These methods provide reliable technical means for the clinical detection of PIC.

[0006] PIC-related antibodies with high specificity and sensitivity play an important role in the diagnosis and detection of PIC, the treatment of diseases associated with imbalances in the fibrinolytic system, and the elucidation of the mechanism of action of PIC. This article focuses on the development path of PIC-related antibodies and the sequences of high-quality antibodies. Since PIC is a complex of plasmin and α-plasmin inhibitor, antibodies against PIC can be prepared directly. Alternatively, as in this patent, one antibody that recognizes plasmin and one that recognizes α-plasmin inhibitor can be prepared. Using a double antibody sandwich assay, PIC can be detected. Summary of the Invention

[0007] To address the problems of the prior art, the present invention provides monoclonal antibodies, kits, and applications for PIC detection. The antibodies of the present invention have high binding activity and affinity for plasmin and α2-plasmin inhibitor, enabling specific, sensitive, reliable, and accurate detection of trace amounts of PIC in samples through a double antibody sandwich approach. The present invention is specifically implemented through the following technical solutions:

[0008] The first object of the present invention is to provide high-quality monoclonal antibodies or conjugates thereof against plasmin and / or α2 plasmin inhibitors, wherein the antibodies or conjugates thereof:

[0009] The first antibody (anti-plasmin antibody) comprises: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID Nos: 1-3; LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID Nos: 4-6;

[0010] The second antibody (anti-α2 plasmin inhibitor antibody) comprises: HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID Nos: 11-13; LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID Nos: 14-16;

[0011] As used herein, "CDR," "CDRs," or "complementarity determining regions" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and are regions comprising one or more, or even all, of the primary amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0012] In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0013] Methods for defining CDRs are well known in the art and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but may still overlap with at least a portion of the Kabat-defined CDR regions, although they may be shortened or lengthened based on predictions or experimental results for specific residues or groups of residues.

[0014] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0015] In the present invention, the antibody conjugate is a conjugate of an antibody and a label or a solid phase carrier. The label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a nanoparticle label, and a radioactive label; and the solid phase carrier is selected from a microsphere, a plate, or a membrane.

[0016] In an alternative embodiment, the aforementioned marker refers to a substance having properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., which can be used to achieve qualitative or quantitative detection of the corresponding target. Examples include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0017] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0018] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0019] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0020] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0021] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0022] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0023] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0024] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0025] In an optional embodiment, the colloidal metal is colloidal gold.

[0026] In an optional embodiment, the above-mentioned antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

[0027] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0028] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0029] In the present invention, the antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

[0030] The amino acid sequence of the heavy chain variable region of the first antibody of the present invention is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0031] The amino acid sequence of the heavy chain variable region of the second antibody of the present invention is shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.18.

[0032] In the present invention, "framework region" or "FR region" includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein, the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0033] In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3

[0034] -LCDR3-LFR4.

[0035] In an alternative embodiment, the antibody further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0036] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies against plasmin and α2 plasmin inhibitors provided by the present invention may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework regions.

[0037] In an optional embodiment, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0038] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0039] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0040] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0041] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0042] In an alternative embodiment, the light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0043] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0044] In an alternative embodiment, the species origin of the constant region is mouse.

[0045] It should be noted that, in other embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region.

[0046] The heavy chain amino acid sequence of the first antibody in the embodiment of the present invention is shown in SEQ ID NO.9; the light chain amino acid sequence is shown in SEQ ID NO.10.

[0047] The heavy chain amino acid sequence of the second antibody in the embodiment of the present invention is shown in SEQ ID NO. 19; the light chain amino acid sequence is shown in SEQ ID NO. 20.

[0048] A second object of the present invention is to provide a reagent or kit for detecting plasmin-α2 plasmin inhibitor, plasmin, or α2 plasmin inhibitor, comprising the aforementioned first and second antibodies. Specifically, the first antibody can be used in a reagent or kit for detecting plasmin, or a reagent or kit for detecting plasmin-α2 plasmin inhibitor; the second antibody can be used in a reagent or kit for detecting α2 plasmin inhibitor, or a reagent or kit for detecting plasmin-α2 plasmin inhibitor.

[0049] Kits for certain detection methodologies include a capture reagent and a detection reagent, each of which includes a primary and secondary antibody or a conjugate thereof. The primary and secondary antibodies are paired and used interchangeably in the kit. That is, when the primary antibody is used as the capture reagent, the secondary antibody is used as the detection reagent, and vice versa.

[0050] For example, the above-mentioned kit can be used for immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, latex immunoturbidimetry, and other assays that utilize the specific binding properties of plasmin or α2 plasmin inhibitors and their antibodies. For example, in fluorescent immunochromatography, the antigen in the sample binds to a fluorescently labeled primary antibody on a conjugate pad. The solution undergoes chromatography due to the siphoning action of the absorbent pad, migrating toward the pad. When the complex moves to the detection line, it binds to the coated antibody on the T line, forming a "sandwich" complex that is enriched on the T line. In chemiluminescence, the antigen in the sample binds to the primary antibody coated on magnetic beads. After washing, it is then bound to an enzyme-labeled secondary antibody (typically horseradish peroxidase (HRP) or alkaline phosphatase (AP)) to form a "solid-phase antibody-antigen-enzyme-labeled antibody" sandwich complex. It should be understood that the first and second antibodies herein do not specifically refer to a specific sequence of the present invention. In theory, as long as the two antibodies forming the "double-antibody sandwich" immune complex bind to different epitopes of the antigen, the two antibodies in the capture and detection reagents are not specific and can be interchanged.

[0051] The third object of the present invention is to provide the use of the first and second antibodies or their conjugates in the preparation of products for detecting plasmin-α2 plasmin inhibitor, plasmin or α2 plasmin inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Clinical relevance of assigned samples DETAILED DESCRIPTION

[0053] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only. If specific conditions are not indicated in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are conventional products that can be purchased commercially.

[0055] Example 1 Preparation of plasmin monoclonal antibody

[0056] (1) Mouse immunization and antibody detection

[0057] Select 5 6-8 week old SPF grade female BALB / c mice, mix Freund's complete adjuvant and plasmin protein (biosynth) at a concentration of 1.2 mg / ml in equal volumes and emulsify. Immunize 6-8 week old SPF grade female BALB / c mice with the emulsified antigen, and inject 40 μg of antigen protein into each mouse by subcutaneous injection. Two weeks after the initial immunization, mix the antigen protein with Freund's complete adjuvant and emulsify it, and inject 15 μg of antigen protein into each mouse again by subcutaneous injection or back injection. Three weeks later, blood was collected through the tail vein, the supernatant was collected by centrifugation, and the serum titer was tested by ELISA. Immunize once every two weeks and test the serum titer. After 2 immunizations, the serum titer was as high as 1.5 or more after a million-fold dilution. Screening serum titer 10 6 Lymph was collected from the above mice to separate lymphocytes for cell fusion.

[0058] (3) Hybridoma antibody screening

[0059] The antibody screening protocol is key to the successful development of the antibodies of the present invention. During the antibody screening phase, an ELISA plate was first coated with plasmin. After the potential target antibody bound to plasmin, the plate was developed using goat anti-mouse HRP in an ELISA colorimetric solution to identify highly reactive hybridoma clones. Subsequently, the plate was coated with PIC protein (produced in-house) and developed using goat anti-mouse HRP in an ELISA colorimetric solution. Ten highly reactive and CRP-specific antibodies were identified, numbered P1-P10.

[0060] (4) Production and purification of monoclonal antibodies

[0061] Twenty 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL paraffin oil to suppress the immune response of the mice. One week after the injection, 0.5 ml of plasminogen activator 1-10 hybridoma cells were injected into the peritoneal cavity of each mouse (two per group). The number of cells was about 1×10 6 Two weeks later, ascites fluid was collected. The collected ascites fluid was precipitated with ammonium sulfate and affinity purified with protein G to obtain the target antibody.

[0062] Example 2 Preparation of Monoclonal Antibodies to α2 Plasmin Inhibitors

[0063] (1) Mouse immunization and antibody detection

[0064] Ten 6-8 week old SPF female BALB / c mice were selected, and Freund's complete adjuvant and α2 plasmin inhibitor (biosynth) at a concentration of 1 mg / ml were mixed and emulsified in equal volumes. 6-8 week old SPF female BALB / c mice were immunized with the emulsified antigen, and 20 μg of antigen protein was injected into each mouse through the foot. Two weeks after the initial immunization, the antigen protein was mixed and emulsified with Freund's complete adjuvant, and 20 μg of antigen protein was injected into each mouse through the foot or back subcutaneous injection. Two weeks later, blood was collected through the tail vein, the supernatant was collected by centrifugation, and the serum titer was tested by ELISA. Immunization was performed every two weeks and the serum titer was tested. After two immunizations, the serum titer was as high as 2.0 or more after a million-fold dilution. Screening serum titer 10 6 Lymph was collected from the above mice to separate lymphocytes for cell fusion.

[0065] (3) Hybridoma antibody screening

[0066] The antibody screening protocol is key to the successful development of the antibodies of the present invention. During the antibody screening phase, an ELISA plate was first coated with plasmin. After the potential target antibody binds to plasmin, color development was performed using goat anti-mouse HRP in an ELISA colorimetric solution. The plate was then coated with PIC protein (produced in-house), and color development was performed using goat anti-mouse HRP in an ELISA colorimetric solution. Highly reactive hybridoma clones were screened, resulting in 10 highly reactive and CRP-specific antibodies, numbered PI1-PI15.

[0067] (4) Production and purification of monoclonal antibodies

[0068] Twenty 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL paraffin oil to suppress the immune response of the mice. One week after the injection, 0.5 ml of plasminogen activator 1-10 hybridoma cells were injected into the peritoneal cavity of each mouse (two per group). The number of cells was about 1×10 6 Two weeks later, ascites fluid was collected. The collected ascites fluid was precipitated with ammonium sulfate and affinity purified with protein G to obtain the target antibody.

[0069] Example 3 Antibody Pairing Evaluation

[0070] The purified antibodies were labeled with HRP, and the P1-P10 and PI1-PI15 antibodies were used as coating antibodies (naked antibodies) and labeled antibodies (antibodies labeled with HRP), respectively. A double antibody sandwich method was used to identify the PIC protein, and antibodies that could be preliminarily used for pairing were screened. The evaluation data are as follows.

[0071] Table 1 Antibody pairing information

[0072]

[0073]

[0074] Table 1 Antibody pairing information (continued)

[0075] PI9 PI10 PI11 PI12 PI13 PI14 PI15 P1 0.0959 0.0471 0.0502 0.0524 1.8932 2.0683 0.2653 P2 0.1034 0.0468 0.0482 0.0754 0.0528 0.0514 0.0464 P3 0.3323 0.0478 0.0548 0.0505 0.0475 0.0548 0.0485 P4 0.4685 0.1377 0.0945 0.0623 0.048 0.0505 0.1556 P5 0.0624 0.0569 1.8243 0.0503 0.7597 0.0482 0.047 P6 0.2463 0.0476 0.1389 0.0588 0.0503 0.0538 0.0485 P7 0.0463 0.5323 0.1549 0.0536 0.0514 0.0475 0.0465 P8 0.1052 0.0742 0.0575 0.0727 0.0496 0.0823 0.3932 P9 1.6848 0.0771 0.0496 0.0485 0.0447 0.0459 0.0459 P10 0.0556 1.6656 0.0581 0.2031 0.0639 0.0486 0.0479

[0076] As can be seen from the above data, antibody pairs with paired antibody OD450 higher than 1.5 were selected, including 9 effective pairings: PI1-P6, PI4-P3, PI5-P8, PI7-P3, PI7-P9, PI10-P10, PI11-P5, PI13-P1, and PI14-P1, involving antibodies P1, P3, P5, P6, P8, P9 and P10 for plasmin, and antibodies PI1, PI4, PI5, PI7, PI10, PI11, PI13 and PI14 for α2 plasmin inhibitor.

[0077] Example 4 Antibody gene sequence cloning and performance testing

[0078] 1. Monoclonal Antibody Subtype Identification and Gene Sequence Cloning

[0079] Southern Biothech's SBA Clonotyping System-HRP kit was used to identify the heavy and light chain isotypes of the monoclonal antibodies P1, P3, P5, P6, P8, P9, and P10 against plasmin, and PI1, PI4, PI5, PI7, PI10, PI11, PI13, and PI14 against α2 plasmin inhibitors, according to the manufacturer's instructions. Specific procedures were as follows:

[0080] a. Dilute the capture antibody to 1 μg / mL using coating solution (0.05M carbonate and bicarbonate buffer, pH 9.5). Add 100 μL / well to the ELISA plate and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20 (plate wash buffer).

[0081] b. Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with diluent (1% BSA, 0.1% PBST), add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibody (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) 1:3000 with diluent;

[0082] After washing the plate three times with plate washer buffer, 100 μL of diluted enzyme-labeled antibody was added to each well and incubated at 37°C for 30 minutes. After washing the plate three more times, the color development solution was added. After about 5 minutes (depending on the strength of the reaction), 2M sulfuric acid was added to terminate the reaction and the OD450 absorbance was read. Based on the antibody subtype results, the antibody gene sequence was cloned using a RACE technology route. Hybridoma cells with good growth status were collected, and total RNA of the hybridoma cells was obtained using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the operating procedures of Takara's SMARTer RACE instructions, and the full-length sequence of the target antibody was amplified.

[0083] 2. Affinity Analysis

[0084] The ELISA method was used to draw a standard curve with the antibody concentration as the abscissa and the OD450 value of the antigen-antibody reaction as the ordinate. The affinity constant (KD represents the equilibrium dissociation constant, i.e., affinity) of the above-mentioned antibody was calculated based on the standard curve and the calculation formula (n[Ab1]-[Ab]) / (n-1). The test results are shown in Tables 2 and 3 below:

[0085] Table 2 Plasmin antibody affinity data

[0086] Antibody No. KD value P1 8.67E-9 P3 7.61E-10 P5 2.58E-9 P6 5.32E-10 P8 4.55E-9 P9 1.87E-10 P10 5.45E-9

[0087] Table 3 Affinity data of α2 plasmin inhibitor antibodies

[0088]

[0089]

[0090] The test results show that the affinity of plasmin antibodies P3, P6, and P9 to the antigen is better; the affinity of α2 plasmin inhibitor antibodies PI4, PI7, PI10, and PI11 to the antigen is better; combined with the pairing information, the three pairs of PI4-P3, PI7-P3, and PI7-P9 were finally selected for subsequent research.

[0091] 2. Activity Identification

[0092] PIC antigen was diluted to 1 μg / mL with 50 mM carbonate buffer for microplate coating, 100 μL per well, incubated at 4°C overnight; the next day, the plates were washed twice with PBST and patted dry; the above antibodies were added, starting from 1000 ng / mL and diluted 3-fold, and the samples were loaded at 100 μL / well and incubated at 37°C for 40 min; the plates were washed five times with PBST and patted dry; horseradish peroxidase-labeled goat anti-mouse IgG was added at 100 μL per well and incubated at 37°C for 30 min; the plates were washed five times with PBST and patted dry; carbamide peroxide (50 μL / well) and tetramethylbenzidine (50 μL / well) were added for 10 min; the reaction was terminated by adding dilute hydrochloric acid (50 μL / well); the OD values were read at 450 nm (reference 630 nm) on a microplate reader; the activity was determined as shown in Table 4 below:

[0093] Table 4 Activity data

[0094]

[0095] The test results show that the antibodies P3, P9, PI4, and PI7 provided by the present invention can still be detected even when the antigen concentration is as low as 1.5 ng / ml, indicating that the antibodies have high activity.

[0096] 3. Stability determination

[0097] The above antibodies were diluted in a predetermined buffer (PBS, 0.05% ProClin TM 300) were heat accelerated at 37°C for 14 days. The accelerated antibodies were evaluated by indirect ELISA, with comparisons to 4°C to determine the antibody's destructive stability. Additionally, five freeze-thaw cycles were performed at -20°C, with the results presented as the deviation between the 4°C and accelerated values. The measurement results are as follows (the detection method used in this experiment is consistent with that used in "Activity Assay").

[0098] Table 5 Stability study

[0099]

[0100]

[0101] The test results show that the antibodies PI4-P3 and PI7-P3 provided by the present invention have good storage stability and freeze-thaw stability.

[0102] 4. Functional testing

[0103] A. Biotinylated Antibodies

[0104] Weigh biotin (NHS-LC-LC-Biotin, from thermo scientific, 21343) and dissolve it in DMSO to 5.677 mg / mL. Take 0.3 mg of antibody (the volume of biotin solution required for every 1 mg of antibody is 3.3 μL). Add biotin to each of the six antibodies and mix well. Connect the antibody and biotin, and react at 25°C in the dark for 4 h.

[0105] B. Alkaline phosphatase labeled antibody

[0106] Dissolve 0.3 mg of antibody in TSE (pH 8.5) buffer. Dissolve 0.66 mg of ALP (Sigma, ALPI12G) in ALP dialysis buffer (pH 7.6) and mix thoroughly to determine the concentration. Dissolve Traut's Reagent 2-Iminothiolane HCl (2-IT, Thermo Scientific, 26101) in TSE (pH 8.5) to 13.76 mg / mL. Dissolve Sulfo-SMCC (Thermo Scientific, PG82085) in purified water to 3.7 mg / mL. Prepare both activator solutions immediately and use within 10 minutes. Calculate the volume of 2-IT solution required in step 1 based on a 5 μL volume per 1 mg of antibody. Add the 2-IT solution to the antibody, mix thoroughly, and allow to react at (25 ± 2)°C for 20 minutes. This is antibody activation. After activation, the activated antibody was exchanged into TSE (pH 7.3) solution and the antibody concentration was determined. Simultaneously, the required volume of Sulfo-SMCC solution was calculated based on a 10 μL volume of SMCC solution per 1 mg of ALP. The Sulfo-SMCC solution was added to the desalted ALP, mixed thoroughly, and allowed to react at (25 ± 2)°C for 20 minutes. After activation, the activated ALP was exchanged into TSMZ (pH 7.3) solution and the ALP concentration was determined. The desalted antibody was diluted to 0.3 mg / mL with TSE (pH 7.3) solution, and the desalted ALP was diluted to 0.4 mg / mL with TSMZ (pH 7.3) solution. The volumes of antibody and ALP used for conjugation were calculated based on a mass ratio of 1:0.91. The antibody and ALP were mixed according to the calculated volumes and allowed to react at 2-8°C for 12-20 hours. Dissolve maleimide in DMSO to 9.7 mg / mL and dilute 10-fold with TSMZ (pH 7.3) to prepare the stop solution. Add 20 μL of the required stop solution per 1 mL of antibody-ALP conjugate to terminate the reaction.

[0107] C. Performance Evaluation

[0108] (1) Linear experiment

[0109] Assay reagents, including R1, R2, and streptavidin magnetic bead solution:

[0110] The R1 includes: biotin-labeled antibody, used at a concentration of 1 μg / mL;

[0111] The R2 includes: alkaline phosphatase labeled antibody, with a concentration of 1 μg / mL.

[0112] The specific experimental steps are as follows:

[0113] a. Experimental preparation: 5 mL each of R1 and R2 working solutions, 4 mL of magnetic bead solution, and calibrators WRSA to WRS F.

[0114] b. Place each component into the kit, then place the kit into the fully automatic chemiluminescence immunoassay analyzer EXI1800 (from Zhongyuan Huiji Biotechnology Co., Ltd.) and select the anti-PIC project (30μL sample, 120μL R1, 30μL R2, 45μL magnetic bead solution) for the experiment. The evaluation data for PI4-P3 are as follows:

[0115] Table 6 Calibration experimental results

[0116]

[0117] From the above data, it can be seen that the pairing of the two antibodies has an obvious gradient within the conventional linear range of PIC.

[0118] (2) Clinical trials

[0119] The specific experimental steps are as follows:

[0120] (1) Experimental preparation: 20 PIC samples were taken out, thawed at room temperature, and then thoroughly mixed on a vortex mixer;

[0121] (2) Test: 20 clinical samples were tested, and the clinical results were as follows Figure 1 As shown:

[0122] Based on the above data, PI4-P3 is preferred for subsequent practical applications. This patent protects the first antibody (P3) and the second antibody (PI4).

[0123] All data, reagents and steps herein should be understood as illustrative and non-restrictive, although described the present invention in conjunction with above-mentioned specific embodiment, many modifications and other variations are apparent to those skilled in the art.All such modifications and other variations also fall within the scheme of the present invention and protection scope.

Claims

1. A monoclonal antibody against plasmin or a conjugate thereof, characterized in that: The monoclonal antibodies include: HCDR1 having an amino acid sequence as shown in SEQ ID No: 1; HCDR2 with the amino acid sequence shown in SEQ ID No: 2; HCDR3 with the amino acid sequence shown in SEQ ID No: 3; LCDR1 having an amino acid sequence as shown in SEQ ID No: 4; LCDR2 having an amino acid sequence as shown in SEQ ID No: 5; LCDR3 having an amino acid sequence as shown in SEQ ID No: 6; The conjugate is composed of the monoclonal antibody coupled with a label or a solid phase carrier.

2. The anti-plasmin monoclonal antibody or its conjugate according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

8.

3. A monoclonal antibody or a conjugate thereof against an α2 plasmin inhibitor, characterized in that: The monoclonal antibodies include: HCDR1 with the amino acid sequence shown in SEQ ID No: 11; HCDR2 with the amino acid sequence shown in SEQ ID No: 12; HCDR3 with the amino acid sequence shown in SEQ ID No: 13; LCDR1 having an amino acid sequence as shown in SEQ ID No: 14; LCDR2 having an amino acid sequence as shown in SEQ ID No: 15; LCDR3 having an amino acid sequence as shown in SEQ ID No: 16; The antibody conjugate is composed of the monoclonal antibody coupled with a label or a solid phase carrier.

4. The anti-α2 plasmin inhibitor monoclonal antibody or its conjugate according to claim 3, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

18.

5. The monoclonal antibody against plasmin or its conjugate according to any one of claims 1-2, or the monoclonal antibody against α2 plasmin inhibitor or its conjugate according to any one of claims 3-4, characterized in that: The monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

6. The monoclonal antibody against plasmin or its conjugate according to any one of claims 1-2, or the monoclonal antibody against α2 plasmin inhibitor or its conjugate according to any one of claims 3-4, characterized in that: The label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, nanoparticle labeling, and radioactive labeling; and the solid phase carrier is selected from microspheres, plates, or membranes.

7. A detection reagent or kit for plasmin or plasmin-α2 plasmin inhibitor complex, characterized in that: The reagent or kit comprises the monoclonal antibody or its conjugate according to claim 1 or 2.

8. A detection reagent or kit for an α2-plasmin inhibitor or a plasmin-α2-plasmin inhibitor complex, characterized in that: The reagent or kit comprises the monoclonal antibody or its conjugate according to claim 3 or 4.

9. A detection reagent or kit for plasmin-α2 plasmin inhibitor complex, characterized in that: The method comprises a capture reagent and a detection reagent, wherein each of the capture reagent and the detection reagent comprises an antibody with a different sequence, and the antibody is selected from one of the monoclonal antibodies according to claim 1 or 2, or one of the monoclonal antibodies according to claim 3 or 4.

10. Use of the monoclonal antibody or conjugate thereof according to claim 1 or 2, or the monoclonal antibody or conjugate thereof according to claim 3 or 4, in the preparation of a product for detecting a plasmin-α2 plasmin inhibitor complex.

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