CN118240085A

CN118240085A - Anti-fibrin degradation product antibody, reagent and kit for detecting fibrin degradation product

Info

Publication number: CN118240085A
Application number: CN202311200671.8A
Authority: CN
Inventors: 孟媛; 钟冬梅; 唐丽娜; 游辉; 曹慧方
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2022-10-08
Filing date: 2023-09-15
Publication date: 2024-06-25

Abstract

The invention discloses an antibody for resisting a fibrin degradation product, a reagent for detecting the fibrin degradation product and a kit, and relates to the field of antibodies. The antibody for resisting the fibrin degradation product disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detecting the fibrin degradation product, and has good affinity or activity.

Description

Anti-fibrin degradation product antibody, reagent and kit for detecting fibrin degradation product

Cross Reference to Related Applications

The present invention claims priority from chinese patent application serial No. 202211222866.8, entitled "anti-fibrin degradation product antibody or functional fragment thereof, reagents and kits for detecting fibrin degradation products" filed at 10/08 of 2022, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the technical field of antibodies, in particular to an antibody for resisting a fibrin degradation product, a reagent for detecting the fibrin degradation product and a kit.

Background

Fibrin degradation products (Fibrin Degradation Products, FDPs) are protein components or polypeptide fragments produced by the hydrolysis of fibrin or fibrinogen by plasmin. Plasmin is formed and acts primarily locally on the endothelial cell surface of the vessel wall to remove small amounts of fibrin formed on the endothelial cell surface. FDPs in blood are normally maintained at a very low level, typically less than 10. Mu.g/mL. There is a substantial increase in the body's fibrinolytic system, which is the body's response to endogenous coagulation. High FDPs concentrations are also found in hyperfibrinolytic or extrinsic coagulation. Therefore, FDPs assays can be used to detect the function of the fibrinolytic system, most commonly in the diagnosis of disseminated intravascular coagulation, hypercoagulable conditions and thrombotic diseases. In addition, elevated FDPs can also be detected when suffering from chronic kidney disease and uremia. The elevation of FDPs can also be seen in liver disease, intracavitary hemorrhage, severe burns, virulent poisoning by mice, protein-lost kidney disease or bowel disease, etc. In addition, FDPs can also be used as markers for tumor detection for colorectal cancer detection.

The biological immunology detection method has the advantages of simplicity, rapidity, low cost and the like, the biological immunology detection method needs antibodies aiming at FDPs, and the field has strong demands for the antibodies which can effectively bind to the FDPs and detect the FDPs.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide an antibody for resisting a fibrin degradation product, a reagent for detecting the fibrin degradation product and a kit.

The invention is realized in the following way:

In a first aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the combination of HCDR1/HCDR2/HCDR3 is the same as the combination of HCDR1/HCDR2/HCDR3 comprised by the heavy chain variable region of any one of SEQ ID NO:18 to SEQ ID NO:21, and wherein the combination of LCDR1/LCDR2/LCDR3 is the same as the combination of LCDR1/LCDR2/LCDR3 comprised by the light chain variable region of either SEQ ID NO:22 or 23.

In alternative embodiments, the CDRs are defined by Kabat, chothia, abM, contact or IMGT systems.

In a second aspect, an embodiment of the present invention provides an antibody against a degradation product of fibrin, the antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3 comprise SEQ ID NOs 1 to 3 in sequence, or are shown in SEQ ID NOs 1 to 3 in sequence; the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 sequentially comprise SEQ ID NO 4-6 or are sequentially shown as SEQ ID NO 4-6.

In a third aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above.

In a fourth aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody comprising a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs 18 to 21 and/or a light chain variable region; or consists of the amino acid sequence shown in any one of SEQ ID NOs 18 to 21; the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 22 or 23; or consists of the amino acid sequence shown in SEQ ID NO. 22 or 23.

In a fifth aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody comprising a heavy chain and a light chain; the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs 26 to 29; or consists of the amino acid sequence shown in any one of SEQ ID NOs 26 to 29; the light chain comprises an amino acid sequence shown in SEQ ID NO. 30 or 31; or consists of the amino acid sequence shown in SEQ ID NO. 30 or 31.

In a sixth aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody that binds to the same epitope in the amino acid sequence of the fibrin degradation product as the antibody described in the previous embodiments; alternatively, the antibody competes with the antibody described in the previous examples for binding to a fibrin degradation product.

In a seventh aspect, embodiments of the invention provide an antibody conjugate comprising an antibody as described in the previous embodiments.

In an eighth aspect, embodiments of the invention provide a reagent or kit comprising an antibody as described in the previous embodiments or an antibody conjugate as described in the previous embodiments.

In a ninth aspect, embodiments of the present invention provide a method of detecting a fibrin degradation product, comprising: the antibody, antibody conjugate, reagent or kit described in the previous examples is contacted with the fibrin degradation product in the sample to be tested to form an immune complex.

In a tenth aspect, embodiments of the present invention provide an isolated nucleic acid encoding an antibody as described in the previous embodiments.

In an eleventh aspect, embodiments of the present invention provide a vector comprising the isolated nucleic acid of the previous embodiments.

In a twelfth aspect, embodiments of the invention provide a cell comprising an isolated nucleic acid as described in the previous embodiments or a vector as described in the previous embodiments.

In a thirteenth aspect, embodiments of the present invention provide a method of preparing an antibody according to the previous embodiments, comprising: the cells described in the previous examples were cultured.

In a fourteenth aspect, embodiments of the present invention provide the use of an antibody, antibody conjugate, reagent or kit as described in the previous embodiments for detecting a fibrin degradation product or for preparing a product for detecting a fibrin degradation product.

The invention has the following beneficial effects:

The anti-fibrin degradation product antibody disclosed herein comprises the heavy chain complementarity determining regions and light chain complementarity determining regions described above, and provides an important source of raw materials for the detection of fibrin degradation products with improved affinity or activity.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of reducing SDS-PAGE of Anti-FDPs 5C1 Rmb1 to Anti-FDPs 5C1 Rmb.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

The HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to the HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody according to the first aspect, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to the LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody according to the first aspect.

For example, the HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 18; the LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO. 22.

In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments so long as they exhibit the desired biological activity. The full-length monoclonal antibody is composed of a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain constant region (CH), and a light chain constant region (CL). In the present invention, the heavy chain variable region, the light chain variable region, the heavy chain constant region and the light chain constant region are represented by VH, VL, CH and CL in this order.

The antigen binding fragments described above generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the teachings herein that the antigen binding fragments described above may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The antigen binding fragments described above are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.

The antigen binding fragments described above may also be obtained synthetically by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it.

In the present invention, the term "heavy chain complementarity determining region", "HCDR" or "HCDRs" refers to complementarity determining regions in one or more or even all of the heavy chain variable regions. The 3 HCDRs contained in the heavy chain variable region include HCDR1, HCDR2 and HCDR3.

In the present invention, the term "light chain complementarity determining regions", "LCDR" or "LCDRs" refers to complementarity determining regions in one or more or even all of the light chain variable regions. The 3 LCDRs contained in the light chain variable region include LCDR1, LCDR2 and LCDR3.

The definition of CDRs is known in the art and may be defined, for example, according to Kabat, chothia, abM, contact or IMGT systems. Still other CDRs definitions may not strictly follow one of the 25 methods described above, but still overlap at least a portion of the CDRs defined by Kabat, although they may be shortened or lengthened depending on the predicted or experimental outcome of a particular residue or group of residues. In the present invention, CDRs may refer to any method known in the art, including combinations of known methods.

As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Exemplary defined CDRs are listed in table 1 below, with slightly different definitions in the different documents. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR. It should be noted that CDRs defined by other methods not limited to table 1 are also within the scope of the disclosure.

Table 1: CDR definition ¹

CDR	Kabat	AbM²	IMGT	Chothia
					HCDR1	H31～H35³	H26～H35³	H26～H33..5⁵	H26～H32..34⁴
HCDR2	H50～H65	H50～H58	H51～H57	H52～H56
					HCDR3	H95～H102	H95～H102	H93～H102	H95～H102
LCDR1	L24～L34	L24～L34	L27～L32	L24～L34
					LCDR2	L50～L56	L50～L56	L50～L51	L50～L56
LCDR3	L89～L97	L89～L97	L89～L97	L89～L97

¹ The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with the amino acid numbers on the heavy chain being indicated by "H+ numbers" and the amino acid numbers on the light chain being indicated by "L+ numbers". The Kabat numbering system can be specifically mapped to any variable region sequence by one of ordinary skill in the art without relying on any experimental data outside of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, U.S. Dept. Of HEALTH AND HumanServices, "Sequence of Proteins of Immunological Interest" (1983).

² The "AbM" as used in table 1 has a lower case "b" referring to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

³ If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if H35A and H35B are present at the same time, then CDR-H1 ends at position 35B.

⁴ If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if H35A and H35B are present at the same time, then CDR-H1 ends at position 34.

⁵ If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or combination of systems Kabat, chothia, IMGT, abM or contacts.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Kabat system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Chothia system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an AbM system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by Kabat, chothia, IMGT, abM or Contact system combinations.

According to an embodiment of the present invention, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, chothia, abM or IMGT system correspond to the following Kabat numbering positions:

CDR	Kabat	AbM	IMGT	Chothia
					HCDR1	H31～H35	H26～H35	H26～H33	H26～H32
HCDR2	H50～H65	H50～H58	H51～H57	H52～H56
					HCDR3	H95～H102	H95～H102	H93～H102	H95～H102
LCDR1	L24～L34	L24～L34	L27～L32	L24～L34
					LCDR2	L50～L56	L50～L56	L50～L51	L50～L56
LCDR3	L89～L97	L89～L97	L89～L97	L89～L97

According to the embodiment of the invention, the HCDR1, HCDR2 and HCDR3 are the amino acid sequences of positions 31 to 35 (SEQ ID No: 1), 50 to 65 (SEQ ID No: 2) and 95 to 100B (SEQ ID No: 3) of the heavy chain variable region numbered by Kabat in sequence; the LCDR1, LCDR2 and LCDR3 comprise or are the amino acid sequences of positions 24-34 (SEQ ID No: 4), 50-56 (SEQ ID No: 5) and 89-95 (SEQ ID No: 6) of the light chain variable region of Kabat numbering in sequence.

In the present invention, the term "framework region" or "FRs" region refers to regions of the antibody heavy and light chain variable regions other than CDRs, including heavy and light chain framework regions. Wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by HCDRs, comprising HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by LCDRs, including LFR1, LFR2, LFR3, and LFR4 framework regions. In the present invention, the heavy chain framework region is represented by HFR or HFRs; the light chain framework region is denoted by LFR or LFRs.

In the present invention, the heavy chain variable region is obtained by ligating the following numbered HCDR with HFR in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region was obtained by ligating the following numbered LCDR with LFR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. The above-mentioned combination fragments are arranged sequentially from the upstream (N-terminal) to the downstream (C-terminal) of the sequence, and the "-" represents a covalent linkage (peptide bond).

In alternative embodiments, the antibody further comprises HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

In alternative embodiments, the HFR1 to HFR4 sequentially comprise the amino acid sequences of SEQ ID NO 7 to SEQ ID NO 10 or at least 80% identity thereto; or, the sequences are shown as SEQ ID NO 7 to SEQ ID NO 10 or the amino acid sequence with at least 80% of the same; the LFR 1-LFR 4 sequentially comprises SEQ ID NO. 11-SEQ ID NO. 14 or an amino acid sequence with at least 80% identity thereto; or, the sequences are shown in sequence from SEQ ID NO. 11 to SEQ ID NO. 14 or the amino acid sequences having at least 80% identity thereto.

In alternative embodiments, the antibody may have a framework region amino acid sequence that is 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 framework regions described above.

In an alternative embodiment, the amino acid sequence of HFR1 is set forth in SEQ ID NO. 15.

In an alternative embodiment, the amino acid sequence of HFR3 is set forth in SEQ ID NO. 16.

In an alternative embodiment, the amino acid sequence of LFR1 is shown as SEQ ID NO. 17.

In a third aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HFR3, LFR4, LFR2, LFR 3.

In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

In a fourth aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody comprising a heavy chain variable region comprising an amino acid sequence of any one of SEQ ID NOs 18 to 21 and/or a light chain variable region; or consists of the amino acid sequence shown in any one of SEQ ID NOs 18 to 21. In alternative embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 22 or 23; or consists of the amino acid sequence shown in SEQ ID NO. 22 or 23.

In alternative embodiments, the antibody further comprises a constant region.

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

In alternative embodiments, the heavy chain constant region is selected from the group consisting of the heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.

In alternative embodiments, the constant region is of any one of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, and human origin.

In an alternative embodiment, the constant region is of murine species origin.

In alternative embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO. 24 or at least 80% identical thereto; or consists of the amino acid sequence shown in SEQ ID NO. 24 or having at least 80% identity thereto.

In alternative embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO. 25 or at least 80% identical thereto; or consists of the amino acid sequence shown in SEQ ID NO. 25 or having at least 80% identity thereto.

In particular, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the above-described constant region (SEQ ID NO:24 or 25).

In a fifth aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody comprising a heavy chain and/or a light chain, said antibody comprising a heavy chain and a light chain; the heavy chain contains the sequence structure of VH-CH, the light chain contains the sequence structure of VL-CL, and the amino acid sequence of VH, VL, CH, CL is VH, VL, CH, CL of any of the above examples. The above-mentioned combination fragments are arranged sequentially from the upstream (N-terminal) to the downstream (C-terminal) of the sequence, and the "-" represents a covalent linkage (peptide bond).

In another aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody comprising a heavy chain and a light chain; the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs 26 to 29; or consists of the amino acid sequence shown in any one of SEQ ID NOs 26 to 29; the light chain comprises an amino acid sequence shown in SEQ ID NO. 30 or 31; or consists of the amino acid sequence shown in SEQ ID NO. 30 or 31.

In another aspect, embodiments of the present invention provide an anti-fibrin degradation product antibody that binds to the same epitope in the amino acid sequence of the fibrin degradation product as the antibody described in any of the embodiments above; alternatively, the antibody competes with an antibody of any of the embodiments above for binding to a fibrin degradation product.

Antibody affinity (KD) assays are widely varied and can be classified into thermodynamic, kinetic and dynamic equilibrium assays based on the principle of detection. Among them, thermodynamic detection methods are common such as Isothermal Titration Calorimetry (ITC); kinetic detection methods are commonly known as Surface Plasmon Resonance (SPR) and biofilm optical interferometry (BLI); dynamic equilibrium detection methods are commonly known as enzyme-linked immunosorbent assay (ELISA) and the like.

In alternative embodiments, the determination of KD employs kinetic detection methods; preferably, the surface plasmon resonance method is used, for example, by using a method such asA biosensor system of the system.

In an alternative embodiment, the determination of KD is performed with reference to the method of the examples of the invention.

In an alternative embodiment, the antibody binds to a fibrin degradation product with an affinity of KD.ltoreq.1.37X10 ^-7 M.

In alternative embodiments, the antibody binds to a fibrin degradation product with an affinity of KD.ltoreq.10 10 ^-07M、KD≤10^-08M、KD≤10^-09M、KD≤10^-10M、KD≤10^-11 M or KD.ltoreq.10 10 ^-12 M.

In an alternative embodiment, the antibody binds adiponectin with an affinity of KD.ltoreq.7.00X10 ^-9 M.

In alternative embodiments, the antibody is selected from any one of F (ab ') ₂, fab', fab, fv, and scFv.

In another aspect, embodiments of the present invention also provide an antibody conjugate comprising an antibody as described in the previous embodiments.

In alternative embodiments, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

In an alternative embodiment, the antibody conjugate further comprises a label conjugated to the antibody.

In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.

In an alternative embodiment, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

In the actual use process, a person skilled in the art can select a suitable marker according to the detection condition or the actual requirement, and no matter what marker is used, the marker belongs to the protection scope of the invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).

In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.

In alternative embodiments, the radioisotope includes, but is 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.

In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloids include, but are not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metal includes, but is not limited to, colloidal gold or colloidal silver.

In an alternative embodiment, the colloidal metal is colloidal gold.

In an alternative embodiment, the antibody conjugate further comprises a solid support coupled to the antibody. In an antibody conjugate, the antibody is conjugated to a solid support.

In alternative embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes.

In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

In an alternative embodiment, the solid support is a nitrocellulose membrane.

In another aspect, embodiments of the invention also provide a reagent or kit comprising an antibody as described in any of the preceding embodiments or an antibody conjugate as described in any of the preceding embodiments.

In alternative embodiments, the reagent or kit has increased detection sensitivity or specificity.

In another aspect, embodiments of the present invention also provide a method of detecting a fibrin degradation product, comprising:

contacting the antibody of any of the preceding embodiments or the antibody conjugate of any of the preceding embodiments or the reagent or kit of any of the preceding embodiments with a fibrin degradation product in a sample to be tested to form an immune complex.

In a preferred embodiment, the immune complex further comprises a second antibody, which binds to the antibody.

In a preferred embodiment, the immune complex further comprises a second antibody, which binds to a fibrin degradation product.

In another aspect, the embodiments of the present invention further provide the use of an antibody or functional fragment according to any of the preceding embodiments or an antibody conjugate according to any of the preceding embodiments or a reagent or kit according to any of the preceding embodiments for detecting a fibrin degradation product or for preparing a product for detecting a fibrin degradation product.

In another aspect, embodiments of the present invention also provide the use of an antibody or functional fragment of any of the embodiments described above or an antibody conjugate of any of the embodiments described above or a reagent or kit of any of the embodiments described above in the preparation of a product having at least one of the following uses, including: diagnosing or aiding in diagnosing a disease associated with the metabolism of a fibrin degradation product, predicting or aiding in predicting at least one of the prognostic efficacy of a disease associated with the metabolism of a fibrin degradation product.

In alternative embodiments, the fibrin degradation product metabolism-related disease comprises disseminated intravascular coagulation, hypercoagulable conditions, thrombosis, renal disease, uremia, liver disease, colorectal cancer disease.

In alternative embodiments, the product comprises a reagent or kit.

In another aspect, embodiments of the invention also provide an isolated nucleic acid encoding an antibody according to any of the preceding embodiments.

In another aspect, embodiments of the invention also provide a vector comprising an isolated nucleic acid as described in any of the previous embodiments.

In another aspect, embodiments of the invention also provide a cell comprising an isolated nucleic acid as described in any of the previous embodiments or a vector as described in any of the previous embodiments.

In another aspect, embodiments of the present invention also provide a method of preparing an antibody according to any of the previous embodiments, comprising: culturing the cells of any of the previous examples.

On the basis of the present disclosure of the amino acid sequence of an antibody, it is easily achieved by a person skilled in the art that the antibody is prepared by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), e.g. isolated and purified from a culture product of recombinant cells capable of recombinantly expressing an antibody according to any of the above, and on the basis of this, the antibody of the present disclosure is within the scope of the present disclosure, regardless of the technique used to prepare the antibody.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one 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 formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.)), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer Vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which are expressly incorporated herein by reference.

EXAMPLE 1 preparation of Anti-FDPs 5C1 monoclonal antibodies

Restriction enzymes, PRIME STAR DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART ^TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by gene sequencing companies.

1. Construction of expression plasmid

1.1 Preparation of Anti-FDPs 5C1 antibody Gene

MRNA is extracted from hybridoma cell strains secreting Anti-FDPs 5C1 monoclonal antibodies, DNA products are obtained through an RT-PCR method, the DNA products are inserted into a pMD-18T vector and are transformed into DH5 alpha competent cells, positive clones of HEAVY CHAIN genes and positive clones of LIGHT CHAIN genes are respectively taken after colony growth, and the positive clones are respectively sequenced by a gene sequencing company.

1.2 Sequence analysis of variable region genes of Anti-FDPs 5C1 antibodies

The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI 11.5.5 software is utilized for analysis to determine that the amplified genes of the heavy chain primer pair and the light chain primer pair are correct, wherein in the LIGHT CHAIN amplified gene fragment, the VL gene sequence is 336bp, and the front side of the VL gene fragment is 57bp leader peptide sequence; in the gene fragment amplified by HEAVY CHAIN primer pair, the VH gene sequence is 360bp, belonging to VH1 gene family, and the front of the gene fragment has 57bp leader peptide sequence.

1.3 Construction of recombinant antibody expression plasmids

pcDNA^TM 3.4Vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is modified to be introduced into a polyclonal enzyme cutting site, and is hereinafter abbreviated as a 3.4A expression vector; according to the result of the gene sequencing of the antibody variable region in pMD-18T, VL and VH gene specific primers of the Anti-FDPs5C1 antibody are designed, restriction enzyme digestion sites and protective bases are respectively arranged at two ends, and a LIGHT CHAIN gene fragment of 0.74KB and a HEAVY CHAIN gene fragment of 1.41KB are amplified by a PCR amplification method.

The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively subjected to double enzyme digestion by adopting restriction enzymes, the 3.4A vector is subjected to double enzyme digestion by adopting restriction enzymes, and the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector to respectively obtain recombinant expression plasmids HEAVY CHAIN and LIGHT CHAIN.

2. Sample preparation of recombinant antibodies

Recovering HEK293 cells in advance, and subculturing to a 200ml system to enable the cell density to reach 3-5×10 ⁶ cells/ml and the cell activity to be more than 95%; cells were washed by centrifugation and reconstituted with medium while the cell density was adjusted to 2.9X10 ⁶ cells/ml as a cell dilution. The medium was used to prepare dilutions of plasmid DNA and transfection reagent, respectively. Adding the transfection reagent diluent into the plasmid DNA diluent, uniformly mixing, standing at room temperature for 15min; the mixture is slowly added into cell dilution within 1min, after uniform mixing, sampling and counting are carried out, the activity of the transfected cells is recorded and observed, and the cells are placed in a constant temperature incubator at 35 ℃ for culture, the rotation speed is 120rmp, the CO2 content is 8%, and the cells are centrifugally collected after 13 days. The centrifuged supernatant was affinity purified using protein A affinity column. 6. Mu.g of purified antibody was subjected to reducing SDS-PAGE, and the electrophoresed pattern was as shown. Two bands were shown after reducing SDS-PAGE, 1 Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).

3. Affinity and Activity optimization

Although the Anti-FDPs 5C1 monoclonal antibody prepared by the method has the capability of combining with a fibrin degradation product antigen, the affinity and the antibody activity are not ideal, so that the applicant carries out directed mutation on the variable region of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a carrier, and preparing the mutant antibody according to the method of the step 2. Monoclonal antibodies with remarkably improved affinity and antibody activity are obtained through screening and are named as Anti-FDPs 5C1RMb1 to Anti-FDPs 5C1RMb7, and the heavy chain and light chain amino acid sequences are shown in the following table respectively.

TABLE 2 antibody sequences

Sample name	Heavy chain sequence number	Light chain sequence number
			Anti-FDPs 5C1RMb1	SEQ ID NO:26	SEQ ID NO:30
Anti-FDPs 5C1RMb2	SEQ ID NO:27	SEQ ID NO:30
			Anti-FDPs 5C1RMb3	SEQ ID NO:28	SEQ ID NO:30
Anti-FDPs 5C1RMb4	SEQ ID NO:28	SEQ ID NO:31
			Anti-FDPs 5C1RMb5	SEQ ID NO:29	SEQ ID NO:30
Anti-FDPs 5C1RMb6	SEQ ID NO:27	SEQ ID NO:31
			Anti-FDPs 5C1RMb7	SEQ ID NO:29	SEQ ID NO:31

Example 2 affinity analysis

The antibody is diluted and purified in advance, and meanwhile, FDPs antigen (purchased from the Phpeng organism) is subjected to gradient dilution; and (3) testing the binding dissociation curve of the antigen antibody on Biacore 8K+ equipment by utilizing a CM5 chip which is coupled with goat anti-mouse IgG in advance, and automatically fitting by an instrument to obtain an affinity constant, a binding rate and a dissociation rate. The results showed that the KD of Anti-FDPs 5C1RMb1 to FDPs 5C1RMb7 were all between 1.0X10 ^-8 M and 1.0X10 ^-11 M, superior to the control (KD indicates equilibrium dissociation constant, i.e., affinity constant; ka indicates binding rate; KD indicates dissociation rate).

Table 3 affinity assay data

Sample name	KD(M)	ka	kd
				Control antibodies	1.37E-07	4.54E+04	6.23E-03
Anti-FDPs 5C1RMb1	6.10E-10	6.83E+05	4.17E-04
				Anti-FDPs 5C1RMb2	3.89E-09	9.40E+04	3.66E-04
Anti-FDPs 5C1RMb3	1.69E-09	4.89E+05	8.24E-04
				Anti-FDPs 5C1RMb4	7.00E-09	4.53E+04	3.17E-04
Anti-FDPs 5C1RMb5	3.50E-09	2.92E+05	1.02E-03
				Anti-FDPs 5C1RMb6	5.00E-09	1.39E+05	6.95E-04
Anti-FDPs 5C1RMb7	5.46E-09	1.82E+05	9.93E-04

EXAMPLE 3 Activity assay

The coating solution (main component NaHCO 3) diluted FDPs antigen (purchased from Figpeng organism) to 2ug/ml, 100uL per well, overnight at 4 ℃; the next day, the washing solution (main component Na ₂ HPO4+Nacl) is washed 2 times and is patted dry; blocking solution (20% BSA+80% PBS) was added and dried at 37℃for 1h in 120uL per well; adding the diluted purified antibody and the control antibody, 100 uL/well, 37 ℃ for 30min; washing with washing liquid for 5 times, and drying; goat anti-mouse IgG-HRP was added at 100uL per well, 37℃for 30min; washing with washing liquid for 5 times, and drying; adding a developing solution A (50 uL/hole) and a developing solution B (50 uL/hole) for 10min; adding a stop solution, 50 uL/well; the OD value is read at 450nm (reference 630 nm) on the enzyme label instrument, and the result shows that the activity reading values of the Anti-FDPs 5C1RMb1 to the Anti-FDPs 5C1RMb7 are all between 1.85 and 2.35, which is superior to the control.

TABLE 4 Activity data

Example 4 assessment of antibody stability

The antibody is placed at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, 7 days, 14 days and 21 days are taken for carrying out state observation, and activity detection is carried out on the 21 days, so that the result shows that no obvious protein state change is seen when the antibody Anti-FDPs5C1RMb3 is placed for 21 days under three examination conditions, the activity also does not have a descending trend along with the increase of the examination temperature, and the stability of the expressed antibody is indicated. Table 5 below shows the results of the detection of OD after 21 days of enzyme-linked immunosorbent assay.

Table 5 stability data

Sample concentration (ng/ml)	250	125	0
				4 ℃,21 Days sample	1.891	1.521	0.012
Sample at-80℃for 21 days	1.921	1.501	0.019
				37 ℃ And 21 days of sample	1.923	1.587	0.013

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The partial amino acid sequences according to the present application are shown in the following Table 6:

Claims

1. An anti-fibrin degradation product antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1/HCDR2/HCDR3 combination is identical to the HCDR1/HCDR2/HCDR3 combination comprising a heavy chain variable region of any one of SEQ ID NOs 18 to 21 and the LCDR1/LCDR2/LCDR3 combination is identical to the LCDR1/LCDR2/LCDR3 combination comprising a light chain variable region of SEQ ID NOs 22 or 23.

2. The antibody of claim 1, wherein the CDRs are defined by Kabat, chothia, abM, contact or IMGT systems.

3. An antibody for resisting a fibrin degradation product, which is characterized by comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 sequentially comprise SEQ ID NO 1-3 or are sequentially shown as SEQ ID NO 1-3; the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 sequentially comprise SEQ ID NO 4-6 or are sequentially shown as SEQ ID NO 4-6.

4. The antibody of any one of claims 1-3, further comprising HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4; the HFR1 to HFR4 sequentially comprise SEQ ID NO:7 to SEQ ID NO 10 or an amino acid sequence having at least 80% identity thereto; or, the sequences are shown as SEQ ID NO 7 to SEQ ID NO 10 or the amino acid sequence with at least 80% of the same; the LFR 1-LFR 4 sequentially comprises SEQ ID NO. 11-SEQ ID NO. 14 or an amino acid sequence with at least 80% identity thereto; or, the sequences are shown in sequence from SEQ ID NO. 11 to SEQ ID NO. 14 or the amino acid sequences having at least 80% identity thereto.

5. An anti-fibrin degradation product antibody comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, LFR3, LFR4 of any one of claims 1 to 3, HFR2, HFR3, HFR4, LFR1, LFR2, LFR4 of LFR 4.

6. An anti-fibrin degradation product antibody comprising a heavy chain variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs 18 to 21; or consists of the amino acid sequence shown in any one of SEQ ID NOs 18 to 21;

The light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 22 or 23; or consists of the amino acid sequence shown in SEQ ID NO. 22 or 23.

7. The antibody of any one of claims 1-6, wherein the antibody further comprises a constant region;

Optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region;

Alternatively, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD; the light chain constant region is selected from a kappa-type or lambda-type light chain constant region;

optionally, the constant region is of any one of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, and human origin;

Alternatively, the constant region is of mouse species origin;

Alternatively, the heavy chain constant region comprises an amino acid sequence shown in SEQ ID NO. 24 or at least 80% identical thereto; or consists of the amino acid sequence shown in SEQ ID NO. 24 or having at least 80% identity thereto;

alternatively, the light chain constant region comprises an amino acid sequence shown in SEQ ID NO. 25 or at least 80% identical thereto; or consists of the amino acid sequence shown in SEQ ID NO. 25 or having at least 80% identity thereto.

8. An anti-fibrin degradation product antibody, wherein said antibody comprises a heavy chain and a light chain; the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NOs 26 to 29; or consists of the amino acid sequence shown in any one of SEQ ID NOs 26 to 29; the light chain comprises an amino acid sequence shown in SEQ ID NO. 30 or 31; or consists of the amino acid sequence shown in SEQ ID NO. 30 or 31.

9. An anti-fibrin degradation product antibody that binds to the same epitope in the amino acid sequence of a fibrin degradation product as the antibody of any one of claims 1-8; or, the antibody competes with the antibody of any one of claims 1-8 for binding to a fibrin degradation product.

10. The antibody of any one of claims 1 to 9, wherein the antibody binds to a fibrin degradation product with an affinity of KD +.1.37 x 10 ^-7 M;

alternatively, the antibody is selected from any one of F (ab ') ₂, fab', fab, fv and scFv.

11. An anti-fibrin degradation product antibody conjugate comprising an antibody according to any one of claims 1 to 10;

Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody;

optionally, the antibody conjugate further comprises a solid support coupled to the antibody;

optionally, the antibody conjugate further comprises a label conjugated to the antibody;

Optionally, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

12. A reagent or kit comprising an antibody according to any one of claims 1 to 10 or an antibody conjugate according to claim 11.

13. A method of detecting a fibrin degradation product, comprising:

Contacting the antibody of any one of claims 1 to 10 or the antibody conjugate of claim 11 or the reagent or kit of claim 12 with a fibrin degradation product in a sample to be tested to form an immune complex;

optionally, the immune complex further comprises a second antibody, the second antibody being bound to the antibody;

optionally, the immune complex further comprises a second antibody, which binds to a fibrin degradation product.

14. An isolated nucleic acid, vector, cell or method of making an antibody according to any one of claims 1 to 10, wherein the nucleic acid encodes an antibody according to any one of claims 1 to 10;

The vector contains the nucleic acid;

the cell contains the nucleic acid or the vector;

the method comprises the following steps: the above-mentioned cells were cultured.

15. Use of an antibody according to any one of claims 1 to 7, an antibody conjugate according to claim 8 or a reagent or kit according to claim 9 for the detection of fibrin degradation products or for the preparation of a product for the detection of fibrin degradation products.