Anti-human matrix metalloproteinase-9 antibodies, antibody pairs, and detection kits

Abstract

The present invention belongs to the technical field of monoclonal antibody preparation, and in particular relates to anti-human matrix metalloproteinase 9 antibodies, antibody pairs and detection kits. The antibody is a first antibody or a second antibody, and the amino acid sequences of the light chain CDR1-3 of the first antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of the heavy chain CDR1-3 are respectively shown as SEQ ID NO.8-10; the amino acid sequences of the light chain CDR1-3 of the second antibody are respectively shown as SEQ ID NO.13-15, and the amino acid sequences of the heavy chain CDR1-3 are respectively shown as SEQ ID NO.18-20. The antibodies and antibody pairs provided by the present invention have high affinity and high specificity for human MMP-9, and are used to develop a double-antibody sandwich enzyme-linked immunosorbent assay system with the advantages of high detection sensitivity, good specificity, strong anti-interference ability, accurate and reliable results, strong system stability and a wide detection concentration range.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody preparation, in particular to an anti-human matrix metalloproteinase 9 antibody, an antibody pair and a detection kit. Background Art

[0002] Matrix metalloproteinases (MMPs) are endopeptidases involved in the formation and remodeling of the extracellular matrix. Matrix metalloproteinase-9 (MMP-9), also known as gelatinase B or gelatinase B, belongs to the gelatinase family of MMPs and catalyzes the degradation of large extracellular matrix components and certain cell surface proteins. MMP-9 substrates include collagen types IV, V, VII, X, and XI, core proteins of proteoglycans, gelatin, fibronectin, laminin, and elastin. Cytokines and their receptors are also substrates for MMP-9. MMP-9 is involved in many physiological processes in the human body, such as tissue modification and the release of growth factors and cytokines. MMP-9 can degrade structural complexes within the respiratory tract and lungs, such as the ECM and basement membrane, and thus participates in the remodeling of the respiratory tract and lungs. It regulates the activity of other proteases and cytokines, degrading α-antitrypsin, protecting neutrophil elastase activity, and enhancing the collagenolytic activity of collagen cells and MMP-13 in collagen colloids. MMP-9 also cleaves a 62-amino acid peptide from interleukin-8 (IL-8), increasing its chemotactic activity toward neutrophils by 10-fold, but it also inhibits other neutrophil chemotactic factors. Furthermore, MMP-9 is involved in numerous pathological processes. Numerous studies have demonstrated that MMP-9 promotes tumor cell growth by promoting angiogenesis and facilitates tumor cell infiltration and invasion by degrading and disrupting the basement membrane. MMP-9 levels are significantly elevated in malignant tumors, including colon, gastric, lung, and cervical cancers, making it a potential target for anti-tumor drugs. Furthermore, studies have shown that elevated MMP-9 activity can damage the corneal epithelium and lead to ocular surface barrier dysfunction. MMP-9 levels in patients with moderate to severe dry eye correlate with clinical examination results and are a non-specific inflammatory marker. Therefore, MMP-9 detection plays an important role in the diagnosis, treatment, and prognosis of many diseases.

[0003] Currently, qualitative and quantitative analysis of MMP-9 relies on immunological methods such as enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, immunoprecipitation, and immunofluorescence. The key to developing these methods is the preparation of specific monoclonal antibodies, the quality of which largely determines the sensitivity and accuracy of the immunoassay. Traditional clinical diagnostics rely on mouse monoclonal antibodies, which generally have lower affinity and specificity than rabbit monoclonal antibodies. Consequently, immunoassay systems developed based on mouse monoclonal antibodies suffer from poor sensitivity and accuracy. Summary of the Invention

[0004] Currently, there are few types of rabbit monoclonal antibodies and detection kits for detecting MMP-9, and there is a lack of diagnostic and treatment tools for related diseases. In order to solve or at least partially alleviate the above technical problems, the present application provides a rabbit monoclonal antibody against human matrix metalloproteinase 9 and an antibody pair thereof, the antibodies having high affinity and high specificity for human MMP-9, and the human MMP-9 double antibody sandwich enzyme-linked immunosorbent assay system developed using the aforementioned antibody pairing has the advantages of high detection sensitivity, good specificity, strong anti-interference ability, accurate and reliable results, strong system stability and a wide range of detection concentrations. Therefore, the present invention provides the use of the aforementioned antibodies and antibody pairs in the preparation of human MMP-9 detection kits, and provides a detection kit containing the aforementioned antibodies and antibody pairs. The present invention is achieved through the following technical solutions:

[0005] In a first aspect, the present invention provides an anti-human matrix metalloproteinase-9 antibody, selected from a first antibody or a second antibody, wherein: the amino acid sequences of CDR1-3 on the light chain variable region of the first antibody are shown as SEQ ID NOs. 3-5, respectively, and the amino acid sequences of CDR1-3 on the heavy chain variable region are shown as SEQ ID NOs. 8-10, respectively; the amino acid sequences of CDR1-3 on the light chain variable region of the second antibody are shown as SEQ ID NOs. 13-15, respectively, and the amino acid sequences of CDR1-3 on the heavy chain variable region are shown as SEQ ID NOs. 18-20, respectively.

[0006] Furthermore, the amino acid sequence of the light chain variable region of the first antibody is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7; the amino acid sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.17.

[0007] Furthermore, the amino acid sequence of the light chain of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6; the amino acid sequence of the light chain of the second antibody is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.16.

[0008] Furthermore, the first antibody and / or the second antibody is a full-length antibody or the antigen-binding region of the full-length antibody; the antigen-binding region is selected from Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv or sc(Fv)2.

[0009] The second aspect of the present invention provides an anti-human matrix metalloproteinase-9 antibody pair, consisting of the first antibody and the second antibody as described above.

[0010] The third aspect of the present invention provides the use of the anti-human matrix metalloproteinase-9 antibody or antibody pair described above in the preparation of a human matrix metalloproteinase-9 detection kit.

[0011] A fourth aspect of the present invention provides a human matrix metalloproteinase-9 detection kit, comprising the first antibody and / or the second antibody as described above.

[0012] Furthermore, the detection kit is an enzyme-linked immunosorbent assay kit, an enzyme-linked immunosorbent assay kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit or a flow cytometry kit.

[0013] Furthermore, the detection kit is a double antibody sandwich enzyme-linked immunosorbent assay kit, comprising a first antibody and a second antibody, wherein the first antibody serves as a capture antibody, the second antibody serves as a detection antibody, and the second antibody is connected to a detection label.

[0014] The fifth aspect of the present invention provides a nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the first antibody or the second antibody as described above.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are:

[0016] The two antibodies provided by the present invention have high affinity and specificity for human matrix metalloproteinase-9 (MMP-9), with affinity constants of 0.432 nM and 0.897 nM, respectively. The antibodies specifically recognize and bind to human MMP-9, with no cross-reactivity with related proteins. The human MMP-9 double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) system developed using these two antibodies offers advantages such as high sensitivity, good specificity, strong anti-interference capabilities, accurate and reliable results, strong system stability, and a wide detection concentration range. This system has significant scientific and practical value for the industrial detection of human MMP-9 and the diagnosis and treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0018] Figure 1 A map of the vector used to construct the rabbit monoclonal antibody expression vector in Example 1 of the present invention, including, from left to right, the pBR322 vector carrying the light chain constant region and the heavy chain constant region;

[0019] Figure 2 This is an affinity curve of the binding of monoclonal antibody 3F5 to human MMP-9 protein in Example 2 of the present invention;

[0020] Figure 3 This is an affinity curve of the monoclonal antibody 3G3 binding to human MMP-9 protein in Example 2 of the present invention;

[0021] Figure 4 This is the standard curve for detecting human MMP-9 protein using a double antibody sandwich ELISA system established based on monoclonal antibodies 3F5 and 3G3 in Example 3 of the present invention;

[0022] Figure 5 This is a graph showing the specificity determination results of the double-antibody sandwich enzyme-linked immunosorbent assay system established based on monoclonal antibodies 3F5 and 3G3 in Example 4 of the present invention;

[0023] Figure 6 This is a graph showing the thermal stability test results of the double antibody sandwich enzyme-linked immunosorbent assay system established based on monoclonal antibodies 3F5 and 3G3 in Example 5 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. The embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] Given the information contained herein, it will be readily apparent to those skilled in the art that various modifications may be made to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the processes, properties, or components defined herein, as these embodiments and other descriptions are intended only to illustrate specific aspects of the present invention. Indeed, various modifications to the embodiments of the present invention that are apparent to those skilled in the art or related fields are intended to be within the scope of the appended claims.

[0026] For a better understanding of the present invention and not to limit the scope of the present invention, all numerals and other numerical values ​​used in the present invention to express amounts, percentages, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant digits and by conventional rounding methods.

[0027] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those skilled in the art.

[0028] The terms "include," "comprising," "containing," "having," and the like are non-restrictive in meaning, i.e., other steps and other ingredients that do not affect the result may be added. The term "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is considered to include the following: (i) A, (ii) B, and (iii) A and B. The terms "first," "second," and the like are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence, and it should be understood that such usage is interchangeable where appropriate.

[0029] The terms "rabbit monoclonal antibody," "monoclonal antibody," "rabbit antibody," and "rabbit monoclonal antibody" have synonymous meanings and, unless otherwise specified, refer to antibodies that specifically bind to human matrix metalloproteinase-9 (MMP-9). The modifier "rabbit" indicates that the complementarity-determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. The terms "human matrix metalloproteinase-9," "human MMP-9," "Human MMP-9," "Human MMP9," and similar terms have synonymous meanings.

[0030] An antibody is an immunoglobulin molecule that is capable of specifically binding to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and genetic or chemical modifications thereof, as long as they exhibit the desired antigen-binding activity.

[0031] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types: kappa (κ) and lambda (λ); heavy chains can be classified into five types: μ, δ, γ, α, and ε, which define antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of heavy and heavy chains vary greatly, while the remaining amino acid sequences are relatively constant. The regions of the light and heavy chains with the most variable amino acid sequences near the N-terminus are called the variable region (V), while the regions with relatively stable amino acid sequences near the C-terminus are called the constant region (C). The heavy chain variable region (VH) and light chain variable region (VL) are generally the most variable parts of antibodies and contain the antigen recognition site. The VH and VL regions can be further subdivided into hypervariable regions (HVRs) and framework regions (FRs). The HVRs, also known as complementarity-determining regions (CDRs), are circular structures. The heavy and light chain CDRs are closely aligned and interact with each other through the FRs, forming a surface that complements the three-dimensional structure of the target antigen or epitope. This determines the antibody's specificity and is the site of antigen recognition and binding. The FRs are the more conserved portions of the VH and VL sequences. They generally follow a β-pleated sheet configuration and are connected by three CDRs that form a connecting loop. Each VH and VL sequence typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0032] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, a cumulative of the Kabat and Chothia definitions, the AbM definition, the contact definition, the IMGT unique numbering definition and / or the conformational definition, or any CDR determination method known in the art. As used herein, the Kabat numbering system is used to define CDRs.

[0033] The light chain constant region (CL) and heavy chain constant region (CH) are not directly involved in antibody-antigen binding, but they exhibit different effector functions, such as antibody-dependent cytotoxicity. The CL lengths of different Ig types (κ or λ) are essentially identical, but the CH lengths of different Ig classes vary. For example, IgG, IgA, and IgD comprise CH1, CH2, and CH3, while IgM and IgE comprise CH1, CH2, CH3, and CH4. The amino acid sequences of the heavy and light chain constant regions of antibodies are well known in the art.

[0034] A full-length antibody is the most complete antibody molecular structure and has a typical Y-shaped molecular structure. Therefore, in the context of the present invention, "full-length antibody", "intact antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0035] Antibody fragments are one or more parts or fragments of a full-length antibody that essentially retain the same biological function or activity as the full-length form. Specifically, an antibody fragment includes at least the same CDR regions as the full-length antibody, and more preferably the same variable regions, thereby retaining complete antigen recognition and binding sites and being able to bind to the same antigen as the full-length antibody, particularly to the same epitope. Typical examples include: Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, and sc(Fv)2. These antibody fragments can be obtained using conventional techniques in the art.

[0036] (i) Fab: An antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable and constant regions) and a portion of the heavy chain (variable and first constant regions). By proteolytic cleavage of the full-length antibody, fragments such as Fab, F(ab')2, and Fab' can be obtained. For example, IgG can be degraded into two Fab fragments and an Fc fragment by papain; and into an F(ab')2 fragment and a pFc' fragment by pepsin. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because Fab contains the antigen-binding region and a portion of the constant region, it not only possesses the same antibody-antigen affinity and excellent tissue penetration as scFv, but also has a more stable structure.

[0037] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0038] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, contains only the variable region, and is composed of the variable regions of a light chain and a heavy chain. It is a dimer of VH and VL non-covalently bound (VH-VL dimer). The three CDRs of each variable region interact with each other to form an antigen binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind to antigens, although the affinity is lower than that of the intact antibody.

[0039] (iv) (Fv)2: Consists of two covalently linked Fv fragments.

[0040] (v) scFv: A single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, consisting of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (typically consisting of 10-25 amino acids). It retains the antigen-binding specificity of the original antibody. The linker in this invention is not particularly limited, as long as it does not hinder the expression of the antibody variable regions connected to it. Compared to full-length antibodies, scFv has a smaller molecular weight, resulting in higher penetration and lower immune side effects.

[0041] (vi) The sc(Fv)2 fragment is composed of two heavy chain variable regions and two light chain variable regions connected by a linker or the like.

[0042] The terms "monoclonal antibody" or "single antibody" and other similar terms are used interchangeably and refer to a homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for a small amount of mutations and / or post-translational modifications (e.g., isomerization, amidation) that may occur naturally. "Monoclonal antibodies" are highly specific and exhibit a single binding specificity and affinity for the same or substantially identical epitope on the antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody population and should not be construed as limiting the source or preparation method of the antibody. The antibody can be prepared by a variety of methods, including but not limited to hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

[0043] The term "specific binding" is a well-known term in the art, and a molecule exhibits "specific binding" if it reacts with a specific target antigen or epitope more frequently, more rapidly, longer-lastingly, and / or with greater affinity than with other target antigens or epitopes. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0044] In order to make the above-mentioned objects and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below.

[0045] The present invention has successfully developed two high-affinity rabbit monoclonal antibodies against human matrix metalloproteinase-9 (MMP-9), with affinity constants K DThe antibodies have high specificity and excellent thermal stability, specifically recognizing and binding to human MMP-9, with no cross-reactivity with related proteins. Protein-protein interaction non-labeled validation demonstrated that they bind to distinct antigenic determinants on the surface of human MMP-9, making them suitable for use as paired antibodies in a double-antibody sandwich ELISA assay for human MMP-9. One of the antibodies is used as a capture antibody to coat a microplate to create a solid-phase antibody. MMP-9 antigen is then added, and the other antibody is biotinylated with the detection antibody to form a double-antibody sandwich ELISA system (capture antibody-antigen-biotinylated detection antibody). After treatment with streptavidin-labeled avidin and development with the substrate TMB, quantitative detection of human MMP-9 is possible with a detection limit as low as 11.53 pg / mL. This assay exhibits high sensitivity, good specificity, strong anti-interference ability, accurate and reliable results, robust system stability, and a wide detection concentration range, making it of great significance for clinical diagnosis and scientific research applications.

[0046] Based on this, one embodiment of the present invention provides an anti-human matrix metalloproteinase-9 antibody, selected from a first antibody or a second antibody, wherein the first antibody and the second antibody both include a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region both include three complementarity determining regions (CDRs), respectively named CDR1, CDR2 and CDR3; wherein: the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. Shown in NO.20.

[0047] The antibodies containing the above-mentioned CDR sequences prepared by the present invention have high affinity and high specificity for human matrix metalloproteinase-9 (MMP-9), providing excellent antibody materials for qualitative or quantitative detection of human MMP-9 levels by immunological means, which is beneficial to significantly improve the accuracy, specificity, sensitivity, stability and reliability of immunoassay results.

[0048] Optionally, the light chain variable region and the heavy chain variable region each include four framework regions (FRs), and the four FRs and three CDRs are arranged in a staggered order to form a variable region. The amino acid sequence of the light chain variable region (VL) of the first antibody is shown in SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO. 7. The amino acid sequence of the light chain variable region (VL) of the second antibody is shown in SEQ ID NO. 12, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO. 17.

[0049] Optionally, the first antibody and the second antibody further comprise a light chain constant region (CL) and a heavy chain constant region (CH), wherein the CL and VL of each antibody constitute a light chain (FL), and the CH and VH constitute a heavy chain (FH). Antibody constant regions can generally be obtained through public searches, such as searching the IMGT online database (www.imgt.org) for rabbit IgG gamma C reign to obtain CH, and searching for rabbit IgG kappa C reign to obtain CL.

[0050] Specifically, the amino acid sequence of the light chain of the first antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 6. The amino acid sequence of the light chain of the second antibody is shown in SEQ ID NO. 11, and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 16.

[0051] Optionally, the first antibody and / or the second antibody is a full-length antibody (having a typical Y-shaped molecular structure) or an antigen-binding region of the full-length antibody; the antigen-binding region refers to a polypeptide that substantially maintains the same biological function or activity as the full-length form of a rabbit monoclonal antibody. Specifically, the antigen-binding region includes the CDR region as described above, more preferably having the variable region as described above, thereby retaining a complete antigen recognition and binding site, and being able to bind to the same antigen as the full-length antibody, especially to the same epitope. Optionally, the antigen-binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv and sc(Fv)2. These antigen-binding regions can be obtained by conventional techniques in the art.

[0052] Based on this, another embodiment of the present invention provides an anti-human matrix metalloproteinase-9 antibody pair, consisting of the first antibody and the second antibody as described above.

[0053] The first antibody and the second antibody of the present invention bind to different antigenic epitopes of human MMP-9 and can be used as paired antibodies in a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) detection system. The double-antibody sandwich ELISA system is constructed with the first antibody as the capture antibody and the second antibody as the detection antibody. The system has the advantages of high detection sensitivity, good specificity, strong anti-interference ability, accurate and reliable results, strong system stability, and a wide detection concentration range. It also effectively improves the minimum detection limit, with the detection concentration as low as 11.53 pg / mL, providing an effective detection means for the high-precision detection of trace levels of human MMP-9 protein in biological samples.

[0054] Based on the same inventive concept as above, another embodiment of the present invention provides the use of the above-mentioned anti-human matrix metalloproteinase-9 antibody or antibody pair in the preparation of a human matrix metalloproteinase-9 detection kit.

[0055] The advantages of using the anti-human MMP-9 antibody or antibody pair in preparing a human MMP-9 detection kit are the same as the advantages of the anti-human MMP-9 antibody or antibody pair over the prior art described above, and will not be repeated here.

[0056] Based on the same inventive concept as above, another embodiment of the present invention provides a human matrix metalloproteinase-9 detection kit, which includes the first antibody and / or the second antibody as described above.

[0057] It should be emphasized that the first antibody and the second antibody can be used separately, together, or in pairs. During detection, if used separately or together, the first antibody and / or the second antibody serve as a primary antibody or capture antibody, the sample to be tested is contacted with the first antibody and / or the second antibody, and then the antibody is detected. In some embodiments, the first antibody and / or the second antibody can be linked (covalently or non-covalently) to a detection label, and qualitative or quantitative detection of MMP-9 is achieved by analyzing the change in the identifiable signal generated by the detection label. In other embodiments, the anti-human MMP-9 antibody (as a primary antibody or capture antibody) is not labeled, and the detection label is linked to a secondary antibody (as a detection antibody) or other molecule that can bind to the primary antibody. For example, if the anti-human MMP-9 antibody is a rabbit IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody, thereby generating a change in the identifiable signal by the secondary antibody linked to the detection label. If used in pairs, one of the first and second antibodies serves as the primary antibody or capture antibody, and the other serves as the secondary antibody or detection antibody.

[0058] The aforementioned detection methods utilize conventional immunological methods, including but not limited to enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB), and flow cytometry (FC). Detection targets include recombinantly expressed MMP-9 protein, cell-secreted MMP-9 protein, or MMP-9 protein in human serum or plasma. Test samples include but are not limited to serum, plasma, urine, cell culture fluid, and tissue homogenate.

[0059] Preferably, the detection kit is a double antibody sandwich enzyme-linked immunosorbent assay kit, comprising a first antibody and a second antibody, wherein the first antibody serves as a capture antibody (or primary antibody), and the second antibody connected to a detection label serves as a detection antibody (or secondary antibody).

[0060] The detection labels for generating a recognizable signal change include, but are not limited to, biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (such as allophycocyanin, phycoerythrin, PerCP and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies or combinations thereof.

[0061] The examples of the present application also disclose a preferred method for preparing the above-mentioned anti-human matrix metalloproteinase-9 antibody, which is obtained by genetic engineering recombination technology; specifically, DNA molecules encoding the heavy chain and light chain genes of the antibody of the present application are obtained by chemical synthesis or PCR amplification, the obtained DNA molecules are inserted into expression vectors, and then transfected into host cells. The transfected host cells are cultured under appropriate conditions to express the antibody of the present invention.

[0062] Based on this, one embodiment of the present invention further provides a nucleic acid molecule, a recombinant vector or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the first antibody and / or the second antibody as described above.

[0063] Nucleic acid molecules can be in the form of DNA (such as cDNA, genomic DNA or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0064] The sequence of the nucleic acid molecule can be derived from the antibody amino acid (AA) sequence using conventional methods, such as codon encoding rules. The full-length sequence of the nucleic acid molecule or fragments thereof can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. The resulting nucleic acid molecule is inserted into an expression vector, then introduced into host cells and cultured under specific conditions to express the antibody.

[0065] The original vector for constructing the recombinant vector is various vectors conventional in the art, as long as it can hold the nucleic acid molecule. Typical vectors include plasmids (e.g., pBR322, pUC series, pET series pGEX series), viral vectors, bacteriophages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), cosmids, and minichromosomes. The vector can be a cloning vector (i.e., for transferring the nucleic acid molecule to a host and multiplying in a large number of host cells) or an expression vector (i.e., comprising the necessary genetic elements to allow the nucleic acid molecule inserted into the vector to be expressed in a host cell). The nucleic acid molecule of the present invention can be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule.

[0066] The nucleic acid molecules encoding the antibodies FL and FH of the present invention can be inserted into two vectors, respectively, which can be introduced into the same or different host cells. When the heavy chain and light chain are expressed in different host cells, each chain can be isolated from the host cell expressing it, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions to form the antibody. In other embodiments, the nucleic acid molecules of the antibodies FL and FH can also be cloned into a single vector, with each nucleic acid sequence linked to a suitable promoter downstream; for example, each nucleic acid sequence encoding the heavy chain and light chain can be operably linked to a different promoter, or the nucleic acid sequence encoding the heavy chain and light chain can be operably linked to a single promoter so that both the heavy chain and the light chain can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0067] Conventional techniques are used to transfect or transform the recombinant vector into host cells. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with CaCl2 or MgCl2. Alternatively, transfection can be accomplished by microinjection, electroporation, or liposome packaging. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, or particle bombardment to achieve gene introduction.

[0068] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present invention include, but are not limited to, Escherichia coli (e.g., DH5α, JM109, BL21, W3110), Bacillus (e.g., Bacillus subtilis, Bacillus thuringiensis), and Enterobacteriaceae strains (e.g., Salmonella typhimurium, Serratia marcescens), and Pseudomonas. Examples of eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining a host cell transfected or transformed with the recombinant vector described above, the antibody can be expressed by culturing under suitable conditions, and then separated to obtain purified antibodies.

[0069] In a preferred embodiment, the recombinant vector is the mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell (293F cell).

[0070] The present invention will be further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer.

[0071] Example 1 Preparation of human MMP-9 rabbit monoclonal antibody

[0072] Based on the monoclonal antibody development technology of single B lymphocyte screening and culture, New Zealand white rabbits were immunized with human matrix metalloproteinase-9 (MMP-9, from ABclonal, catalog number RP00103) as the immunogen; B lymphocytes were sorted from the rabbit spleen and cultured; RNA was extracted from the antigen-specific B lymphocytes and reverse transcribed into cDNA; the cDNA was amplified by PCR to obtain the genes of the heavy chain variable region (VH) and light chain variable region (VL) of the naturally paired rabbit monoclonal antibody; these genes were loaded into expression vectors containing the heavy chain constant region and the light chain constant region, respectively, and the vectors were transfected into host cells. The host cells were cultured and the target antibodies 3F5 and 3G3 were isolated and purified.

[0073] The antibodies were sequenced by Jinkairui Biotechnology Co., Ltd. The amino acid (AA) sequences of the antibodies are shown in Tables 1-2. For ease of description, the light chain complementary determining regions (CDRs) 1-3 are designated LCDRs 1-3, and the heavy chain complementary determining regions (CDRs) 1-3 are designated HCDRs 1-3.

[0074] Table 1 Sequence information of rabbit monoclonal antibody 3F5 in this example

[0075]

[0076]

[0077] Table 2 Sequence information of rabbit monoclonal antibody 3G3 in this example

[0078]

[0079] In the above embodiment, the specific steps include:

[0080] 1. Animal Immunization: Each rabbit was immunized with 200 μg of antigen (from ABclonal, Catalog No. RP00103). Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple points on the rabbit's abdomen and back. After the first immunization, 100 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple points on the rabbit's abdomen and back every three weeks for two booster immunizations. After three immunizations, rabbit serum samples were collected and their titers against human MMP-9 were determined by ELISA. Rabbits with high serum titers were boosted once with 200 μg of the immunogen by multiple subcutaneous injections. Three days later, the animals were sacrificed and their spleens were removed.

[0081] 2. Isolate spleen cells: Aseptically remove a culture dish in a safety cabinet, add 30-40 mL of basal culture medium (RPMI Medium 1640basic + 1% Pen Strep; RPMI 1640 purchased from Gibco, product number C11875500BT; PenStrep purchased from Gibco, product number 15140-163), place a cell sieve, remove the spleen and place it in the cell sieve, cut off excess connective tissue and fat on the rabbit spleen tissue, cut the spleen tissue into pieces and place it in the cell sieve for grinding, take a clean grinding rod, and use the end of the pressing part to crush and grind the tissue to free the cells in the membrane. After passing through the cell sieve, suspend them in the culture dish solution; wash the cell sieve with 10 mL of basal culture medium, and collect the basal culture medium outside the cell sieve. Centrifuge at room temperature for 5 minutes at 400 g, remove the supernatant, retain the cells, add 13 mL of room temperature RBC red blood cell lysis solution (purchased from BioGems), gently blow off the cell clusters with a pipette and time for 1 minute to perform red blood cell lysis, add 37 mL of basal culture medium and mix well to terminate red blood cell lysis, centrifuge at room temperature for 5 minutes at 400 g, remove the supernatant, retain the cells, add 40 mL of basal culture medium placed at room temperature, gently blow off the cell clusters with a pipette, resuspend the cells, complete the first wash, centrifuge at room temperature for 5 minutes at 400 g, remove the supernatant, retain the cells, add 20 mL of basal culture medium placed at room temperature, gently blow off the cell clusters with a pipette, and resuspend the cells; filter the resuspended cells again through a cell sieve to remove the clumping cells, and then count the cells.

[0082] 3. Sorting B lymphocytes: Conventional methods are used to isolate B lymphocytes from the spleen and sort them to obtain antigen-specific B lymphocytes. For related methods, please refer to the patent "Method for Efficiently Isolating Single Antigen-Specific B Lymphocytes from Spleen Cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)" and the patent "A B Lymphocyte In Vitro Culture System and Application (Publication No.: CN111518765A, Publication Date: 2020-08-11)".

[0083] 4. Cloning of rabbit monoclonal antibody gene: The supernatant of cultured B lymphocytes was used to identify positive clones by ELISA coated with MMP-9 antigen. The positive clone cells were collected and lysed and then analyzed by Quick-RNA TM RNA was extracted using a Micro Prep Kit (ZYMO, Cat. No. R1051) and reverse transcribed into cDNA. Using this cDNA as a template, naturally paired rabbit monoclonal antibody light chain variable regions (VL) and heavy chain variable regions (VH) were amplified from the cDNA of the corresponding positive clones using PCR and sequenced.

[0084] The PCR reaction system consisted of 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2× Gloria HiFi (ABclonal, Cat. No. RK20717), and 6.5 μL HO. The PCR amplification procedure included initial denaturation at 98°C for 30 seconds, followed by 40 cycles of 98°C for 10 seconds, 64°C for 30 seconds, and 72°C for 30 seconds, followed by a final hold at 72°C for 5 minutes. The resulting reaction solution was stored at 4°C. The primer sequences (5'-3') for amplifying the VL and VH genes are shown in Table 3, where F and R represent the forward and reverse primers, respectively.

[0085] Table 3 Primer sequence information for amplifying the rabbit monoclonal antibody of this example

[0086] Primer name Sequence information VL-F tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO. 21) VL-R cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 22) VH-F tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 23) VH-R gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 24)

[0087] 5. Production and Purification of Rabbit Monoclonal Antibodies: To obtain multiple rabbit monoclonal antibodies that recognize human MMP-9, the mammalian expression vector pBR322, carrying the light chain constant region (CL) and heavy chain constant region (CH) genes, was linearized using XbaI and NheI restriction endonucleases, respectively. The PCR-amplified VL and VH genes containing the signal peptides were purified and then constructed into the aforementioned expression vectors by homologous recombination to obtain light chain and heavy chain gene expression vectors. Sequencing confirmed the successful construction of the expression vectors.

[0088] The expression patterns of the above-mentioned vectors are shown in Figure 1 The pBR322 origin and f1 origin are replication promoters, Ampcillin is the resistance gene, CMV promoter is the transcription promoter, SV40 PAterminator is the tailing signal, Light chain constant is the nucleotide sequence of the light chain constant region (left), and Heavy chain constant is the nucleotide sequence of the heavy chain constant region (right). The CL and CH genes were obtained by searching the IMGT online database (www.imgt.org) for rabbit IgG gamma C reign to obtain CH, and rabbit IgG kappa C reign to obtain CL.

[0089] The signal peptide of this embodiment can adopt the antibody expression signal peptide sequence commonly used in the art, such as the patent "Rabbit monoclonal antibody against human interferon α2 and its application (publication number: CN116063487A, publication date: 2023-05-05)" and the patent "High-affinity Human IL-5 rabbit monoclonal antibody and its application (publication number: CN115819578A, publication date: 2023-03-21)". The light chain variable region upstream has a signal peptide "MDTRAPTQLLGLLLLWLPGATF" or "MDTRAPTQLLGLLLLWLPGARC", and the heavy chain variable region upstream has a signal peptide "METGLRWLLLVAVLKGVQC".

[0090] Expression vectors containing both the light and heavy chain genes were transfected into 293F cells and cultured for 72-96 hours. The culture supernatant contained recombinant rabbit monoclonal antibodies that recognized human MMP-9. The target antibody was purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, Catalog No. SA023100). The purified antibody was aliquoted and stored at -20°C until ready for use.

[0091] Example 2 Affinity determination of rabbit monoclonal antibodies 3F5 and 3G3

[0092] Antibody affinity was assessed using the ProbeLife Gator Biomolecular Interaction Analyzer (Gator Prime) using a Protein A probe. Test antibodies 3F5 and 3G3 were immobilized on the Protein A probe at concentrations of 8.72 μg / mL for 3F5 and 10.1 μg / mL for 3G3. Affinity curves were then generated for the two antibodies using recombinant human MMP-9 at concentrations of 75 nM and 150 nM, respectively.

[0093] The affinity curves of antibodies 3F5 and 3G3 binding to human MMP-9 are shown in Figure 2-3 The ordinate represents the change in thickness of the conjugate after the probe binds to the antibody and protein, the abscissa represents the binding time, the dark gray curve represents the real-time binding value curve, and the light gray curve represents the fitted average value curve. The affinity constants calculated by curve fitting are shown in Table 4.

[0094] Table 4 Affinity-related parameter determination results of rabbit monoclonal antibodies

[0095] Antibody <![CDATA[K off (1 / s)]]> <![CDATA[K on (1 / Ms)]]> <![CDATA[K D (M)]]> 3F5 <![CDATA[1.37×10 -4 ]]> <![CDATA[3.16×10 5 ]]> <![CDATA[4.32×10 -10 ]]> 3G3 <![CDATA[2.94×10 -4 ]]> <![CDATA[3.28×10 5 ]]> <![CDATA[8.97×10 -10 ]]>

[0096] The results showed that the dissociation rate constants of antibodies 3F5 and 3G3 against human matrix metalloproteinase-9 were 1.37×10 -4, 2.94×10 -4 ; The binding rate constants were 3.16×10 5 , 3.28×10 5 ; The dissociation equilibrium constant is 4.32×10 -10 , 8.97×10 -10 , showing that the antibody has a high affinity for human MMP-9.

[0097] Example 3 Establishment of a double-antibody sandwich enzyme-linked immunosorbent assay based on antibodies 3F5 and 3G3 and its sensitivity determination

[0098] Detection antibody 3G3 biotin labeling: Take 100μg of antibody 3G3, add 10μg of biotin (purchased from Biosai Biotechnology, product number B5064), mix thoroughly after addition, incubate at 4℃ overnight, and set aside.

[0099] A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established using antibody 3F5 as the capture antibody and biotin-labeled antibody 3G3 as the detection antibody. The steps are as follows:

[0100] 1) Coating with capture antibody 3F5: Dilute antibody 3F5 to 2 μg / mL with 1× PBS, mix thoroughly on a vortexer, and add 100 μL / well to a 96-well microplate. Cover with film and incubate at 4°C for 16-20 hours.

[0101] 2) Washing: After incubation, discard the liquid in the wells, wash the plate once with 1× PBST, add 300 μL of sample, let it stand for 40 seconds, then discard the liquid in the wells and pat dry on a tissue paper;

[0102] 3) Blocking: Add 200 μL / well of blocking solution (1× PBS containing 2% BSA, 5% sucrose, 0.05% Tween 20, and 0.1% proclin 300, pH 7.2) to each well. Cover with a cover film and block at 37°C for 2 h. After blocking, discard the blocking solution, pat the plate dry, and dry it in a 37°C oven for 0.5-2 h before removing it for later use.

[0103] 4) Adding Antigen Protein: Human MMP-9 protein (purchased from RD, Catalog No. DY911, Component No. 841030) was serially diluted in blocking buffer (1× PBS containing 2% BSA, 0.05% Tween 20, and 0.1% proclin 300, pH 7.2) to the following concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, and 0 pg / mL. The dilutions were then added to the ELISA plate at 100 μL / well, covered with a cover film, and incubated at 37°C for 2 h.

[0104] 5) Washing the plate: Same as step 2);

[0105] 6) Add detection antibody 3G3: Dilute the biotin-labeled antibody 3G3 (3G3-biotin) to 0.25 μg / mL and add 100 μL / well to the ELISA plate. Cover with a cover film and incubate at 37°C for 1 hour.

[0106] 7) Washing the plate: Same as step 2);

[0107] 8) Adding SA-HRP: Dilute 100× SA-HRP (horseradish peroxidase-labeled streptavidin, purchased from Wuhan Sanying Biotechnology Co., Ltd., catalog number SA00001-0) concentrate 100-fold and add 100 μL / well to the ELISA plate. Cover with a cover film and incubate at 37°C for 0.5 h.

[0108] 9) Washing the plate: Same as step 2);

[0109] 10) Add color development solution: Add 3,3',5,5'-tetramethylbenzidine (TMB) color development solution (purchased from Tetramethylpyridamole, product number 4ATMB1000) to the ELISA plate at 100 μL / well, cover with cover film, and incubate at 37°C for 15 min;

[0110] 11) Reading: After incubation, remove the microplate and add 50 μL of stop solution (1 mol / L hydrochloric acid) to each well. Immediately read the plate at 450 nm using a microplate reader.

[0111] The concentration of human MMP-9 is used as the horizontal axis, and the absorbance value OD 450nm As the vertical axis, the standard curve is obtained (see Figure 4 ), demonstrating good linearity and high accuracy in detecting human MMP-9. The sensitivity of the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was determined by substituting the average absorbance value of 16 blank wells (where the antibody solution was replaced with diluent) and twice the standard deviation into the standard curve. The resulting concentration was the sensitivity of the double-antibody sandwich enzyme-linked immunosorbent assay for human MMP-9. The sensitivity of this antibody pair was 11.53 pg / mL. The results are shown in Table 5.

[0112] Table 5 Sensitivity test data of double antibody sandwich ELISA based on antibodies 3F5 and 3G3

[0113]

[0114] Example 4 Cross-reaction test of a double antibody sandwich ELISA system based on antibodies 3F5 and 3G3

[0115] A variety of proteins similar to Human MMP-9 (purchased from RD, product number DY911, component number 841030) were used for cross-reaction to determine the antigen recognition specificity of the double antibody sandwich ELISA detection system. The cross-reaction test proteins are: Human NGAL / Lipocalin-2 (from ABclonal, product number RP01823), Human MMP-2 (purchased from RD, product number DY902, component number 843029), Human MMP-3 (from ABclonal, product number RP00220) and Human TIMP-4 (purchased from RD, product number 974-TSF-010). The double antibody sandwich ELISA method in the above Example 3 was used for detection, and the concentration of the standard protein was 2000pg / mL. The results are shown in Figure 5 , in the figure, Positive Control represents Human MMP-9.

[0116] like Figure 5 As shown, when the rabbit monoclonal antibodies 3F5 and 3G3 of the present invention were used to perform double antibody sandwich ELISA detection on multiple proteins, the reaction system only bound to human MMP-9 and did not produce any cross-reactions with other proteins, indicating that antibodies 3F5 and 3G3 have high specificity for human MMP-9 protein.

[0117] Example 5 Thermal stability test of a double antibody sandwich ELISA system based on antibodies 3F5 and 3G3

[0118] The ELISA plate coated with capture antibody 3F5, freeze-dried human MMP-9 protein, and 100× concentrated detection antibody 3G3 were sealed and stored at -20°C and 37°C for 7 days, respectively. The double antibody sandwich ELISA method in Example 3 was used for detection. The coefficient of variation of the standard curves established under different treatment conditions was compared, and the thermal stability of the detection system constructed with antibodies 3F5 and 3G3 was analyzed. The results are shown in Tables 6 and Figure 6 .

[0119] Table 6 Stability test data of double antibody sandwich ELISA based on antibodies 3F5 and 3G3

[0120]

[0121] The results showed that the coefficient of variation of the concentration of Human MMP-9 protein detected by antibodies 3F5 and 3G3 treated at -20°C and 37°C, respectively, was less than 10%, indicating that the detection system of the present invention has strong thermal stability.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-human matrix metalloproteinase-9 antibody, characterized in that: is a primary antibody or a secondary antibody, wherein: The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the first antibody are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively; The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, respectively.

2. The anti-human matrix metalloproteinase-9 antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the first antibody is shown in SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7; The amino acid sequence of the light chain variable region of the second antibody is shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

17.

3. The anti-human matrix metalloproteinase-9 antibody according to claim 2, characterized in that The amino acid sequence of the light chain of the first antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6; The amino acid sequence of the light chain of the second antibody is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

16.

4. The anti-human matrix metalloproteinase-9 antibody according to claim 1, characterized in that The first antibody and / or the second antibody is a full-length antibody or an antigen-binding region of the full-length antibody; The antigen binding region is selected from Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv or sc(Fv)2.

5. An anti-human matrix metalloproteinase-9 antibody pair, characterized in that: The method comprises the first antibody and the second antibody as described in any one of claims 1 to 4.

6. Use of the anti-human matrix metalloproteinase-9 antibody according to any one of claims 1 to 4 or the anti-human matrix metalloproteinase-9 antibody pair according to claim 5 in preparing a human matrix metalloproteinase-9 detection kit.

7. A human matrix metalloproteinase-9 detection kit, characterized in that: The method comprises the anti-human matrix metalloproteinase-9 antibody according to any one of claims 1 to 4 or the anti-human matrix metalloproteinase-9 antibody pair according to claim 5.

8. The human matrix metalloproteinase-9 detection kit according to claim 7, characterized in that: The detection kit is an enzyme-linked immunosorbent assay kit, an enzyme-linked immunosorbent assay kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit or a flow cytometry kit.

9. The human matrix metalloproteinase-9 detection kit according to claim 8, characterized in that: The detection kit is a double antibody sandwich enzyme-linked immunosorbent assay kit, comprising a first antibody and a second antibody, wherein the first antibody serves as a capture antibody, the second antibody serves as a detection antibody, and the second antibody is connected to a detection label.

10. A nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the anti-human matrix metalloproteinase-9 antibody according to any one of claims 1 to 4.

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