Antibodies against PIC, kits, and methods for detecting PIC

Abstract

The present invention provides an anti-PIC antibody, a kit and a method for detecting PIC. The anti-PIC antibody comprises 6 CDRs, and the 6 CDRs are selected from (i) or (ii), wherein (i) is selected from the amino acid sequences shown in SEQ ID NOs: 1-6, and (ii) is selected from the amino acid sequences shown in SEQ ID NOs: 7-12. The anti-PIC antibody with 6 CDRs of the present application has higher affinity and specificity for the PIC antigen. Therefore, when using the antibody of the present application for sandwich immunoassay to detect a sample, one-step detection can be achieved, which not only has high specificity and accuracy, but also greatly shortens the detection time and improves the detection speed and efficiency.

Description

Technical Field

[0001] The present invention relates to the field of blood detection, and more particularly, to an antibody against PIC, a kit and a method for detecting PIC. Background Art

[0002] The process of fibrin formed during blood coagulation being decomposed and liquefied is called fibrinolysis (abbreviated as fibrinolysis). Fibrinolysis is an important anticoagulation process in the body. Like the blood coagulation process, it is also a protective physiological reaction of the body. The main function of the fibrinolysis system is to remove fibrin deposited on the blood vessel wall, dissolve blood clots and maintain blood flow. Among them, plasmin and α2-plasmin inhibitor (α2-AP) play an important role in the formation and dissolution of thrombus in the body.

[0003] Plasmin is transformed from plasminogen (Plg) under the action of plasminogen activator. It has similar antigenicity to plasminogen and belongs to serine protease. It can degrade cross-linked fibrinogen or fibrin to form fibrin degradation products, which is beneficial to the degradation of thrombus. α2-plasmin inhibitor is the most important plasmin-specific inhibitor in plasma. It can react with plasmin to form a covalent enzyme inhibitor, inactivate plasmin, thereby controlling the activation degree of the fibrinolysis system, regulating the balance of the coagulation and fibrinolysis systems, and is used to inhibit the excessive hydrolysis of fibrin and avoid excessive blood loss.

[0004] The half-life of plasmin in blood is extremely short (about 10 minutes), making it difficult to directly measure. α2-plasmin inhibitor can quickly neutralize plasmin, so that active plasmin cannot be detected in plasma. Plasmin-α2-plasmin inhibitor complex (PIC) is a complex formed by plasmin and α2-plasmin inhibitor (α2-AP) in a ratio of 1:1, that is, the product of the combination of fibrinolysis and antifibrinolysis substances in the body, and is a marker for detecting the activation of the fibrinolysis system, which helps to comprehensively understand the coagulation and fibrinolysis status of the body. The half-life of PIC in blood is relatively long, about 6 hours.

[0005] The concentrations of the precursor molecules plasminogen and α2-AP in normal plasma are about 100 times or more that of PIC. To avoid the interference of these precursor molecules, the specificity of the antibody is particularly important for the determination of PIC. In the prior art, the existing antibodies and detection methods for detecting PIC, such as the detection method disclosed in Monoclonal Antibodies to Discrete Regions in a2-PlasminInhibitor: connecting an anti-plasminogen antibody through a solid-phase carrier and peroxidase-labeling an anti-α2 plasmin antibody, and detecting in two steps.

[0006] In the above detection method, when using the existing pairing scheme of Plasmin antibody and anti-α2plasmin antibody for detection, the detection effect is not good: the blood concentration of plasminogen is about 0.2 mg / ml, and that of α2-plasmin inhibitor is about 0.07 mg / ml, which is more than 100 times that of PIC. Plasmin and plasminogen have similar antigenicity. When using Plasmin antibody as the capture antibody to capture the analyte PIC, it will be interfered by a large amount of plasminogen present in the plasma, resulting in a decrease in detection sensitivity. At the same time, the concentration of plasminogen fluctuates within a certain range among different individuals. When the Plasmin antibody scheme is used to detect the PIC levels of different individuals, fluctuations are likely to occur and the anti-interference ability is poor. Similarly, when using anti-α2plasmin antibody as the detection antibody, it will also be interfered by a large amount of α2-plasmin inhibitor present in the blood. To reduce the interference of α2-AP, step-by-step detection must be carried out. After the Plasmin antibody is incubated with the sample, the α2-AP in the sample is washed away, and then the anti-α2plasmin antibody labeled with a luminescent substance is added to complete the detection. Therefore, in the above technical solution, in addition to being interfered by the high-concentration precursor substances in the sample itself, reducing the detection sensitivity and accuracy, the step-by-step method must also be used, and the reaction time is long.

[0007] In addition, although there are also patent documents in the prior art that disclose the use of complex antibodies for detection, no relevant commercially available products have been found so far. Therefore, there is still a need to develop new detection products that can specifically detect PIC. Summary of the Invention

[0008] The main object of the present invention is to provide an antibody against PIC, a kit and a method for detecting PIC, so as to provide a PIC detection product with high specificity.

[0009] To achieve the above object, according to one aspect of the present invention, an antibody against PIC is provided. The antibody against PIC includes 6 CDRs, and the 6 CDRs are selected from (i) or (ii): wherein (i) CDR1-VL1 has the sequence shown in SEQ ID NO: 1: RASESVDSFGYSFX 1 H, where X 1 is M or L; CDR2-VL1 has the sequence shown in SEQ ID NO: 2: RASNLES; CDR3-VL1 has the sequence shown in SEQ ID NO: 3: QQTNEDPX 2 T, where X 2 is Y or F or W; CDR1-VH1 has the sequence shown in SEQ ID NO: 4: SSWMN; CDR2-VH1 has the sequence shown in SEQ ID NO: 5: RIYPGDGX 3TNYNGKFKG, where X 3 is D or H; and CDR3-VH1, having the sequence shown in SEQ ID NO: 6: G X 4 PTTAYAMDY, where X 4 is N, S or R; or (ii) CDR1-VL2, having the sequence shown in SEQ ID NO: 7: KASQDIKX 5 FLN, where X 5 is S or N; CDR2-VL2, having the sequence shown in SEQ ID NO: 8: YAX 6 SLAD, where X 6 is R or T; CDR3-VL2, having the sequence shown in SEQ ID NO: 9: LQHGESPFT; CDR1-VH2, having the sequence shown in SEQ ID NO: 10: SX 7 WMH, where X 7 is S or Y; CDR2-VH2, having the sequence shown in SEQ ID NO: 11: X 8 IDPYDSETHYNQKFKD, where X 8 is Y or R or N; and CDR3-VH2, having the sequence shown in SEQ ID NO: 12: GYDRSFDX9, where X9 is Y or F.

[0010] Furthermore, the anti-PIC antibody is selected from the following wild-type or any one of the following mutant combinations:

[0011]

[0012]

[0013] Furthermore, the anti-PIC antibody is selected from the following wild-type or any one of the following mutant combinations:

[0014]

[0015]

[0016] Furthermore, the anti-PIC antibody also includes the framework regions of the light chain variable region and the heavy chain variable region. Among them, the framework region of the light chain variable region includes FR1-VL1, FR2-VL1, FR3-VL1, and FR4-VL1, and the framework region of the heavy chain variable region includes FR1-VH1, FR2-VH1, FR3-VH1, and FR4-VH1. Alternatively, the framework region of the light chain variable region includes FR1-VL2, FR2-VL2, FR3-VL2, and FR4-VL2, and the framework region of the heavy chain variable region includes FR1-VH2, FR2-VH2, FR3-VH2, and FR4-VH2. Among them, FR1-VL1 has the amino acid sequence of SEQ ID NO: 13 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 13; FR2-VL1 has the amino acid sequence of SEQ ID NO: 14 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 14; FR3-VL1 has the amino acid sequence of SEQ ID NO: 15 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 15; FR4-VL1 has the amino acid sequence of SEQ ID NO: 16 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 16; FR1-VH1 has the amino acid sequence of SEQ ID NO: 17 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 17; FR2-VH1 has the amino acid sequence of SEQ ID NO: 18 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 18; FR3-VH1 has the amino acid sequence of SEQ ID NO: 19 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 19; FR4-VH1 has the amino acid sequence of SEQ ID NO: 20 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 20; FR1-VL2 has the amino acid sequence of SEQ ID NO: 21 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 21; FR2-VL2 has the amino acid sequence of SEQ ID NO: 22 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 22;FR3-VL2 has the amino acid sequence of SEQ ID NO: 23 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 23; FR4-VL2 has the amino acid sequence of SEQ ID NO: 24 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 24; FR1-VH2 has the amino acid sequence of SEQ ID NO: 25 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 25; FR2-VH2 has the amino acid sequence of SEQ ID NO: 26 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 26; FR3-VH2 has the amino acid sequence of SEQ ID NO: 27 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 27; FR4-VH2 has the amino acid sequence of SEQ ID NO: 28 or a sequence having a homology of more than 90%, preferably more than 95%, more preferably more than 99% with SEQ ID NO: 28; SEQ ID NO: 13: DIVLTQSPASLAVSLGQRATLSC; SEQ ID NO: 14: WYQQNPGQPPKLLIY; SEQ ID NO: 15: GIPARFSGSGSRTDFTLTINPVEADDVATYFC; SEQ ID NO: 16: FGGGAKLEIK; SEQ ID NO: 17: QVQLQQSGPELVKPGASVKLSCKASGNAFS; SEQ ID NO: 18: WVKQRPGQGLEWIG; SEQ ID NO: 19: KATLTVDKSSSTVYMQLSSLTSVDSAVYFCGR; SEQ ID NO: 20: WGQGTSVTVSS; SEQ ID NO: 21: DIKMTQSPSSMFASLGERVTITC; SEQ ID NO: 22: WYQQKPWRSPKTLIY; SEQ ID NO: 23: GVPSRFSGSGSGQDFSLTISSLESDDAATYYC; SEQ ID NO: 24: FGGGTKLEIK; SEQ ID NO: 25: QVQLQQPGAELVRPGASVKLSCKASGYTFT; SEQ ID NO: 26: WVKQRPEQGLEWIG; SEQ ID NO: 27: KAILTVDKSSSTAYMQLSSLTSEDSAVYYCAR; SEQ ID NO: 28: WGQGTTLTVSS;

[0017] Furthermore, the anti-PIC antibody has any one of the following light chain variable regions and heavy chain variable regions: (i) the light chain variable region VL1 shown in SEQ ID NO: 29 and the heavy chain variable region VH1 shown in SEQ ID NO: 30; SEQ ID NO: 29: DIVLT QSPASLAVSLGQRATLSC RASESVDSFGYSFMH WYQQNPGQPPKLLIY RASNLES GIPARFSGSGSRTDFTLTINP VEADDVATYFC QQTNEDPYT FGGGAKLEIK ; SEQ ID NO: 30: QVQLQQSGPELVKPGASVKLSCKASGNAFS SSWMN WVKQRPGQGLEWIG RIYPGDGDTNYNGKFKG KATLTVDKSSSTVYMQLSSLTSVDSAVYFCGR GNPTTAYAMDY WGQGTSVTVSS ; (ii) the light chain variable region VL2 shown in SEQ ID NO: 31 and the heavy chain variable region VH2 shown in SEQ ID NO: 32; SEQ ID NO: 31:

[0018] DIKMTQSPSSMFASLGERVTITC KASQDIKSFLN WYQQKPWRSPKTLIY YATSLAD GVPSRFSGSGSG QDFSLTISSLESDDAATYYC LQHGESPFT FGGGTKLEIK; SEQ ID NO: 32: QVQLQQPGAELVRPGASVKLSCK ASGYTFT SSWMH WVKQRPEQGLEWIG YIDPYDSETHYNQKFKD KAILTVDKSSSTAYMQLSSLTSEDSAVYYCAR GYDRSFDY WGQGTTLTVSS; (iii) a light chain variable region having a homology of 90% or more, preferably 95% or more, more preferably 99% or more with the sequence shown in SEQ ID NO: 29 and a heavy chain variable region having a homology of 90% or more, preferably 95% or more, more preferably 99% or more with the sequence shown in SEQ ID NO: 30; (iv) a light chain variable region having a homology of 90% or more, preferably 95% or more, more preferably 99% or more with the sequence shown in SEQ ID NO: 31 and a heavy chain variable region having a homology of 90% or more, preferably 95% or more, more preferably 99% or more with the sequence shown in SEQ ID NO: 32.

[0019] Further, the antibody is a full-length antibody, and the full-length antibody further includes a light chain constant region CL and a heavy chain constant region CH. Among them, the sequence of the light chain constant region CL is: 1) SEQ ID NO: 33; 2) a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with the sequence shown in SEQ ID NO: 33; the sequence of the heavy chain constant region CH is: 1) SEQ ID NO: 34; 2) a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with the sequence shown in SEQ ID NO: 34.

[0020] Further, the anti-PIC antibody has an affinity with PIC of 4.30*10 -11 ≤Kd(M)≤7.58*10 -9 ; preferably, the anti-PIC antibody is human, humanized or chimeric.

[0021] According to the second aspect of the present application, an isolated nucleic acid molecule is provided, and the isolated nucleic acid molecule encodes any one of the above anti-PIC antibodies.

[0022] According to the third aspect of the present application, an expression vector is provided, and the expression vector includes the above isolated nucleic acid molecule.

[0023] According to the fourth aspect of the present application, a host cell is provided, and the host cell is transfected with the above expression vector.

[0024] According to the fifth aspect of the present application, a PIC detection kit is provided, and the kit includes any one of the above anti-PIC antibodies.

[0025] Further, the kit includes two anti-PIC antibodies, one of which is an anti-PIC antibody coated on magnetic beads, and the other is a labeled anti-PIC antibody; preferably, the labeled anti-PIC antibody is an ABEI-labeled anti-PIC antibody; preferably, the kit further includes a calibrator and / or a quality control product.

[0026] According to the fifth aspect of the present application, a method for detecting PIC is provided, and the method includes: contacting a sample from a subject with any one of the above anti-PIC antibodies according to the one-step double antibody sandwich method to perform a binding reaction; detecting whether an immune complex is produced in the binding reaction, and if an immune complex is produced, it indicates the presence of PIC.

[0027] Further, the method is selected from any one or more of the following: fluorescence immunoassay, chemiluminescence immunoassay, colloidal gold immunoassay, radioimmunoassay or enzyme-linked immunoassay; preferably, the chemiluminescence immunoassay is a chemiluminescence immuno double antibody sandwich method; preferably, the method is analyzed using a semi-automatic immunoanalyzer or a fully automatic immunoanalyzer.

[0028] Furthermore, the sample is selected from at least one of whole blood, serum or plasma. Preferably, the whole blood, serum or plasma is derived from peripheral blood.

[0029] Applying the technical solution of the present invention, the anti-PIC antibody containing the CDRs of the present application has higher affinity and specificity for PIC. Therefore, when the anti-PIC antibody combination provided by the present application is applied to the sandwich ELISA for detecting PIC, it can detect whether PIC is contained in the sample to be tested through a one-step method. On the premise of high detection specificity and accuracy, it can also greatly improve the detection speed and efficiency. Detailed implementation manners

[0030] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0031] Term explanation:

[0032] Plg (Plasminogen);

[0033] Plm (Plasmin);

[0034] α2-AP (α2-antiplasmin);

[0035] PIC (plasmin-α2-plasmin inhibitor complex);

[0036] RNA (Ribonucleic Acid);

[0037] PCR (Polymerase Chain Reaction);

[0038] VL (variable region of light chain);

[0039] VH (variable region of heavy chain);

[0040] CLIA (Chemiluminescence Immunoassay);

[0041] BSA (Bovine Serum Albumin);

[0042] NaN 3 (Sodium azide);

[0043] ABEI [N-(4-aminobutyl)-N-ethylisoluminol];

[0044] TMB (Tetramethylbenzidine);

[0045] HRP (Horseradish Peroxidase);

[0046] ELISA (Enzyme-Linked Immunosorbent Assay);

[0047] RLU (Relative Light Unit).

[0048] As mentioned in the background art, although there have been some reports on specific antibodies against PIC in the prior art, there is still a market demand for more specific detection products. Therefore, in this application, total RNA was extracted from lymphocytes of PIC-immunized mice, VL and VH fragments were amplified separately by PCR, a vector was constructed to form a phage display library, and a novel site antibody with high affinity and high specificity against PIC was screened by using phage display technology.

[0049] Based on the above research results, in a typical embodiment of this application, an antibody against PIC is provided. The antibody against PIC comprises 6 CDRs, and these 6 CDRs are selected from (i) or (ii): wherein, (i) CDR1-VL1 has the sequence shown in SEQ ID NO: 1: RASESVDSFGYSFX 1 H, where X 1 is M or L; CDR2-VL1 has the sequence shown in SEQ ID NO: 2: RASNLES; CDR3-VL1 has the sequence shown in SEQ ID NO: 3: QQTNEDPX 2 T, where X 2 is Y or F or W; CDR1-VH1 has the sequence shown in SEQ ID NO: 4: SSWMN; CDR2-VH1 has the sequence shown in SEQ ID NO: 5: RIYPGDGX 3 TNYNGKFKG, where X 3 is D or H; and CDR3-VH1 has the sequence shown in SEQ ID NO: 6: GX 4 PTTAYAMDY, where X 4 is N, S or R; or (ii) CDR1-VL2 has the sequence shown in SEQ ID NO: 7: KASQDIKX 5 FLN, where X 5 is S or N; CDR2-VL2 has the sequence shown in SEQ ID NO: 8: YAX 6 SLAD, where X 6is R or T; CDR3-VL2 has the sequence shown in SEQ ID NO: 9: LQHGESPFT; CDR1-VH2 has the sequence shown in SEQ ID NO: 10: SX 7 WMH, where X 7 is S or Y; CDR2-VH2 has the sequence shown in SEQ ID NO: 11: X 8 IDPYDSETHYNQKFKD, where X 8 is Y or R or N; and CDR3-VH2 has the sequence shown in SEQ ID NO: 12: GYDRSFDX 9 , where X 9 is Y or F.

[0050] The anti-PIC antibody containing the above CDRs has higher affinity and specificity for PIC. Therefore, when the anti-PIC antibody combination provided in this application is applied to the sandwich ELISA for detecting PIC, it can detect whether PIC is contained in the sample to be tested through a one-step method. On the premise of high detection specificity and accuracy, it can also greatly improve the detection speed and efficiency.

[0051] Among the CDRs of the above antibodies, the amino acids at the X positions on different CDRs have the same or similar effects. Therefore, the amino acids at the X positions on different CDRs of different antibodies can be arbitrarily combined. For example, when X1 in CDR1-VL1 is M, X2 in CDR3-VL1 can be Y or F or W; similarly, when X1 in CDR1-VL1 is L, X2 in CDR3-VL1 can be Y or F or W. Therefore, a variety of different anti-PIC antibodies can be combined.

[0052] In some preferred embodiments, the anti-PIC antibody is selected from any one of the following table:

[0053]

[0054]

[0055] In some preferred embodiments, the anti-PIC antibody is selected from any one of the following table:

[0056]

[0057]

[0058] It should be noted that the above-mentioned antibodies of the present application include antibody fragments (including but not limited to Fab, Fab′, F(ab’)2, Fv, diabody, scFv, scFv-Fcs, single domain antibody, single-chain antibody, single heavy chain antibody and single light chain antibody) or full-length antibodies, provided that they exhibit the desired biological activity (such as epitope binding). They can be monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, chimeric antibodies, covalently modified antibodies or antibody conjugates (such as antibodies conjugated to detectable markers). Among them, Fab, scFv, Fv, whether monovalent or divalent, as long as they can contain the structures of VL and VH simultaneously, can achieve binding to antigen PIC. The difference between monovalent and divalent is only some differences in binding strength and stability.

[0059] In some preferred embodiments, the anti-PIC antibody further includes the framework regions of the light chain variable region and the heavy chain variable region. Among them, the framework region of the light chain variable region includes FR1-VL1, FR2-VL1, FR3-VL1, and FR4-VL1, and the framework region of the heavy chain variable region includes FR1-VH1, FR2-VH1, FR3-VH1, and FR4-VH1. Alternatively, the framework region of the light chain variable region includes FR1-VL2, FR2-VL2, FR3-VL2, and FR4-VL2, and the framework region of the heavy chain variable region includes FR1-VH2, FR2-VH2, FR3-VH2, and FR4-VH2. Among them, FR1-VL1 has the amino acid sequence of SEQ ID NO: 13 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 13; FR2-VL1 has the amino acid sequence of SEQ ID NO: 14 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 14; FR3-VL1 has the amino acid sequence of SEQ ID NO: 15 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 15; FR4-VL1 has the amino acid sequence of SEQ ID NO: 16 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 16; FR1-VH1 has the amino acid sequence of SEQ ID NO: 17 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 17; FR2-VH1 has the amino acid sequence of SEQ ID NO: 18 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 18; FR3-VH1 has the amino acid sequence of SEQ ID NO: 19 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 19; FR4-VH1 has the amino acid sequence of SEQ ID NO: 20 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 20; FR1-VL2 has the amino acid sequence of SEQ ID NO: 21 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 21; FR2-VL2 has the amino acid sequence of SEQ ID NO: 22 or a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 22;FR3-VL2 has the amino acid sequence of SEQ ID NO: 23 or a sequence with more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 23; FR4-VL2 has the amino acid sequence of SEQ ID NO: 24 or a sequence with more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 24; FR1-VH2 has the amino acid sequence of SEQ ID NO: 25 or a sequence with more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 25; FR2-VH2 has the amino acid sequence of SEQ ID NO: 26 or a sequence with more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 26; FR3-VH2 has the amino acid sequence of SEQ ID NO: 27 or a sequence with more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 27; FR4-VH2 has the amino acid sequence of SEQ ID NO: 28 or a sequence with more than 90%, preferably more than 95%, more preferably more than 99% homology with SEQ ID NO: 28.;

[0060] Among them, SEQ ID NO: 13: DIVLTQSPASLAVSLGQRATLSC; SEQ ID NO: 14: WYQQNPGQPPKLLIY; SEQ ID NO: 15: GIPARFSGSGSRTDFTLTINPVEADDVATYFC; SEQ ID NO: 16: FGGGAKLEIK; SEQ ID NO: 17: QVQLQQSGPELVKPGASVKLSCKASGNAFS; SEQ ID NO: 18: WVKQRPGQGLEWIG; SEQ ID NO: 19: KATLTVDKSSSTVYMQLSSLTSVDSAVYFCGR; SEQ ID NO: 20: WGQGTSVTVSS; SEQ ID NO: 21: DIKMTQSPSSMFASLGERVTITC; SEQ ID NO: 22: WYQQKPWRSPKTLIY; SEQ ID NO: 23: GVPSRFSGSGSGQDFSLTISSLESDDAATYYC; SEQ ID NO: 24: FGGGTKLEIK; SEQ ID NO: 25: QVQLQQPGAELVRPGASVKLSCKASGYTFT; SEQ ID NO: 26: WVKQRPEQGLEWIG; SEQ ID NO: 27: KAILTVDKSSSTAYMQLSSLTSEDSAVYYCAR; SEQ ID NO: 28: WGQGTTLTVSS.

[0061] In some other preferred embodiments, the anti-PIC antibody has any one of the following light chain variable regions and heavy chain variable regions: (i) the light chain variable region VL1 shown in SEQ ID NO: 29 and the heavy chain variable region VH1 shown in SEQ ID NO: 30; or (ii) the light chain variable region VL2 shown in SEQ ID NO: 31 and the heavy chain variable region VH2 shown in SEQ ID NO: 32; or (iii) a light chain variable region having more than 90%, preferably more than 95%, more preferably more than 99% homology with the sequence shown in SEQ ID NO: 29 and a heavy chain variable region having more than 90%, preferably more than 95%, more preferably more than 99% homology with the sequence shown in SEQ ID NO: 30; or (iv) a light chain variable region having more than 90%, preferably more than 95%, more preferably more than 99% homology with the sequence shown in SEQ ID NO: 31 and a heavy chain variable region having more than 90%, preferably more than 95%, more preferably more than 99% homology with the sequence shown in SEQ ID NO: 32.

[0062] SEQ ID NO: 29:

[0063] DIVLTQSPASLAVSLGQRATLSC RASESVDSFGYSFMH WYQQNPGQPPKLLIY RASNLES GIPARFSG SGSRTDFTLTINPVEADDVATYFC QQTNEDPYT FGGGAKLEIK ;

[0064] SEQ ID NO: 30:

[0065] QVQLQQSGPELVKPGASVKLSCKASGNAFS SSWMN WVKQRPGQGLEWIG RIYPGDGDTNYNGKFKG KA TLTVDKSSSTVYMQLSSLTSVDSAVYFCGR GNPTTAYAMDY WGQGTSVTVSS ;

[0066] SEQ ID NO: 31:

[0067] DIKMTQSPSSMFASLGERVTITC KASQDIKSFLN WYQQKPWRSPKTLIY YATSLAD GVPSRFSGSGSG QDFSLTISSLESDDAATYYC LQHGESPFT FGGGTKLEIK;

[0068] SEQ ID NO: 32:

[0069] QVQLQQPGAELVRPGASVKLSCKASGYTFT SSWMH WVKQRPEQGLEWIG YIDPYDSETHYNQKFKD KA ILTVDKSSSTAYMQLSSLTSEDSAVYYCAR GYDRSFDY WGQGTTLTVSS。

[0070] In some preferred embodiments, the affinity of the anti-PIC antibody for PIC is 4.30*10 -11 ≤Kd(M)≤7.58*10 -9 .

[0071] The antibodies in the present application can be derived from any species. In an exemplary embodiment, the antibody is a human antibody. In other embodiments, the antibody is a mouse antibody. The full-length antibodies in the present application can have a human Fc, i.e., IgG1, IgG2, IgG3 or IgG4, or can have a mouse Fc, i.e., IgG1, IgG2a, IgG2b or IgG3.

[0072] In a preferred embodiment, the anti-PIC antibody is a full-length antibody, further comprising a light chain constant region CL and a heavy chain constant region CH; more preferably, the light chain constant region CL is selected from C κ or C λ; Preferably, the heavy chain constant region CH is selected from the light chain constant regions of human or murine immunoglobulins; preferably, the human immunoglobulin is selected from any one of the following: IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD or IgE; preferably, the murine immunoglobulin is selected from any one of the following: IgGl, IgG2a, IgG2b or IgG3.

[0073] In a preferred embodiment, the anti-PIC antibody is a full-length antibody, and the anti-PIC antibody further includes a light chain constant region CL and a heavy chain constant region CH. Among them, the sequence of the light chain constant region CL is: (1) SEQ ID NO: 33; (2) a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with the sequence shown in SEQ ID NO: 33; the sequence of the heavy chain constant region CH is: (1) SEQ ID NO: 34; (2) a sequence having more than 90%, preferably more than 95%, more preferably more than 99% homology with the sequence shown in SEQ ID NO: 34.

[0074] Homology, in this application, refers to that a polynucleotide or polypeptide has a certain percentage of sequence homology with another polynucleotide or polypeptide, which means that when aligning, when comparing two sequences, the percentage of bases or amino acids is the same and at the same relative position. To determine sequence homology, various convenient methods and computer programs (such as BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) can be used to align the sequences, and the methods and computer programs can be obtained on websites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / .

[0075] In a preferred embodiment, the above anti-PIC antibody has an affinity with PIC of Kd(M) ≥ 4.30*10 -11 . The anti-PIC antibody formed by the sequence having the above homology, if its affinity with PIC satisfies the condition of Kd(M) ≤ 4.30*10 -11 , is also within the protection scope of this application.

[0076] The source of the above anti-PIC antibody is not particularly limited and can be human, humanized or chimeric. More preferably, it is humanized or human, which helps to improve specificity.

[0077] In the second typical embodiment of the present application, a nucleic acid molecule is provided, which encodes the above-mentioned anti-PIC antibody or a fragment of the antibody, such as a light chain or a heavy chain. A nucleic acid molecule combination is also provided, which includes a nucleic acid molecule encoding the light chain of the above-mentioned anti-PIC antibody and a nucleic acid molecule encoding the heavy chain of the above-mentioned anti-PIC antibody.

[0078] In the third typical embodiment of the present application, a recombinant expression vector is provided, and the recombinant plasmid includes the above-mentioned nucleic acid molecule or nucleic acid molecule combination.

[0079] In the fourth typical embodiment of the present application, a host cell is provided, and the host cell is transfected with the above-mentioned recombinant plasmid including the nucleic acid molecule combination.

[0080] In the fifth typical embodiment of the present application, a PIC detection kit is provided, and the kit includes the above-mentioned anti-PIC antibody.

[0081] In a preferred embodiment, the kit includes two anti-PIC antibodies, one of which is the anti-PIC antibody coated on magnetic beads and the other is the labeled anti-PIC antibody. Preferably, the labeled anti-PIC antibody is the ABEI-labeled anti-PIC antibody; more preferably, the kit further includes any one or more of a calibrator, a quality control product, an anti-human immunoglobulin antibody labeled with a luminescent marker, and a sample diluent; more preferably, the luminescent marker is luminol, isoluminol, isoluminol derivative, horseradish peroxidase or alkaline phosphatase, and the anti-human immunoglobulin antibody is one or more of anti-human IgA, anti-human IgM, anti-human IgG, anti-human LgD or anti-human LgE; more preferably, the reagent buffer is Tris-HCl buffer, phosphate buffer, carbonate buffer or borate buffer; more preferably, the sample diluent is phosphate buffer, Tris-HCl buffer, carbonate buffer, borate buffer or physiological saline.

[0082] In some preferred embodiments, the working concentrations of the anti-PIC antibody coated on magnetic beads and the ABEI-labeled anti-PIC antibody are the same, such as 1.1 ng / mL.

[0083] In some embodiments, the loading mass ratio of the anti-PIC antibody coated on magnetic beads to the labeled anti-PIC antibody is the same, such as 1:1.

[0084] In some embodiments, the antibody coated on magnetic beads can be in relative excess, and those skilled in the art can adjust it according to the actual situation. For example, the loading mass ratio of the anti-PIC antibody coated on magnetic beads to the labeled anti-PIC antibody is 1.1:1 - 20:1.

[0085] In the sixth typical embodiment of the present application, a method for detecting PIC is provided, which includes: contacting a sample from a subject with the above-mentioned anti-PIC antibody according to the one-step double-antibody sandwich method to carry out a binding reaction; detecting whether an immune complex is produced in the binding reaction, and if an immune complex is produced, it indicates the presence of PIC.

[0086] The antibodies of the present application have higher affinity and specificity. Therefore, when using the antibody combination of the present application to detect a sample by the double-antibody sandwich method, only the one-step method is required for detection to achieve the detection of PIC antigen. Compared with the two-step detection method in the prior art, on the basis of high detection specificity and accuracy, the detection time is greatly shortened, and the detection speed and efficiency are improved.

[0087] In a preferred embodiment, the above method is selected from any one or more of the following: fluorescence immunoassay, chemiluminescence immunoassay, colloidal gold immunoassay, radioimmunoassay or enzyme-linked immunoassay; preferably, the chemiluminescence immunoassay is chemiluminescence immunoassay double-antibody sandwich method; preferably, the method is analyzed using a semi-automatic immunoanalyzer or a fully automatic immunoanalyzer.

[0088] In a preferred embodiment, the sample is selected from at least one of whole blood, serum or plasma, wherein the whole blood, serum or plasma is derived from peripheral blood.

[0089] The solution of the present application will be further described below with specific examples.

[0090] Example 1 Antibody Screening

[0091] The antibody screening in the following examples was carried out by hybridoma fusion technology. The principle of hybridoma fusion technology is to fuse the lymph B cells of the immunized mouse spleen with the myeloma cells of the mouse, so that the lymph B cells obtain the ability of infinite proliferation. By detecting the secreted antibodies in the cell supernatant by ELISA, the cell wells expressing specific antibodies are screened, and further subcloned to obtain monoclonal antibodies. After a complete in vivo immunization process, the animal body can produce high-affinity antibodies, combined with electrofusion to ensure a high fusion rate and a large number of fusions, and excellent target antibodies can be screened.

[0092] The specific steps of this example are as follows:

[0093] 1. Immunize mice

[0094] Purchase plasmin (Meridian, A50192H) and anti-α2plasmin (Meridian, A50112H), synthesize PIC as an immunogen. Take 6-8-week-old SPF-grade Balb / c mice, fully emulsify the antigen containing 200 μg / 500 μl of PIC with an equal volume of Freund's complete adjuvant, and inject subcutaneously at multiple points. After 3 weeks, emulsify the antigen with Freund's incomplete adjuvant and inject subcutaneously at multiple points. Strengthen the immunization subcutaneously 2 times, and the last injection is intraperitoneal injection for boosting.

[0095] 2. Cell fusion and subclonal screening

[0096] Take the spleen of the immunized mice, grind and separate to obtain dispersed single spleen cells. Mix the spleen cells and myeloma cells using PEG. After the medium is terminated, centrifuge and resuspend, then spread on a 96-well plate. Change the medium after one week, take the supernatant and detect it by indirect ELISA. The coated antigens are Plg / α2-AP / PIC. Select the high-value positive wells respectively targeting the three coated antigens, and further perform subcloning by the limiting dilution method. After culturing for one week, continue to detect by indirect ELISA, select the high-value wells for limiting dilution, and repeat 3-4 times until all the detection wells are positive and the cells in the wells are single colonies, then select the cell well with the highest OD value for expansion culture to obtain a series of specific hybridoma cell lines, including the hybridoma cell line 1C2B4 (denoted as WT1) and 5B3F6 (denoted as WT2) targeting PIC.

[0097] 3. Preparation of ascites from positive cell lines

[0098] Expand the culture of single colony cells, inject them into mice pre-inoculated with IFA to prepare ascites, collect the ascites to obtain antibodies against different epitopes, and purify them by the SPA affinity purification method.

[0099] Example 2 Construction of expression plasmid

[0100] 1. Gene retrieval

[0101] Amplify and culture the 1C2B4 and 5B3F6 hybridoma cell lines, extract mRNA, obtain cDNA products by reverse transcription. After adding A to the products with rTaq DNA polymerase, insert them into the pMD-18T vector, and transform them into DH5α competent cells. Take 10 plaques each for the Heavy chain and Light chain gene clones and send them to a gene sequencing company for sequencing.

[0102] 2. Sequence analysis of antibody variable region genes

[0103] Analyze the gene sequences obtained from the above sequencing in the IMGT antibody database and use VNTI11.5 software for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs are correct.

[0104] The sequencing results are as follows:

[0105] Nucleotide sequence:

[0106] SEQ ID NO: 41 (VL1)

[0107] gacattgtgctgacccaatctccagccagcctggccgtgagcctgggccagagggccaccctgagctgcagggccagcgagagcgtggacagcttcggctacagcttcatgcactggtaccagcagaaccccggccagccccccaagctgctgatctacagggccagcaacctggagagcggcatccccgccaggttcagcggcagcggcagcaggaccgacttcaccctgaccatcaaccccgtggaggccgacgacgtggccacctacttctgccagcagaccaacgaggacccctacaccttcggcggcggcgccaagctggagatcaag。

[0108] SEQ ID NO: 42 (VH1)

[0109] caggttcagctgcagcagtctggccccgagctggtgaagcccggcgccagcgtgaagctgagctgcaaggccagcggcaacgccttcagcagcagctggatgaactgggtgaagcagaggcccggccagggcctggagtggatcggcaggatctaccccggcgacggcgacaccaactacaacggcaagttcaagggcaaggccaccctgaccgtggacaagagcagcagcaccgtgtacatgcagctgagcagcctgaccagcgtggacagcgccgtgtacttctgcggcaggggcaaccccaccaccgcctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc。

[0110] SEQ ID NO: 43 (VL2)

[0111] gacatcaagatgacccagtctccatctagcatgttcgccagcctgggcgagagggtgaccatcacctgcaaggccagccaggacatcaagagcttcctgaactggtaccagcagaagccctggaggagccccaagaccctgatctactacgccaccagcctggccgacggcgtgcccagcaggttcagcggcagcggcagcggccaggacttcagcctgaccatcagcagcctggagagcgacgacgccgccacctactactgcctgcagcacggcgagagccccttcaccttcggcggcggcaccaagctggagatcaag。

[0112] SEQ ID NO: 44 (VH2)

[0113] caggtccaactgcagcagcctggcgccgagctggtgaggcccggcgccagcgtgaagctgagctgcaaggccagcggctacaccttcaccagcagctggatgcactgggtgaagcagaggcccgagcagggcctggagtggatcggctacatcgacccctacgacagcgagacccactacaaccagaagttcaaggacaaggccatcctgaccgtggacaagagcagcagcaccgcctacatgcagctgagcagcctgaccagcgaggacagcgccgtgtactactgcgccaggggctacgacaggagcttcgactactggggccagggcaccaccctgaccgtgagcagc。

[0114] The corresponding amino acid sequence is as follows:

[0115] SEQ ID NO: 29: (VL1)

[0116] DIVLTQSPASLAVSLGQRATLSC RASESVDSFGYSFMH WYQQNPGQPPKLLIY RASNLES GIPARFSG SGSRTDFTLTINPVEADDVATYFC QQTNEDPYT FGGGAKLEIK 。

[0117] SEQ ID NO: 30: (VH1)

[0118] QVQLQQSGPELVKPGASVKLSCKASGNAFS SSWMN WVKQRPGQGLEWIG RIYPGDGDTNYNGKFKG KA TLTVDKSSSTVYMQLSSLTSVDSAVYFCGR GNPTTAYAMDY WGQGTSVTVSS 。

[0119] SEQ ID NO: 31: (VL2)

[0120] DIKMTQSPSSMFASLGERVTITC KASQDIKSFLN WYQQKPWRSPKTLIY YATSLAD GVPSRFSGSGSG QDFSLTISSLESDDAATYYC LQHGESPFT FGGGTKLEIK。

[0121] SEQ ID NO: 32: (VH2)

[0122] QVQLQQPGAELVRPGASVKLSCKASGYTFT SSWMH WVKQRPEQGLEWIG YIDPYDSETHYNQKFKD KA ILTVDKSSSTAYMQLSSLTSEDSAVYYCAR GYDRSFDY WGQGTTLTVSS。

[0123] SEQ ID NO: 33: (CL - constant region of light chain)

[0124] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0125] SEQ ID NO: 34: (CH - constant region of heavy chain)

[0126] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

[0127] 3. Construction of Recombinant Antibody Expression Plasmid

[0128] Using pFastBac TM dual as the vector to construct a recombinant antibody expression vector. This expression vector has introduced multiple cloning enzyme digestion sites such as BamHI / SalI / XhoI / KpnI, and is simply referred to as the pFD vector. According to the antibody variable region gene sequencing results in the above pMD-18T, VL and VH gene-specific primers of the primers were designed (see Table 1). Both segments carry corresponding enzyme digestion sites and protection bases, and a 0.73 kb Light chain gene fragment and a 1.4 kb Heavy chain gene fragment were amplified by PCR amplification method.

[0129] First, Heavy chain and pFastBacTMdual were digested with BamHI / SalI double enzymes. After recovering the target fragments and vectors and purifying them, they were ligated and transformed. After verification, the vector ligated with Heavy chain was extracted, simply referred to as pFD-H. Then, this vector and the Light chain gene fragment were digested with XhoI / KpnI double enzymes. After recovering the target fragments and vectors and purifying them, they were ligated and transformed. After verification, the vector ligated with Heavy and light chain was extracted, simply referred to as pFD-HL. The recombinant plasmid was transformed into E. coli DH10Bac (containing Bacmid of AcNPV and helper plasmid) competent cells, and spread on an LB agar plate containing kanamycin (50 μg / mL), tetracycline (10 μg / mL), gentamicin (7 μg / mL), IPTG (40 μg / mL), and X-gal (100 μg / mL). White colonies were picked by blue-white screening, and the recombinant bacmid was extracted using the Omega BAC / PAC extraction kit.

[0130] Table 1:

[0131] Primer Number Base Sequence F-H1 5’-gtacGGATCCcaggttcagctgcagcagtct-3’(SEQ ID NO:35) F-L1 5’-gtacCTCGAGgacattgtgctgacccaatctcca-3’(SEQ ID NO:36) F-H2 5’-gtacGGATCCcaggtccaactgcagcagcct-3’(SEQ ID NO:37) F-L2 5’-gtacCTCGAGgacatcaagatgacccagtctccat-3’(SEQ ID NO:38) R-H 5’-gtacGTCGACtttaccaggagagtgggagaggc-3’(SEQ ID NO:39) R-L 5’-gtacGGTACCacactcattcctgttgaagctcttg-3’(SEQ ID NO:40)

[0132] Example 3 Recombinant Antibody Expression

[0133] Transfect the recombinant bacmid extracted in Example 2 into Sf9 cells according to the instruction manual of Gibco liposome transfection, with a passage density of 1*10^6 / ml, culture at 28 °C for 6 days. Under light-shielded conditions, filter the obtained supernatant through a 0.22 μm filter, and the collected supernatant is the first-generation (P1) virus solution. Infect Sf9 cells with the P1 virus again to obtain a high-titer recombinant virus solution, and thus obtain the P2 / P3 virus solution. Finally, transfer the P3-generation virus solution into HF cells, culture at 28 °C for 3 days, and collect the supernatant.

[0134] Purify the above expression supernatant using an SPA column according to the operation instructions of the GE AKTA Pure protein separation and purification system to obtain the purified recombinant antibody.

[0135] Example 4 Verification of Antibody Specificity

[0136] Detect the specificity of the antibody by ELISA method, and the results are shown in the following table. In the following table, "+" indicates that the antibody has a specific reaction with the antigen, and "-" indicates that the antibody does not react with the antigen.

[0137] Table 2 Specificity Detection of Different Antibodies

[0138]

[0139]

[0140] Source of Plg antibody: meridian, H86431M;;

[0141] Source of AP antibody: meridian, H63710M;

[0142] (The sources of Plg antibody and AP antibody involved below are the same as this.)

[0143] Example 5 Verification of Antibody Binding Affinity

[0144] The BIAcore technology was used to detect the binding affinity of 2 strains of antibodies and commercially purchased Plg and α2-AP antibodies (as shown in Table 3) with the PIC antigen. The experimental procedure was carried out according to the instructions of BiacoreR 3000 and Biacore AB. The specific affinity F(ab') fragment of goat anti-mouse IgG Fc region (Jackson ImmunoResearch) was immobilized on the CM5 sensor chip through amine coupling chemistry. HBS-EP was selected as the flowing buffer with a flow rate of 10 μL / min. After injecting 10 μL of the antibody (4 μg / mL), different concentrations of PIC antigen were titrated at a flow rate of 30 μL / min. The antibody affinity was measured at four gradients of PIC antigen concentrations: 2.1 nM, 4.2 nM, 8.4 nM, and 16.8 nM. The equilibrium dissociation constant Kd (M) represents the dissociation degree of antigen-antibody at the equilibrium state. The smaller the Kd (M), the stronger the antigen-antibody affinity.

[0145] Table 3 Equilibrium dissociation constant Kd (M) of different antibodies against PIC complex

[0146] <![CDATA[Ka(M -1 s -1 )]]> <![CDATA[Kd(s -1 )]]> Kd(M) Plg Antibody <![CDATA[4.69*10 5 > <![CDATA[6.85*10 -5 > <![CDATA[1.46*10 -10 > α2-AP Antibody <![CDATA[4.24*10 5 > <![CDATA[6.42*10 -5 > <![CDATA[1.51*10 -10 > PIC Antibody (WT1) <![CDATA[6.59*10 4 > <![CDATA[8.54*10 -5 > <![CDATA[1.29*10 -9 > PIC Antibody (WT2) <![CDATA[7.89*10 4 > <![CDATA[9.54*10 -5 > <![CDATA[1.21*10 -9 >

[0147] Example 6 Verification of anti-interference of self-produced antibodies against precursor molecules plasminogen and α2-plasmin inhibitor

[0148] Detection principle: The chemiluminescence immunological double-antibody sandwich method was adopted. One strain of monoclonal antibody was coated on magnetic beads, and another strain of monoclonal antibody was labeled with ABEI. The ratio of coated antibody to labeled antibody was 10:1. The sample, magnetic microspheres, and ABEI were added to the reaction cup in sequence and incubated at 37 °C for 10 min. After precipitation by an external magnetic field, the supernatant was removed, and the precipitated complex was washed 3 times with the washing solution and directly entered the sample measurement chamber. The instrument automatically pumped in luminescent substrates 1 and 2 and automatically monitored the relative light intensity (RLU) emitted within 3 s. (The principle of the kits used in Examples 7-11 is the same, only the antibodies are replaced)

[0149] A PIC zero-concentration plasma basic sample was selected; by adding PIC enterprise calibrator (PIC concentration was 40 μg / mL, 10 mmol / L pH 7.4, PBS buffer solution) to the zero-concentration basic sample, basic samples with theoretical concentrations of 0.200 μg / mL, 1.00 μg / mL, and 20.0 μg / mL were prepared, with 6 mL of each sample.

[0150] Preparation of plasminogen stock solution: 30 mg of plasminogen was added to 1 mL of 10 mmol / L pH 7.4, PBS buffer solution;

[0151] Preparation of α2-plasmin inhibitor stock solution: 10 mg of human plasminogen was added to 1 mL of 10 mmol / L pH 7.4, PBS buffer solution;

[0152] Preparation of interference samples: Divide the three-level basic sample into 4 parts, and add the corresponding volume of the interference substance stock solution (referring to the above mother liquor) and the solvent for preparing the interference substance stock solution (i.e., 10 mmol / L pH 7.4, PBS buffer solution) to 3 of them to obtain 3 analytical samples (C1, C2, C3) with different interference substance concentration levels. The sample without any addition is denoted as C0. The added volumes of the interference substance stock solution, solvent, and basic sample are shown in the following table:

[0153] Table 4:

[0154]

[0155] Calculate the interference rate according to formulas (14) and (15):

[0156]

[0157]

[0158] Note: *Concentration restored to the mean value of the control group sample results

[0159] ① When preparing the sample for interference evaluation at a ratio of 1:99, the concentration restored to the mean value of the control group sample results is equal to the mean value of the determination results of the control group sample divided by 0.99;

[0160] ② When preparing the sample for interference evaluation at a ratio of 1:9, the concentration restored to the mean value of the control group sample results is equal to the mean value of the determination results of the control group sample divided by 0.90.

[0161] If the absolute value of the interference rate ≤ 10%, it indicates that there is no obvious interference to the test result; if the absolute value of the interference rate > 10%, it indicates that there is interference to the test result.

[0162] Table 5 Anti-interference verification of different antibody combination schemes for precursor molecules

[0163]

[0164]

[0165] Result analysis: By adding different concentrations of precursor molecules (plasminogen and α2-plasmin inhibitor) to the zero-concentration diluent, the anti-interference ability of six combination schemes for precursor molecules was verified. It was found that the detection scheme composed of two monoclonal antibodies against the PIC complex had the strongest anti-interference ability and the best effect on plasminogen and α2-plasmin inhibitor, and was hardly interfered.

[0166] Example 7. Influence of different antibody combination schemes on the accuracy of samples near the cut-off value

[0167] Select the PIC zero-concentration plasma basic sample, and configure 5 PIC plasma samples A1 - A5 with the final concentration near the cut-off value (0.8 μg / mL) by adding the PIC complex (PIC concentration is 40 μg / mL, 10 mmol / L pH 7.4, PBS buffer). Configure the kit with different self-produced antibody combination schemes (the antibody combination method is the same as that set in Example 6), detect the PIC concentration in each sample respectively, calculate the average concentration, and judge the positive and negative.

[0168] Table 6 Influence of different antibody combination schemes on the accuracy of samples near the cut-off value

[0169]

[0170] Result analysis: By comparing the six combination schemes in parallel, it is found that the precursor molecules plasminogen and α2-plasmin inhibitor present in the plasma sample will interfere with the detection scheme composed of Plg antibody or α2-AP antibody, resulting in low accuracy of sample detection. The higher the concentration of the precursor molecule, the greater the interference, which will seriously affect the accuracy of samples near the cut-off value and lead to misjudgment of positive and negative.

[0171] Example 8. Influence of different antibody combination schemes on the sensitivity of low-value samples

[0172] Select the PIC zero-concentration plasma basic sample, and configure samples with theoretical concentrations of 0.025 μg / mL, 0.050 μg / mL, 0.100 μg / mL, 0.200 μg / mL, 0.400 μg / mL, and 0.800 μg / mL by adding the PIC enterprise calibration product (PIC concentration is 40 μg / mL, 10 mmol / L pH 7.4, PBS buffer) to the zero-concentration sample to obtain 6 low-level samples. Configure the kit with different self-produced antibody combination schemes (the antibody combination method is the same as that set in Example 6), and detect the 6 low-level samples respectively. ) ,Configure samples with theoretical concentrations of 0.025 μg / mL, 0.050 μg / mL, 0.100 μg / mL, 0.200 μg / mL, 0.400 μg / mL, and 0.800 μg / mL to obtain 6 low-level samples. Configure the kit with different self-produced antibody combination schemes (the antibody combination method is the same as that set in Example 6), and detect the 6 low-level samples respectively.

[0173] Table 7 Verification of the influence of different antibody combination schemes on the sensitivity of low-value samples

[0174]

[0175] Result analysis: By comparing the six combination schemes in parallel, it is found that only the scheme of PIC monoclonal antibody (WT1) combined with PIC monoclonal antibody (WT2) can best distinguish from the zero-concentration standard at 0.025 μg / mL, and the larger the ratio of sample 6 to the zero-concentration calibration product (indicating better discrimination of low values and higher sensitivity of low values); while other schemes cannot meet the requirements. It is analyzed that it is interfered by the high concentration of plasminogen and α2-plasmin inhibitor present in the plasma sample, resulting in insufficient sensitivity of the detected low-value samples.

[0176] Example 9, Linear Range

[0177] Select a PIC zero-concentration plasma basic sample and prepare samples within the linear range with different theoretical concentrations, which are 0.100 μg / mL, 0.500 μg / mL, 1.000 μg / mL, 10.0 μg / mL, 20.0 μg / mL, and 40.0 μg / mL respectively. Also, detect the diluent sample. Detect the samples using each matching scheme (the antibody matching method is the same as that set in Example 6). It can be found that the matching scheme of the PIC antibody has better sample discrimination, stronger anti-interference ability, and better linear correlation coefficient.

[0178] Table 8 Comparison of Linear Ranges of Different Antibody Matching Schemes

[0179]

[0180] Example 10: HOOK Effect

[0181] Select a PIC zero-concentration plasma basic sample and prepare high-concentration PIC samples using a PIC enterprise calibrator (PIC concentration is 400 μg / mL, PBS buffer with 10 mmol / L and pH 7.4). The theoretical concentrations are 40 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL respectively. Then, detect using the pairing scheme of the Plg antibody and α2-AP antibody purchased in the market. An obvious HOOK effect appears above 100 μg / mL, while the PIC antibody pairing scheme of this application does not show an obvious HOOK effect within the concentration range of 400 μg / mL; this shows that the pairing scheme for detecting the PIC complex antibody has better detection effect.

[0182] Table 9 Comparison of HOOK Effects of Different Antibody Matching Schemes (the antibody matching method is the same as that set in Example 6)

[0183]

[0184] Example 11, Comparison between the Self-produced PIC Antibody Matching Scheme and a Commercial Scheme

[0185] Compare the performance of the self-produced PIC antibody (WT1, labeled antibody) and PIC antibody (WT2, coated antibody) matching scheme with a commercial product. The results are shown in Table 10. Compared with a commercial kit, the one-step method used in this application can save consumables, has higher detection efficiency, the time for the first result is 15 min, the detection time is shorter, and the result is obtained faster. In addition, the LoB of this application can reach 0.03 μg / mL, and the precision is less than 5%, and the reagent performance is better.

[0186] Table 10 Comparison Results between the New Industry and Sysmex Commercial Reagents

[0187]

[0188] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The high-affinity PIC new antibody prepared by the phage display technology of the present invention specifically recognizes the PIC complex with high affinity and does not cross-react with plasminogen, plasmin, and α2-plasmin inhibitor, solving the interference of the large amount of precursor molecules plasminogen and α2-plasmin inhibitor present in the blood, and improving the anti-interference ability, sensitivity, and accuracy of the detection. At the same time, the present invention uses a one-step method to complete the detection, which can significantly improve the inspection efficiency.

[0189] Example 12 PIC Antibody Mutants

[0190] Based on the above new anti-PIC antibodies of the present application, in order to further improve their affinity with PIC, the applicant further mutated the light chain CDR and heavy chain CDR of the above two wild-type antibodies. And the specificity of the antibodies obtained after various mutations to the PIC complex and the antibody affinity were detected. The mutant antibodies with specific mutation site combinations are shown in Tables 11 and 12 below.

[0191] Table 11: Mutation Sites Related to the Affinity of Anti-PIC Antibody (WT1)

[0192]

[0193]

[0194] Table 12: Mutation Sites Related to the Affinity of Anti-PIC Antibody (WT2)

[0195]

[0196]

[0197] The specificity of the above mutant strains was detected by ELISA, and it was found that the above mutant strains could specifically react with the PIC complex and did not react with free Plg and α2-AP.

[0198] Table 13:

[0199]

[0200]

[0201]

[0202] The affinity of the above mutant antibodies was detected:

[0203] The binding affinity of the antibody and mutant antibody in Example 1 with the PIC antigen was detected using BIAcore technology. The experimental procedure was carried out according to the instructions of BiacoreR 3000, Biacore AB. The specific affinity F(ab') fragment of goat anti-mouse IgG Fc region (Jackson ImmunoResearch) was immobilized on the CM5 sensor chip through amine coupling chemistry. HBS-EP was selected as the flowing buffer with a flow rate of 10 μL / min. After injecting 10 μL of the self-produced antibody (4 μg / mL), different concentrations of PIC antigen were titrated at a flow rate of 30 μL / min. The antibody affinity was measured at four gradients of PIC antigen concentrations: 2.1 nM, 4.2 nM, 8.4 nM, and 16.8 nM. The equilibrium dissociation constant Kd (M) represents the dissociation degree of the antigen-antibody at the equilibrium state. The smaller the Kd (M), the stronger the antigen-antibody affinity.

[0204] Table 14:

[0205]

[0206]

[0207]

[0208] From the detection results of the affinity between the above mutant antibodies and the PIC antigen, it can be seen that these mutant antibodies also all have relatively high affinity.

[0209] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Sequence Listing <110> Shenzhen New Industries Biomedical Engineering Co., Ltd. <120> Antibody Against PIC, Kit and Method for Detecting PIC <130> PN163100SZSW <160> 44 <170> SIPOSequenceListing 1.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (14)..(14) <223> Xaa is M or L <400> 1 Arg Ala Ser Glu Ser Val Asp Ser Phe Gly Tyr Ser Phe Xaa His 1 5 10 15 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (1)..(7) <223> CDR2-VL1 <400> 2 Arg Ala Ser Asn Leu Glu Ser 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (8)..(8) <223> Xaa is Y or F or W <400> 3 Gln Gln Thr Asn Glu Asp Pro Xaa Thr 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (1)..(5) <223> CDR1-VH1 <400> 4 Ser Ser Trp Met Asn 1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (8)..(8) <223> Xaa is D or H <400> 5 Arg Ile Tyr Pro Gly Asp Gly Xaa Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15 Gly <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (2)..(2) <223> Xaa is N, S or R; <400> 6 Gly Xaa Pro Thr Thr Ala Tyr Ala Met Asp Tyr 1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (8)..(8) <223> Xaa is S or N <400> 7 Lys Ala Ser Gln Asp Ile Lys Xaa Phe Leu Asn 1 5 10 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (3)..(3) <223> Xaa is R or T <400> 8 Tyr Ala Xaa Ser Leu Ala Asp 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (1)..(9) <223> CDR3-VL2 <400> 9 Leu Gln His Gly Glu Ser Pro Phe Thr 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (2)..(2) <223> Xaa is S or Y <400> 10 Ser Xaa Trp Met His 1 5 <210> 11 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (1)..(1) <223> Xaa is Y or R or N <400> 11 Xaa Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (8)..(8) <223> Xaa is Y or F <400> 12 Gly Tyr Asp Arg Ser Phe Asp Xaa 1 5 <210> 13 <211> 23 <212> PRT <213> Mus musculus <400> 13 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Leu Ser Cys 20 <210> 14 <211> 15 <212> PRT <213> Mus musculus <400> 14 Trp Tyr Gln Gln Asn Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 15 <211> 32 <212> PRT <213> Mus musculus <400> 15 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Phe Cys 20 25 30 <210> 16 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 16 Phe Gly Gly Gly Ala Lys Leu Glu Ile Lys 1 5 10 <210> 17 <211> 30 <212> PRT <213> Mouse (Mus musculus) <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Asn Ala Phe Ser 20 25 30 <210> 18 <211> 14 <212> PRT <213> Mouse (Mus musculus) <400> 18 Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 <210> 19 <211> 32 <212> PRT <213> Mouse (Mus musculus) <400> 19 Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Val Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys Gly Arg 20 25 30 <210> 20 <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> 20 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 21 <211> 23 <212> PRT <213> Mouse (Mus musculus) <400> 21 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Phe Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys 20 <210> 22 <211> 15 <212> PRT <213> Mouse (Mus musculus) <400> 22 Trp Tyr Gln Gln Lys Pro Trp Arg Ser Pro Lys Thr Leu Ile Tyr 1 5 10 15 <210> 23 <211> 32 <212> PRT <213> Mouse (Mus musculus) <400> 23 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Phe Ser 1 5 10 15 Leu Thr Ile Ser Ser Leu Glu Ser Asp Asp Ala Ala Thr Tyr Tyr Cys 20 25 30 <210> 24 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 24 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 25 <211> 30 <212> PRT <213> Mouse (Mus musculus) <400> 25 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210> 26 <211> 14 <212> PRT <213> Mouse (Mus musculus) <400> 26 Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly 1 5 10 <210> 27 <211> 32 <212> PRT <213> Mouse origin (Mus musculus) <400> 27 Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 28 <211> 11 <212> PRT <213> Mouse origin (Mus musculus) <400> 28 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <210> 29 <211> 111 <212> PRT <213> Mouse origin (Mus musculus) <400> 29 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Phe 20 25 30 Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Asn Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Phe Cys Gln Gln Thr Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Ala Lys Leu Glu Ile Lys 100 105 110 <210> 30 <211> 120 <212> PRT <213> Mouse (Mus musculus) <400> 30 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Asn Ala Phe Ser Ser Ser 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys 85 90 95 Gly Arg Gly Asn Pro Thr Thr Ala Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 31 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 31 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Phe Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Ser Phe 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Trp Arg Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Tyr Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Phe Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Asp Asp Ala Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 32 <211> 117 <212> PRT <213> Mouse origin (Mus musculus) <400> 32 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Asp Arg Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 33 <211> 107 <212> PRT <213> Mouse origin (Mus musculus) <400> 33 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 34 <211> 324 <212> PRT <213> Mouse (Mus musculus) <400> 34 Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala 1 5 10 15 Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 100 105 110 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 115 120 125 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 130 135 140 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 145 150 155 160 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 165 170 175 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 180 185 190 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 195 200 205 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 210 215 220 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 225 230 235 240 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 245 250 255 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 260 265 270 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 275 280 285 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 290 295 300 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 305 310 315 320 Ser Pro Gly Lys <210> 35 <211> 31 <212> DNA <213> Mouse (Mus musculus) <400> 35 gtacggatcc caggttcagc tgcagcagtc t 31 <210> 36 <211> 34 <212> DNA <213> Mouse (Mus musculus) <400> 36 gtacctcgag gacattgtgc tgacccaatc tcca 34 <210> 37 <211> 31 <212> DNA <213> Mouse (Mus musculus) <400> 37 gtacggatcc caggtccaac tgcagcagcc t 31 <210> 38 <211> 35 <212> DNA <213> Mouse (Mus musculus) <400> 38 gtacctcgag gacatcaaga tgacccagtc tccat 35 <210> 39 <211> 33 <212> DNA <213> Mouse (Mus musculus) <400> 39 gtacgtcgac tttaccagga gagtgggaga ggc 33 <210> 40 <211> 35 <212> DNA <213> Mouse (Mus musculus) <400> 40 gtacggtacc acactcattc ctgttgaagc tcttg 35 <210> 41 <211> 333 <212> DNA <213> Mouse (Mus musculus) <400> 41 gacattgtgc tgacccaatc tccagccagc ctggccgtga gcctgggcca gagggccacc 60 ctgagctgca gggccagcga gagcgtggac agcttcggct acagcttcat gcactggtac 120 cagcagaacc ccggccagcc ccccaagctg ctgatctaca gggccagcaa cctggagagc 180 ggcatccccg ccaggttcag cggcagcggc agcaggaccg acttcaccct gaccatcaac 240 cccgtggagg ccgacgacgt ggccacctac ttctgccagc agaccaacga ggacccctac 300 accttcggcg gcggcgccaa gctggagatc aag 333 <210> 42 <211> 360 <212> DNA <213> Mouse (Mus musculus) <400> 42 caggttcagc tgcagcagtc tggccccgag ctggtgaagc ccggcgccag cgtgaagctg 60 agctgcaagg ccagcggcaa cgccttcagc agcagctgga tgaactgggt gaagcagagg 120 cccggccagg gcctggagtg gatcggcagg atctaccccg gcgacggcga caccaactac 180 aacggcaagt tcaagggcaa ggccaccctg accgtggaca agagcagcag caccgtgtac 240 atgcagctga gcagcctgac cagcgtggac agcgccgtgt acttctgcgg caggggcaac 300 cccaccaccg cctacgccat ggactactgg ggccagggca ccagcgtgac cgtgagcagc 360 <210> 43 <211> 321 <212> DNA <213> Mouse (Mus musculus) <400> 43 gacatcaaga tgacccagtc tccatctagc atgttcgcca gcctgggcga gagggtgacc 60 atcacctgca aggccagcca ggacatcaag agcttcctga actggtacca gcagaagccc 120 tggaggagcc ccaagaccct gatctactac gccaccagcc tggccgacgg cgtgcccagc 180 aggttcagcg gcagcggcag cggccaggac ttcagcctga ccatcagcag cctggagagc 240 gacgacgccg ccacctacta ctgcctgcag cacggcgaga gccccttcac cttcggcggc 300 ggcaccaagc tggagatcaa g 321 <210> 44 <211> 351 <212> DNA <213> Mouse (Mus musculus) <400> 44 caggtccaac tgcagcagcc tggcgccgag ctggtgaggc ccggcgccag cgtgaagctg 60 agctgcaagg ccagcggcta caccttcacc agcagctgga tgcactgggt gaagcagagg 120 cccgagcagg gcctggagtg gatcggctac atcgacccct acgacagcga gacccactac 180 aaccagaagt tcaaggacaa ggccatcctg accgtggaca agagcagcag caccgcctac 240 atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc caggggctac 300 gacaggagct tcgactactg gggccagggc accaccctga ccgtgagcag c 351

Claims

1. An antibody against PIC, characterized in that, the antibody comprises 6 CDRs, and the 6 CDRs are selected from (i) or (ii): (i) CDR1-VL1, with the sequence SEQ ID NO: 1: RASESVDSFGYSFX 1 H, where X 1 is M or L; CDR2-VL1, with the sequence of SEQ ID NO: 2: RASNLES; CDR3-VL1, with the sequence SEQ ID NO: 3: QQTNEDPX 2 T, where X 2 is Y or F or W; CDR1-VH1, with the sequence of SEQ ID NO: 4: SSWMN; CDR2-VH1, with the sequence SEQ ID NO: 5: RIYPGDGX 3 TNYNGKFKG, where X 3 is D or H; and CDR3-VH1, with the sequence SEQ ID NO: 6: GX 4 PTTAYAMDY, where X 4 is N, S or R; or (ii) CDR1-VL2, with the sequence SEQ ID NO: 7: KASQDIKX 5 FLN, where X 5 is S or N; CDR2-VL2, with the sequence SEQ ID NO: 8: YAX 6 SLAD, where X 6 is R or T; CDR3-VL2, with the sequence of SEQ ID NO: 9: LQHGESPFT; CDR1-VH2, with the sequence SEQ ID NO: 10: SX 7 WMH, where X 7 is S or Y; CDR2-VH2, with the sequence SEQ ID NO: 11: X 8 IDPYDSETHYNQKFKD, where X 8 is Y or R or N; and CDR3-VH2, with the sequence SEQ ID NO: 12: GYDRSFDX 9 , where X 9 is Y or F; wherein, the PIC is plasmin-α2 plasmin inhibitor complex.

2. The antibody against PIC according to claim 1, characterized in that, the antibody against PIC is selected from the following wild type or any one of the following mutant combinations:

3. The antibody against PIC according to claim 1, characterized in that, the antibody against PIC is selected from the following wild type or any one of the following mutant combinations:

4. The antibody against PIC according to any one of claims 1 to 3, characterized in that, the antibody against PIC further comprises the framework region of the light chain variable region and the framework region of the heavy chain variable region. Among them, the framework region of the light chain variable region comprises FR1-VL1, FR2-VL1, FR3-VL1 and FR4-VL1, and the framework region of the heavy chain variable region comprises FR1-VH1, FR2-VH1, FR3-VH1 and FR4-VH1. Or, the framework region of the light chain variable region comprises FR1-VL2, FR2-VL2, FR3-VL2 and FR4-VL2, and the framework region of the heavy chain variable region comprises FR1-VH2, FR2-VH2, FR3-VH2 and FR4-VH2, wherein, the amino acid sequence of FR1-VL1 is SEQ ID NO: 13; the amino acid sequence of FR2-VL1 is SEQ ID NO: 14; the amino acid sequence of FR3-VL1 is SEQ ID NO: 15; the amino acid sequence of FR4-VL1 is SEQ ID NO: 16; the amino acid sequence of FR1-VH1 is SEQ ID NO: 17; the amino acid sequence of FR2-VH1 is SEQ ID NO: 18; the amino acid sequence of FR3-VH1 is SEQ ID NO: 19; the amino acid sequence of FR4-VH1 is SEQ ID NO: 20; the amino acid sequence of FR1-VL2 is SEQ ID NO: 21; the amino acid sequence of FR2-VL2 is SEQ ID NO: 22; the amino acid sequence of FR3-VL2 is SEQ ID NO: 23; the amino acid sequence of FR4-VL2 is SEQ ID NO: 24; the amino acid sequence of FR1-VH2 is SEQ ID NO: 25; the amino acid sequence of FR2-VH2 is SEQ ID NO: 26; the amino acid sequence of FR3-VH2 is SEQ ID NO: 27; the amino acid sequence of FR4-VH2 is SEQ ID NO: 28; SEQ ID NO: 13: DIVLTQSPASLAVSLGQRATLSC; SEQ ID NO: 14: WYQQNPGQPPKLLIY; SEQ ID NO: 15: GIPARFSGSGSRTDFTLTINPVEADDVATYFC; SEQ ID NO: 16: FGGGAKLEIK; SEQ ID NO: 17: QVQLQQSGPELVKPGASVKLSCKASGNAFS; SEQ ID NO: 18: WVKQRPGQGLEWIG; SEQ ID NO: 19: KATLTVDKSSSTVYMQLSSLTSVDSAVYFCGR; SEQ ID NO: 20: WGQGTSVTVSS; SEQ ID NO: 21: DIKMTQSPSSMFASLGERVTITC; SEQ ID NO: 22: WYQQKPWRSPKTLIY; SEQ ID NO: 23: GVPSRFSGSGSGQDFSLTISSLESDDAATYYC; SEQ ID NO: 24: FGGGTKLEIK; SEQ ID NO: 25: QVQLQQPGAELVRPGASVKLSCKASGYTFT; SEQ ID NO: 26: WVKQRPEQGLEWIG; SEQ ID NO: 27: KAILTVDKSSSTAYMQLSSLTSEDSAVYYCAR; SEQ ID NO: 28: WGQGTTLTVSS.

5. The anti-PIC antibody according to any one of claims 1 to 3, characterized in that the anti-PIC antibody further comprises a framework region of the light chain variable region and a framework region of the heavy chain variable region, wherein the framework region of the light chain variable region comprises FR1-VL1, FR2-VL1, FR3-VL1 and FR4-VL1, and the framework region of the heavy chain variable region comprises FR1-VH1, FR2-VH1, FR3-VH1 and FR4-VH1, or the framework region of the light chain variable region comprises FR1-VL2, FR2-VL2, FR3-VL2 and FR4-VL2, and the framework region of the heavy chain variable region comprises FR1-VH2, FR2-VH2, FR3-VH2 and FR4-VH2, wherein the amino acid sequence of FR1-VL1 is a sequence having more than 90% homology with SEQ ID NO: 13; the amino acid sequence of FR2-VL1 is a sequence having more than 90% homology with SEQ ID NO: 14; the amino acid sequence of FR3-VL1 is a sequence having more than 90% homology with SEQ ID NO: 15; the amino acid sequence of FR4-VL1 is a sequence having more than 90% homology with SEQ ID NO: 16; The amino acid sequence of the FR1-VH1 is a sequence having more than 90% homology with SEQ ID NO: 17; The amino acid sequence of the FR2-VH1 is a sequence having more than 90% homology with SEQ ID NO: 18; The amino acid sequence of the FR3-VH1 is a sequence having more than 90% homology with SEQ ID NO: 19; The amino acid sequence of the FR4-VH1 is a sequence having more than 90% homology with SEQ ID NO: 20; or The amino acid sequence of the FR1-VL2 is a sequence having more than 90% homology with SEQ ID NO: 21; The amino acid sequence of the FR2-VL2 is a sequence having more than 90% homology with SEQ ID NO: 22; The amino acid sequence of the FR3-VL2 is a sequence having more than 90% homology with SEQ ID NO: 23; The amino acid sequence of the FR4-VL2 is a sequence having more than 90% homology with SEQ ID NO: 24; The amino acid sequence of the FR1-VH2 is a sequence having more than 90% homology with SEQ ID NO: 25; The amino acid sequence of the FR2-VH2 is a sequence having more than 90% homology with SEQ ID NO: 26; The amino acid sequence of the FR3-VH2 is a sequence having more than 90% homology with SEQ ID NO: 27; The amino acid sequence of the FR4-VH2 is a sequence having more than 90% homology with SEQ ID NO:

28.

6. The anti-PIC antibody according to any one of claims 1 to 3, wherein, the anti-PIC antibody further comprises a framework region of the light chain variable region and a framework region of the heavy chain variable region, wherein the framework region of the light chain variable region comprises FR1-VL1, FR2-VL1, FR3-VL1 and FR4-VL1, and the framework region of the heavy chain variable region comprises FR1-VH1, FR2-VH1, FR3-VH1 and FR4-VH1, or the framework region of the light chain variable region comprises FR1-VL2, FR2-VL2, FR3-VL2 and FR4-VL2, and the framework region of the heavy chain variable region comprises FR1-VH2, FR2-VH2, FR3-VH2 and FR4-VH2, wherein, the amino acid sequence of the FR1-VL1 is a sequence having more than 95% homology with SEQ ID NO: 13; the amino acid sequence of the FR2-VL1 is a sequence having more than 95% homology with SEQ ID NO: 14; the amino acid sequence of the FR3-VL1 is a sequence having more than 95% homology with SEQ ID NO: 15; the amino acid sequence of the FR4-VL1 is a sequence having more than 95% homology with SEQ ID NO: 16; the amino acid sequence of the FR1-VH1 is a sequence having more than 95% homology with SEQ ID NO: 17; The amino acid sequence of the FR2-VH1 is a sequence having a homology of more than 95% with SEQ ID NO:18; The amino acid sequence of the FR3-VH1 is a sequence having a homology of more than 95% with SEQ ID NO:19; The amino acid sequence of the FR4-VH1 is a sequence having a homology of more than 95% with SEQ ID NO:20; or The amino acid sequence of the FR1-VL2 is a sequence having a homology of more than 95% with SEQ ID NO:21; The amino acid sequence of the FR2-VL2 is a sequence having a homology of more than 95% with SEQ ID NO:22; The amino acid sequence of the FR3-VL2 is a sequence having a homology of more than 95% with SEQ ID NO:23; The amino acid sequence of the FR4-VL2 is a sequence having a homology of more than 95% with SEQ ID NO:24; The amino acid sequence of the FR1-VH2 is a sequence having a homology of more than 95% with SEQ ID NO:25; The amino acid sequence of the FR2-VH2 is a sequence having a homology of more than 95% with SEQ ID NO:26; The amino acid sequence of the FR3-VH2 is a sequence having a homology of more than 95% with SEQ ID NO:27; The amino acid sequence of the FR4-VH2 is a sequence having a homology of more than 95% with SEQ ID NO:

28.

7. The anti-PIC antibody according to any one of claims 1 to 3, wherein, the anti-PIC antibody further comprises a framework region of the light chain variable region and a framework region of the heavy chain variable region, wherein the framework region of the light chain variable region comprises FR1-VL1, FR2-VL1, FR3-VL1 and FR4-VL1, and the framework region of the heavy chain variable region comprises FR1-VH1, FR2-VH1, FR3-VH1 and FR4-VH1, or the framework region of the light chain variable region comprises FR1-VL2, FR2-VL2, FR3-VL2 and FR4-VL2, and the framework region of the heavy chain variable region comprises FR1-VH2, FR2-VH2, FR3-VH2 and FR4-VH2, wherein, the amino acid sequence of the FR1-VL1 is a sequence having a homology of more than 99% with SEQ ID NO:13; the amino acid sequence of the FR2-VL1 is a sequence having a homology of more than 99% with SEQ ID NO:14; the amino acid sequence of the FR3-VL1 is a sequence having a homology of more than 99% with SEQ ID NO:15; the amino acid sequence of the FR4-VL1 is a sequence having a homology of more than 99% with SEQ ID NO:16; the amino acid sequence of the FR1-VH1 is a sequence having a homology of more than 99% with SEQ ID NO:17; the amino acid sequence of the FR2-VH1 is a sequence having a homology of more than 99% with SEQ ID NO:18; The amino acid sequence of said FR3-VH1 is a sequence having a homology of more than 99% with SEQ ID NO:19; The amino acid sequence of said FR4-VH1 is a sequence having a homology of more than 99% with SEQ ID NO:20; or The amino acid sequence of said FR1-VL2 is a sequence having a homology of more than 99% with SEQ ID NO:21; The amino acid sequence of said FR2-VL2 is a sequence having a homology of more than 99% with SEQ ID NO:22; The amino acid sequence of said FR3-VL2 is a sequence having a homology of more than 99% with SEQ ID NO:23; The amino acid sequence of said FR4-VL2 is a sequence having a homology of more than 99% with SEQ ID NO:24; The amino acid sequence of said FR1-VH2 is a sequence having a homology of more than 99% with SEQ ID NO:25; The amino acid sequence of said FR2-VH2 is a sequence having a homology of more than 99% with SEQ ID NO:26; The amino acid sequence of said FR3-VH2 is a sequence having a homology of more than 99% with SEQ ID NO:27; The amino acid sequence of said FR4-VH2 is a sequence having a homology of more than 99% with SEQ ID NO:

28.

8. The anti-PIC antibody according to claim 4, wherein, said anti-PIC antibody has any one of the following light chain variable regions and heavy chain variable regions: (i) The light chain variable region VL1 shown in SEQ ID NO:29 and the heavy chain variable region VH1 shown in SEQ ID NO:30; SEQ ID NO:29: DIVLTQSPASLAVSLGQRATLSC RASESVDSFGYSFMH WYQQNPGQPPKLLIY RASNLES GIPARFSGSGSR TDFTLTINPVEADDVATYFC QQTNEDPYT FGGGAKLEIK ; SEQ ID NO:30: QVQLQQSGPELVKPGASVKLSCKASGNAFS SSWMN WVKQRPGQGLEWIG RIYPGDGDTNYNGKFKG KATLTV DKSSSTVYMQLSSLTSVDSAVYFCGR GNPTTAYAMDY WGQGTSVTVSS ; (ii) The light chain variable region VL2 shown in SEQ ID NO:31 and the heavy chain variable region VH2 shown in SEQ ID NO:32; SEQ ID NO:31: DIKMTQSPSSMFASLGERVTITC KASQDIKSFLN WYQQKPWRSPKTLIY YATSLAD GVPSRFSGSGSGQDFS LTISSLESDDAATYYC LQHGESPFT FGGGTKLEIK; SEQ ID NO:32: QVQLQQPGAELVRPGASVKLSCKASGYTFT SSWMH WVKQRPEQGLEWIG YIDPYDSETHYNQKFKD KAILTV DKSSSTAYMQLSSLTSEDSAVYYCAR GYDRSFDY WGQGTTLTVSS.

9. The anti-PIC antibody according to claim 4, It is characterized in that the anti-PIC antibody has any one of the following light chain variable regions and heavy chain variable regions: (iii) a light chain variable region having more than 90% homology with the sequence shown in SEQ ID NO: 29 and a heavy chain variable region having more than 90% homology with the sequence shown in SEQ ID NO: 30; (iv) a light chain variable region having more than 90% homology with the sequence shown in SEQ ID NO: 31 and a heavy chain variable region having more than 90% homology with the sequence shown in SEQ ID NO:

32.

10. The anti-PIC antibody according to claim 4, It is characterized in that the anti-PIC antibody has any one of the following light chain variable regions and heavy chain variable regions: (iii) a light chain variable region having more than 95% homology with the sequence shown in SEQ ID NO: 29 and a heavy chain variable region having more than 95% homology with the sequence shown in SEQ ID NO: 30; (iv) a light chain variable region having more than 95% homology with the sequence shown in SEQ ID NO: 31 and a heavy chain variable region having more than 95% homology with the sequence shown in SEQ ID NO:

32.

11. The anti-PIC antibody according to claim 4, It is characterized in that the anti-PIC antibody has any one of the following light chain variable regions and heavy chain variable regions: (iii) a light chain variable region having more than 99% homology with the sequence shown in SEQ ID NO: 29 and a heavy chain variable region having more than 99% homology with the sequence shown in SEQ ID NO: 30; (iv) a light chain variable region having more than 99% homology with the sequence shown in SEQ ID NO: 31 and a heavy chain variable region having more than 99% homology with the sequence shown in SEQ ID NO:

32.

12. The anti-PIC antibody according to claim 4, It is characterized in that the antibody is a full-length antibody, and the full-length antibody further includes a light chain constant region CL and a heavy chain constant region CH, wherein the sequence of the light chain constant region CL is SEQ ID NO: 33; the sequence of the heavy chain constant region CH is SEQ ID NO:

34.

13. The anti-PIC antibody according to claim 4, It is characterized in that the antibody is a full-length antibody, and the full-length antibody further includes a light chain constant region CL and a heavy chain constant region CH, wherein the sequence of the light chain constant region CL is a sequence having more than 90% homology with the sequence shown in SEQ ID NO: 33; and / or the sequence of the heavy chain constant region CH is a sequence having more than 90% homology with the sequence shown in SEQ ID NO:

34.

14. The anti-PIC antibody according to claim 4, It is characterized in that the antibody is a full-length antibody, and the full-length antibody further includes a light chain constant region CL and a heavy chain constant region CH, wherein the sequence of the light chain constant region CL is a sequence having more than 95% homology with the sequence shown in SEQ ID NO: 33; and / or The sequence of the heavy chain constant region CH is a sequence having a homology of more than 95% with the sequence shown in SEQ ID NO:

34.

15. The anti-PIC antibody according to claim 4, wherein, the antibody is a full-length antibody, and the full-length antibody further includes a light chain constant region CL and a heavy chain constant region CH, wherein, the sequence of the light chain constant region CL is a sequence having a homology of more than 99% with the sequence shown in SEQ ID NO: 33; and / or the sequence of the heavy chain constant region CH is a sequence having a homology of more than 99% with the sequence shown in SEQ ID NO:

34.

16. The anti-PIC antibody according to claim 4, wherein, The anti-PIC antibody has an affinity for PIC of 4.30*10 -11 ≤Kd(M)≤7.58*10 -9 .

17. The anti-PIC antibody according to any one of claims 1 to 3, wherein, the anti-PIC antibody is humanized or chimeric.

18. An isolated nucleic acid molecule, wherein, the isolated nucleic acid molecule encodes the anti-PIC antibody according to any one of claims 1 to 17.

19. An expression vector, wherein, the expression vector includes the nucleic acid molecule according to claim 18.

20. A host cell, wherein, the host cell is transfected with the expression vector according to claim 19.

21. A PIC detection kit, wherein, the kit includes the anti-PIC antibody according to any one of claims 1 to 17.

22. The kit according to claim 21, wherein, the kit includes two of the anti-PIC antibodies, one of which is the anti-PIC antibody coated on magnetic beads and the other is the labeled anti-PIC antibody.

23. The kit according to claim 22, wherein, the labeled anti-PIC antibody is the ABEI-labeled anti-PIC antibody.

24. The kit according to claim 21, wherein, the kit further includes a calibrator and / or a quality control product.

25. Use of the anti-PIC antibody according to any one of claims 1 to 17 in the preparation of a detection product for detecting PIC.

26. The use according to claim 25, wherein, the detection product is a product detected by any one or more of the following methods: fluorescence immunoassay, chemiluminescence immunoassay, colloidal gold immunoassay, radioimmunoassay or enzyme-linked immunosorbent assay.

27. The use according to claim 26, wherein, the chemiluminescence immunoassay is a chemiluminescence immunoassay double antibody sandwich method.

28. The use according to claim 26, wherein, the use further includes detecting PIC using a semi-automatic immunoanalyzer or a fully automatic immunoanalyzer.

29. The use according to claim 25, wherein, the sample to be detected is selected from at least one of whole blood, serum or plasma.

30. The use according to claim 29, wherein, the whole blood, serum or plasma is derived from peripheral blood.

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