Antibodies and antibody pairs for detecting mouse monocyte chemoattractant protein 1 and their applications

Abstract

The present invention belongs to the field of immunological detection technology, and particularly relates to antibodies and antibody pairs for detecting mouse monocyte chemoattractant protein 1 and their applications. The antibody is a first antibody or a second antibody, and the amino acid sequences of CDR1-3 on the light chain of the first antibody are shown in SEQ ID NO.3-5, respectively, and the amino acid sequences of CDR1-3 on the heavy chain are shown in SEQ ID NO.8-10, respectively; the amino acid sequences of CDR1-3 on the light chain of the second antibody are shown in SEQ ID NO.13-15, respectively, and the amino acid sequences of CDR1-3 on the heavy chain are shown in SEQ ID NO.18-20, respectively. The antibody provided by the present invention has high affinity and high specificity for mouse monocyte chemoattractant protein 1, and has the advantages of good detection specificity, extremely high detection sensitivity and good accuracy for immune pairing detection of mouse monocyte chemoattractant protein 1.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and in particular to an antibody and an antibody pair for detecting mouse monocyte chemotactic protein 1 and applications thereof. Background Art

[0002] Monocyte chemotactic protein 1 (MCP-1), also known as chemokine (CCL2), is a small molecular weight cytokine of the CC chemokine family. MCP-1 is located on chromosome 17 in the human genome and encodes a 99 amino acid monomer polypeptide with a theoretical molecular weight of 11KD. Mouse MCP-1 consists of 148aa and has a theoretical size of 16KD. Both human and mouse MCP-1 have glycosylation modification sites. MCP-1 is anchored to the plasma membrane of endothelial cells through the mucopolysaccharide side chains of proteoglycans. It is mainly secreted by monocytes, macrophages and dendritic cells. Astrocytes and microglia are also considered to be the source of MCP-1. The main biological function of MCP-1 is to recruit monocytes, memory T cells and dendritic cells to the inflammatory sites caused by tissue damage or infection. This migration process plays an important role in physiological and pathological processes such as inflammatory response, tissue repair and immune response.

[0003] MCP-1 plays an important role in a variety of pathological processes. In the adaptive immune response, inflammatory stimuli activate the expression of MCP-1, maintain and aggravate the recruitment of Th17 cells to specific locations, and subsequently produce inflammatory cytokines. Therefore, MCP-1 is associated with many inflammatory and neurodegenerative diseases, such as atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy. For example, the inflammatory response of patients with heart disease and diabetes often leads to the massive production and release of MCP-1, thereby inducing the chemotaxis of monocytes and macrophages to the site of skin lesions, ultimately leading to tissue damage and organ damage. In addition, MCP-1 has a malignant effect in the tumor microenvironment. MCP-1 and its receptor CCR2 are stimulated by TNF-α from tumor cells during tumor development, further recruiting tumor-associated macrophages, helping cancer cells escape the immune system and promoting tumor progression. For example, overexpression of MCP-1 can enhance the growth, invasion and metastasis of cancer cells in the breast cancer mouse model 4T1; MCP-1 overexpression has been detected in prostate cancer, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, renal cell carcinoma, lung cancer, colon cancer and papillary thyroid cancer. Therefore, quantitative detection of MCP-1 levels in biological samples such as peripheral blood and urine has certain clinical value for early diagnosis, prognosis and efficacy observation of certain diseases.

[0004] The immunological detection of MCP-1 is an effective method for qualitative and quantitative analysis of MCP-1 in biological samples, which is established on the basis of specific anti-MCP-1 monoclonal antibodies. By applying different monoclonal antibody markers and detection techniques, a variety of detection methods can be established, such as enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), etc. However, the anti-MCP-1 antibodies prepared in the prior art have problems such as low antibody specificity, low detection sensitivity and insufficient reliability, and are often not effective when applied to the above detection methods. Therefore, screening out monoclonal antibodies with high specificity and high affinity binding to Mouse MCP-1 has very important practical application value. Summary of the invention

[0005] In view of the problems of low antibody specificity, low detection sensitivity and / or detection reliability in the prior art, the present invention provides two high-affinity and high-specificity anti-mouse MCP-1 rabbit-derived antibodies and an antibody pair composed of the same, which are used for immune pairing detection of mouse monocyte chemoattractant protein 1 and have the advantages of good detection specificity, extremely high detection sensitivity and good accuracy. Therefore, the present invention also provides the use of the aforementioned antibodies and antibody pairs in the preparation of a mouse MCP-1 detection kit, and provides a detection kit containing the aforementioned antibodies and antibody pairs.

[0006] The present invention is specifically implemented through the following technical solutions:

[0007] The first aspect of the present invention provides an antibody for detecting mouse monocyte chemoattractant protein 1, which is 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 respectively shown as SEQ ID NO.3-5, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NO.8-10; the amino acid sequences of CDR1-3 on the light chain variable region of the second antibody are respectively shown as SEQ ID NO.13-15, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NO.18-20.

[0008] 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.

[0009] Furthermore, the amino acid sequence of the light chain of the first antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO.6; the amino acid sequence of the light chain of the second antibody is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO.16.

[0010] Furthermore, the light chain constant region of the first antibody and the second antibody is a κ chain, and the heavy chain constant region is an IgG1 type.

[0011] Furthermore, 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.

[0012] The second 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.

[0013] The third aspect of the present invention provides an antibody pair for detecting mouse monocyte chemoattractant protein 1, which consists of the first antibody and the second antibody as described above.

[0014] The fourth aspect of the present invention provides the use of the above-mentioned antibody or antibody pair for detecting mouse monocyte chemoattractant protein 1 in the preparation of a mouse monocyte chemoattractant protein 1 detection kit.

[0015] The fifth aspect of the present invention provides a detection kit for detecting mouse monocyte chemoattractant protein 1, wherein the detection kit comprises the antibody or antibody pair for detecting mouse monocyte chemoattractant protein 1 as described above.

[0016] 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, and the second antibody modified with a detection marker serves as a detection antibody.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are:

[0018] 1. The antibody containing the above CDR sequence provided by the present invention can specifically recognize and bind to mouse monocyte chemoattractant protein 1, and the antibody has high affinity, and the affinity constant K D All of them were at the 0.1nM level, providing key antibody materials for establishing quantitative detection of mouse MCP-1 protein.

[0019] 2. The two antibodies provided by the present invention recognize different antigenic epitopes on the surface of mouse monocyte chemoattractant protein-1 protein, and can form a double antibody sandwich enzyme-linked immunosorbent assay system, which provides a feasible and reliable method for accurately and efficiently detecting trace levels of mouse MCP-1 protein in samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 The vector map used to construct the rabbit monoclonal antibody expression vector in Example 1 of the present invention, from left to right are the pBR322 vector maps carrying the light chain constant region and the heavy chain constant region;

[0022] Figure 2 This is an affinity curve diagram of the binding of rabbit monoclonal antibody 6G7 to mouse MCP-1 in Example 2 of the present invention;

[0023] Figure 3 This is an affinity curve diagram of the rabbit monoclonal antibody 2H2 binding to mouse MCP-1 in Example 2 of the present invention;

[0024] Figure 4 This is a graph showing the recognition of MCP-1 antigen epitopes by rabbit monoclonal antibodies 6G7 and 2H2 in Example 2 of the present invention;

[0025] Figure 5 This is the standard curve for detecting mouse MCP-1 using the double antibody sandwich ELISA method established based on rabbit monoclonal antibodies 6G7 and 2H2 in Example 3 of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes 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 defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0028] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, 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 according to the reported significant figures and by conventional rounding methods.

[0029] 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 of ordinary skill in the art.

[0030] The meaning of the terms "including", "comprising", "containing", "having" and the like is non-restrictive, that is, other steps and other ingredients that do not affect the results can be added. The term "and / or" should be regarded 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 regarded as including the following situations: (i) A, (ii) B and (iii) A and B. The terms "first", "second", etc. are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that such usage can be interchangeable under appropriate circumstances.

[0031] The terms "rabbit monoclonal antibody", "monoclonal antibody", "rabbit antibody" and "rabbit monoclonal antibody" and other similar terms have the same meaning. Unless otherwise specified, they all refer to antibodies that specifically bind to mouse monocyte chemoattractant protein 1. The modifier "rabbit" indicates that the complementarity determining region (CDR) of the antibody is derived from a rabbit immunoglobulin sequence. The terms "mouse monocyte chemoattractant protein 1", "Mouse MCP-1", "Mouse CCL2" and other similar terms have the same meaning.

[0032] An antibody is an immunoglobulin molecule that can specifically bind 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 their genetic or chemical modifications, as long as they exhibit the desired antigen-binding activity.

[0033] 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, namely κ chain and λ chain; heavy chains can be classified into five types, namely μ, δ, γ, α and ε chains, and antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. The amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. The regions near the N-terminus of the light and heavy chains where the amino acid sequences vary greatly are called variable regions (V), and the regions near the C-terminus where the amino acid sequences are relatively stable are called constant regions (C). The heavy chain variable region (VH) and the light chain variable region (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable regions are also called complementarity determining regions (CDRs), which are ring-shaped structures. The heavy chain CDR and the light chain CDR are closely together and cooperate with each other through the FR region, forming a surface that can complement the three-dimensional structure of the target antigen or epitope, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. The FR region is the more conservative part of VH and VL. They are generally in a β-folded configuration and are connected by three CDRs that form a connecting loop. Each VH and VL is usually composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0034] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the accumulation of both the Kabat definition and the Chothia definition, 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.

[0035] The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity of antibodies. The length of CL of different types of Ig (κ or λ) is basically the same, but the length of CH of different classes of Ig is different, such as IgG, IgA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the heavy chain and light chain constant regions of antibodies are well known in the art.

[0036] 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", "complete antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0037] Antibody fragments are one or more parts or fragments of a full-length antibody, which substantially retain the same biological function or activity as the full-length form. Specifically, antibody fragments at least include the same CDR region as the full-length antibody, and more preferably have the same variable region, thereby retaining complete antigen recognition and binding sites, and can bind to the same antigen as the full-length antibody, especially to the same epitope. In typical examples, antibody fragments include: Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, sc(Fv)2, and these antibody fragments can be obtained by conventional techniques in the art.

[0038] (i) Fab: Antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable region and constant region) and a part of the heavy chain (variable region and the first constant region). By proteolytic cleavage of the full-length antibody, fragments such as Fab, F(ab')2, and Fab' can be obtained. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab')2 fragment and one pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab' fragments. Since Fab has an antigen-binding region and a part of the constant region, it not only has the same antibody-antigen affinity and excellent tissue penetration as scFv, but also has a more stable structure.

[0039] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0040] (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.

[0041] (iv) (Fv)2: consists of two Fv fragments covalently linked together.

[0042] (v) scFv: Single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, which is composed of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (generally composed of 10-25 amino acids), which retains the binding specificity of the original antibody to the antigen. The linker in the present invention is not particularly limited as long as it does not hinder the expression of the antibody variable regions connected to its two ends. Compared with full-length antibodies, scFv has the characteristics of small molecular weight, so it has higher penetration and lower immune side effects.

[0043] (vi) The sc(Fv)2 fragment is composed of two heavy chain variable regions and two light chain variable regions connected via a linker or the like.

[0044] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include a complementary determining region (CDR) and a framework region (FR) from a rabbit immunoglobulin sequence. In other embodiments, the antibody may include amino acid residues encoded by non-rabbit immunoglobulin sequences, for example, mouse-derived antibodies, chimeric antibodies, etc., to reduce the body's rejection reaction while maintaining the required specificity and affinity. The term "chimeric antibody" refers to a portion of an antibody derived from a specific source or species, while the rest is derived from a different source or species. The term "mouse-derived antibody" is a chimeric antibody of a non-mouse antibody such as a rabbit antibody's CDR region and a mouse FR region. In some cases, the variable region of a non-mouse antibody is combined with a mouse antibody's constant region, such as a mouse-rabbit chimeric antibody; in other cases, the CDR region of a non-mouse antibody is combined with a FR region and a constant region derived from a mouse antibody sequence, that is, the CDR region of a non-mouse antibody is grafted onto a mouse antibody framework (FR) sequence, and this framework sequence is derived from a single or multiple other mouse antibody variable region framework sequences. In the present invention, the CDR region in the chimeric antibody or murinized antibody is derived from a rabbit CDR region.

[0045] The terms "monoclonal antibody" or "single antibody" and the like are used interchangeably and refer to a homogeneous antibody population, i.e., the individual antibodies constituting 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 epitopes on an 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.

[0046] 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.

[0047] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0048] The embodiment of the present invention provides an antibody for detecting mouse monocyte chemoattractant protein 1, wherein the antibody is 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, wherein the light chain variable region and the heavy chain variable region both include three complementary determining regions (CDRs), which are named CDR1, CDR2 and CDR3, respectively; wherein: the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are shown as 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 as 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 as 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 as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, respectively. As shown in ID NO.20.

[0049] The present invention successfully developed a high-affinity rabbit monoclonal antibody that can specifically recognize and bind to mouse monocyte chemoattractant protein 1 (MCP-1) by using single B cell screening and culture technology, wherein the affinity constant KD of the first antibody is 4.96×10 -10 (M), the affinity constant KD of the second antibody is 1.98×10 -10(M), providing key antibody materials for establishing quantitative detection of mouse MCP-1 protein. The first antibody and the second antibody have different antigenic determinant recognition sites, and can be used as paired antibodies for the double antibody sandwich enzyme-linked immunosorbent assay (ELISA) system. Taking the first antibody as the capture antibody and the second antibody as the detection antibody as an example, the detection limit of mouse MCP-1 protein in biological samples is as low as 3.38pg / mL, with good detection specificity and extremely high detection sensitivity, providing an effective way to accurately, efficiently and reliably detect extremely trace levels of mouse MCP-1 protein, which is of great significance in clinical diagnosis and scientific research applications.

[0050] Optionally, the light chain variable region and the heavy chain variable region each include 4 framework regions (FRs), and the 4 FRs and 3 CDRs are arranged alternately in sequence 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.

[0051] 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). The constant region of an antibody can usually be obtained through public inquiries, such as searching for rabbit IgG gamma C reign to obtain CH, and searching for rabbit IgG Kappa C reign to obtain CL through the IMGT online database (www.imgt.org).

[0052] 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. The light chain constant region of the first antibody and the second antibody is a κ chain, and the heavy chain constant region is an IgG1 type.

[0053] 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 the antibody, specifically, the antigen binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining a complete antigen recognition and binding site, and can 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.

[0054] Yet another embodiment of the present invention 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.

[0055] Nucleic acid molecules can be in the form of DNA (such as cDNA or genomic DNA or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can also be a coding strand or a non-coding strand.

[0056] The sequence of the nucleic acid molecule can be derived from the antibody AA sequence by conventional means such as codon coding rules. The full-length sequence of the nucleic acid molecule or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. The obtained nucleic acid molecule is inserted into an expression vector, then introduced into a host cell, and cultured under specific conditions to express the antibody.

[0057] 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., used to transfer the nucleic acid molecule to a host and multiply in a host cell) or an expression vector (i.e., containing 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.

[0058] 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 the light chain are expressed in different host cells, each chain can be separated from the host cell expressing it, and the separated heavy chain and light chain are mixed and incubated under suitable conditions to form antibodies. In other embodiments, the nucleic acid molecules of antibodies FL and FH can also be cloned into a vector, and each nucleic acid sequence is connected to a suitable promoter downstream; for example, each nucleic acid sequence encoding the heavy chain and the light chain can be operably connected to different promoters, or the nucleic acid sequence encoding the heavy chain and the light chain can be operably connected 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.

[0059] The recombinant vector is transfected or transformed into the host cell using conventional techniques. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA are harvested after the exponential growth period and treated with CaCl2 or MgCl2; they can also be transfected 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.

[0060] 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 a recombinant vector as described above, the antibody can be expressed by culturing under suitable conditions, and then separated to obtain a purified antibody.

[0061] Preferably, the recombinant vector is a mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell (293F cell).

[0062] Another embodiment of the present invention provides an antibody pair for detecting mouse monocyte chemoattractant protein 1, which consists of the first antibody and the second antibody as described above.

[0063] The first antibody and the second antibody provided by the present invention recognize and bind to different epitopes of the mouse MCP-1 protein, and are used to develop a double antibody sandwich enzyme-linked immunosorbent assay system for the mouse MCP-1 protein, which has the advantages of high specificity, excellent sensitivity, and extremely low detection limit, and has a wide detection linear range, and can meet the requirements of high-accuracy detection of the mouse MCP-1 protein level in biological samples.

[0064] Still another embodiment of the present invention provides the use of the above-mentioned antibody or antibody pair for detecting mouse monocyte chemoattractant protein 1 in the preparation of a mouse monocyte chemoattractant protein 1 detection kit.

[0065] The advantages of using the antibody or antibody pair for detecting mouse monocyte chemoattractant protein 1 in preparing a mouse monocyte chemoattractant protein 1 detection kit are the same as the advantages of the antibody or antibody pair for detecting mouse monocyte chemoattractant protein 1 over the prior art as described above, and will not be repeated here.

[0066] Based on the same inventive concept as above, an embodiment of the present invention further provides a detection kit for detecting mouse monocyte chemoattractant protein 1, wherein the detection kit comprises the first antibody and / or the second antibody as described above.

[0067] It should be emphasized that the first antibody and the second antibody can be used separately, together, or in pairs. When detecting, if used separately or together, the first antibody and / or the second antibody are used as a primary antibody or a 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 coupled to a detection label, and the qualitative or quantitative detection of MCP-1 is achieved by analyzing the changes in the identifiable signals generated by the detection label. In other embodiments, the first antibody and / or the second antibody (as a primary antibody or a capture antibody) against mouse MCP-1 is not labeled, and the detection label is coupled to a secondary antibody (as a detection antibody) or other molecules that can bind to the primary antibody. For example, if the anti-mouse MCP-1 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 coupling the secondary antibody of the detection label. When used in pairs, one of the first antibody and the second antibody is used as a primary antibody or a capture antibody, and the other is used as a secondary antibody or a detection antibody.

[0068] The above-mentioned detection method adopts common immunological methods, including but not limited to: enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB), flow cytometry (FC), etc. The detection object includes recombinantly expressed MCP-1, naturally secreted or expressed MCP-1 protein. The detection sample includes but is not limited to MCP-1 protein in serum, plasma, urine, cell culture fluid, tissue homogenate, etc.

[0069] 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), the second antibody serves as a detection antibody (or secondary antibody), and the second antibody is modified with a detection label.

[0070] The above-mentioned detection labels for generating identifiable signal changes 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.

[0071] Preferably, the detectable label is biotin.

[0072] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the conditions recommended by the manufacturer.

[0073] Example 1 Preparation of rabbit monoclonal antibodies

[0074] Commercial recombinant Mouse MCP-1 protein (from ABclonal, catalog number RP01622) was used as an antigen to immunize New Zealand white rabbits. Then, based on the monoclonal antibody development technology of single B lymphocyte screening and culture, single antigen-specific B lymphocytes were sorted out, and the antibody encoding gene in the cell was amplified by PCR technology, and recombinant expression was performed to obtain candidate antibodies. Finally, the target antibody 6G7 and 2H2 strains were screened by affinity testing and antigen epitope identification. The antibody sequencing work was completed by Jinkairui Biotechnology Co., Ltd. The amino acids (AA) of the antibodies are shown in Tables 1-2, where LCDR1-3 represent the light chain complementary determining regions CDR1-3, and HCDR1-3 represent the heavy chain complementary determining regions CDR1-3.

[0075] Table 1 Sequence information of rabbit monoclonal antibody 6G7 in this example

[0076]

[0077]

[0078] Table 2 Sequence information of rabbit monoclonal antibody 2H2 in this example

[0079]

[0080] The preparation method of rabbit antibody 6G7 and antibody 2H2 specifically includes the following steps:

[0081] 1. Animal immunization: Two New Zealand white rabbits were immunized with Mouse MCP-1; 200 μg of each white rabbit was immunized. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to prepare 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 prepare an emulsion every 3 weeks, which was injected subcutaneously at multiple points on the rabbit's abdomen and back, and booster immunization was performed twice; after three immunizations, 200 μg of the immunogen was injected subcutaneously at multiple points to boost immunization once, and the animals were sacrificed and the spleen was taken out three days later.

[0082] 2. Isolate spleen cells: Take out a culture dish in a safety cabinet under aseptic operation, add 30-40mL of basal culture medium (RPMI Medium 1640basic+1% Pen Strep; RPMI 1640 purchased from Gibco, item number C11875500BT; PenStrep purchased from Gibco, item number 15140-163), put a cell sieve, take out the spleen and put it in the cell sieve, cut off the excess connective tissue and fat on the rabbit spleen tissue, cut the spleen tissue into pieces and put it into the cell sieve for grinding, take a clean grinding rod, and use the end of the pressing part to crush the tissue. The cells in the grinding membrane will slowly free themselves, and after passing through the cell sieve, they will be suspended in the culture dish solution; wash the cell sieve with 10mL of basal culture medium, and collect the basal culture medium outside the cell sieve. Centrifuge at 400g for 5min at room temperature, remove the supernatant, retain the cells, add 13mL of RBC lysis solution at room temperature (purchased from BioGems), gently blow away the cell clusters with a pipette and then time for 1min to lyse the red blood cells, add 37mL of basal culture medium and mix well to terminate the red blood cell lysis, centrifuge at 400g for 5min at room temperature, remove the supernatant, retain the cells, add 40mL of basal culture medium placed at room temperature, gently blow away the cell clusters with a pipette, resuspend the cells, complete the first wash, centrifuge at 400g for 5min at room temperature, remove the supernatant, retain the cells, add 20mL of basal culture medium placed at room temperature, gently blow away 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.

[0083] 3. B lymphocyte sorting and culture: Refer to the patent "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells (publication number: CN110016462A, publication date: 2019-07-16)" and the patent "A B lymphocyte in vitro culture system and application (publication number: CN111518765A, publication date: 2020-08-11)" to obtain antigen-specific B lymphocytes.

[0084] 4. Cloning of the gene encoding rabbit monoclonal antibody: The cultured B lymphocyte supernatant was used to identify positive clones by antigen-coated ELISA. The positive clone cells were collected and lysed and then analyzed by Quick-RNA TMRNA was extracted according to the instructions of MicroPrep kit (purchased from ZYMO, item number R1051) and reverse transcribed into cDNA. The reverse transcription system includes: Oligo (dT) 12-18 primer (Life) 1 μL, dNTPs (10 mM) 1 μL, RNA 11 μL; after 65 ° C, 5 min in the PCR instrument, 5 × FS Buffer (from ABconal) 4 μL, DTT (100 mM) 1 μL, RNase OUT (40 U / μL) 1 μL, ABScript Ⅱ RT (200 U / μL, from ABconal) 1 μL were added to the above products, mixed and reacted at 42 ° C, 1 h, 85 ° C, 5 min to obtain cDNA. Using the above cDNA as a template, the naturally paired rabbit monoclonal antibody light chain variable region (VL) and heavy chain variable region (VH) were amplified from the cDNA of the corresponding positive clone by PCR and sequenced. The PCR reaction system included: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria HiFi (from ABclonal, catalog number RK20717) and 6.5 μL H2O; the PCR amplification program included: 98°C pre-denaturation for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s, and finally kept at 72°C for 5 min, and the resulting reaction solution was stored at 4°C. The primer sequences (5'-3') for amplifying VL and VH genes are shown in Table 3, where F and R represent forward primers and reverse primers, respectively.

[0085] Table 3 Primer sequence information for amplifying rabbit monoclonal antibodies in 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: In order to produce anti-mouse MCP-1 antibodies on a large scale, the mammalian expression vector pBR322 (pBR322 OSLIC Lc and pBR322 OSLIC Hc) carrying the light chain constant region (CL) and heavy chain constant region (CH) genes were linearized with XbaI and NheI restriction enzymes respectively. The VL and VH genes containing the signal peptide amplified by the above PCR were purified and then constructed into the above expression vectors by homologous recombination to obtain light chain gene and heavy chain gene expression vectors. The expression vectors were successfully constructed and verified by sequencing. The expression map of the vector used is shown in Figure 1, pBR322 origin and f1 origin are replication promoters, Ampcillin is a resistance gene, CMV promoter is a transcription promoter, SV40 PAterminator is a tailing signal, Light chain constant is the nucleic acid sequence of the light chain constant region (left), Heavy chain constant is the nucleic acid sequence of the heavy chain constant region (right). CL and CH genes were obtained by searching the IMGT online database (www.imgt.org) to obtain CH from rabbit IgG gamma C reign, and CL from rabbit IgG Kappa C reign.

[0088] 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".

[0089] The constructed expression vectors containing the light chain (FL) gene and the heavy chain (FH) gene were transfected into 293F cells together. After transfection, the cells were cultured for 72-96 hours to obtain the culture supernatant containing the recombinant rabbit monoclonal antibody that recognizes Mouse MCP-1. Protein A affinity gel resin (purchased from Tiandi Renhe, catalog number SA023100) was used to purify the target antibody from the culture supernatant. Using 12% SDS-PAGE gel electrophoresis for detection, the concentration of antibody 6G7 was 1.53 mg / mL, and the concentration of antibody 2H2 was 1.79 mg / mL, and the purity was greater than 95%. The purified antibodies were divided and stored at -20°C for later use.

[0090] Example 2 Performance test of rabbit-derived 6G7 and 2H2 antibody strains in recognizing MCP-1 antigen

[0091] 1) The affinity of rabbit 6G7 and 2H2 antibody strains to Mouse MCP-1 protein was measured using the Probe Life Gator biomolecular interaction analyzer; the specific operation was as follows: Mouse MCP-1 protein was immobilized on the HFC (Anti-H IgG FC, purchased from Gator Bio, catalog number 160003) probe at a concentration of 3 μg / mL, and the probes with immobilized antigens were placed in 6G7 and 2H2 antibody dilution solutions, respectively, to test the affinity of the antibody binding to the antigen, and to obtain the affinity curve; wherein, the working concentration of the 6G7 antibody strain was 6.25 μg / mL, and the working concentration of the 2H2 antibody strain was 5.08 μg / mL.

[0092] The affinity curves of 6G7 antibody strain and 2H2 antibody strain binding to Mouse MCP-1 are shown in Figure 2-3 , where the ordinate represents the change in the thickness of the conjugate after the probe binds to the antibody and protein, the abscissa represents the binding time, the dark gray curve is the real-time binding value curve, and the light gray curve is the fitted average curve. The affinity constants obtained by curve fitting and calculation are shown in Table 4, the dissociation coefficient K off The constant used to characterize the dissociation rate between antibody and antigen, the binding coefficient K on A constant used to characterize how quickly an antibody binds to its target, the affinity constant K D K off / K on The ratio of , represents the equilibrium dissociation constant between the antibody and its antigen.

[0093] Table 4 Affinity-related parameter determination results of rabbit monoclonal antibodies

[0094] Antibody <![CDATA[K on (1 / Ms)]]> <![CDATA[K on Error]]> <![CDATA[K off (1 / s)]]> <![CDATA[K off Error]]> <![CDATA[K D (M)]]> 6G7 <![CDATA[3.54×10 5 ]]> 0.00 <![CDATA[1.75×10 -4 ]]> <![CDATA[1.04×10 -6 ]]> <![CDATA[4.96×10 -10 ]]> 2H2 <![CDATA[9.77×10 5 ]]> 0.00 <![CDATA[1.93×10 -4 ]]> <![CDATA[9.86×10 -7 ]]> <![CDATA[1.98×10 -10 ]]>

[0095] Depend on Figure 2-3 As shown in Table 4, the affinity constants K of antibodies 6G7 and 2H2 for Mouse MCP-1 protein are D They are 4.96×10 -10 (M) and 1.98×10 -10 (M), the antibody recognizes and binds the antigen with high affinity.

[0096] 2) Identification of antigen recognition epitopes: The epitopes of the mouse MCP-1 protein recognized by the rabbit 6G7 and 2H2 antibody strains were identified using the Probe Life Gator biomolecular interaction analyzer. The material used was HFC (Anti-Human IgG FC, purchased from Gator Bio, catalog number 160003), the mouse MCP-1 protein immobilization concentration was 3 μg / mL, and the concentrations of the antibodies to be tested, 6G7 and 2H2, were both 3 μg / mL.

[0097] The antigen epitopes recognized by 6G7 antibody strain and 2H2 antibody strain on Mouse MCP-1 are shown in Figure 4 , where the ordinate represents the change in thickness of the conjugate after the probe binds to the antibody and protein, and the abscissa represents the binding time. The results show that the probe after solidifying MouseMCP-1 can still significantly bind to the 2H2 antibody after binding to the 6G7 antibody, indicating that the two antibody strains recognize different epitopes of the Mouse MCP-1 protein and the binding sites do not interfere with each other. Therefore, antibodies 6G7 and 2H2 can be used as paired antibodies for double antibody sandwich enzyme-linked immunosorbent assay.

[0098] Example 3 Establishment of a double antibody sandwich ELISA system based on antibodies 6G7 and 2H2 and its sensitivity test

[0099] Antibody 2H2 biotin labeling: Anti-Mouse MCP-1 rabbit monoclonal 2H2 was prepared into a 1 mg / mL solution, and NHS-LC-biotin was prepared into a 60 mg / mL solution with DMSO; 200 μL of 1 mg / mL 2H2 antibody solution was taken, and 10 μL of 60 mg / mL NHS-LC-biotin solution was added; after mixing, the mixture was placed at room temperature for 30 minutes, and 50 μg of 500 mM Tris solution (pH 9.0) was added to terminate the reaction; finally, a large amount of 1×PBS buffer solution at pH 7.4 was added, and the mixture was centrifuged using a centrifugal column with an exclusion limit of 30KD to remove excess biotin molecules and balance the buffer system to obtain biotin-labeled antibody 2H2 (2H2-biotin).

[0100] The antibody 6G7 was used as the capture antibody and the biotin-labeled antibody 2H2-biotin was used as the detection antibody to establish a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method, and the steps are as follows: 1) Coating the capture antibody 6G7: dilute the antibody 6G7 to 2 μg / mL with 1×PBS, mix it on a vortexer, add it to a 96-well microplate at 100 μL / well, cover it with a cover film, and incubate it in a 4°C refrigerator for 16-20 hours; 2) Washing the plate: after the incubation is completed, discard the liquid in the well, wash the plate once with 1×PBST, add 300 μL of the sample, let it stand for 40 seconds, then discard the liquid in the well, and pat the liquid in the well dry on a flat paper; 3) Blocking: Add E013 blocking solution (1×PBS containing 2% BSA, 5% sucrose, 0.05% Tween 20 and 0.1% proclin 300, pH 5.0) to the plate. 7.2) Add 200 μL / well to the plate wells, cover with a cover film, and block at 37°C for 2 hours. After blocking, discard the blocking solution, pat the ELISA plate dry, place it in a 37°C oven to dry for 0.5-2 hours, and take it out for use; 4) Add antigen protein MCP-1: Mouse MCP-1 protein is blocked with E003 blocking solution (1×PBS containing 2% BSA, 0.05% Tween 20, 0.1% proclin 300, pH7.2) for gradient dilution, dilution concentration: 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 and 0pg / mL, then add different concentrations to the ELISA plate at 100μL / well, cover with cover film, and incubate at 37℃ for 2h; 100μL / well of E003 dilution without MCP-1 protein was used as blank control; 5) Washing: Same as step 2); 6) Adding detection antibody 2H2: After diluting 2H2-biotin to 0.125μg / mL, add 100μL / well to the ELISA plate in turn, cover with cover film, and incubate at 37℃ for 1h; 7) Washing: Same as step 2); 8) Adding SA-HR P: Dilute 100×SA-HRP (horseradish peroxidase labeled streptavidin, purchased from Wuhan Sanying Biotechnology Co., Ltd., catalog number SA00001-0) concentrate 100 times, add 100 μL / well to the ELISA plate, cover with cover film, and incubate at 37°C for 0.5h; 9) Wash the plate: Same as step 2); 10) Add TMB colorimetric solution: Add 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution to the ELISA plate at 100 μL / well, cover with cover film, and incubate at 37°C for 15min; 11) Read: After the incubation is completed, take out the ELISA plate, add 50 μL of stop solution (1 mol / L hydrochloric acid) to each well, and immediately read with an ELISA reader.

[0101] The concentration of mouse MCP-1 protein was used as the horizontal axis, and the correction value of the absorbance value was Y1 (Y1 = OD 450nm -OD 630nm) as the ordinate, and the standard curve was obtained by using the four-parameter fitting of the Logistic curve (see Figure 5 ), the fitting parameters are shown in Table 5, indicating that the standard curve equation is: Y = (AD) / (1 + (X / C) ^B) + D, A = 35.34880, B = -0.90231, C = 9127.67971, D = 0.04727, R 2 =0.9999.

[0102] Table 5 Sensitivity test data of double antibody sandwich ELISA system based on antibodies 6G7 and 2H2

[0103]

[0104] The sensitivity of the double antibody sandwich ELISA system was determined by taking the lowest mouse MCP-1 concentration at which the average absorbance value was greater than three times the average absorbance value of the blank control. The results showed that the detection sensitivity of the double antibody sandwich ELISA system established based on anti-Mouse MCP-1 protein rabbit monoclonal antibodies 6G7 and 2H2 reached 3.38 pg / mL, which can achieve high sensitivity and high specificity detection of extremely trace levels of MCP-1 protein in biological samples to be tested.

[0105] 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 protection scope of the present invention.

Claims

1. An antibody for detecting mouse monocyte chemoattractant protein 1, It is characterized in that Selected from the first antibody or the second antibody, wherein: The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the first antibody are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively; The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the light chain variable region of the second antibody are shown as SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, respectively.

2. The antibody for detecting mouse monocyte chemoattractant protein 1 according to claim 1, It is 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 antibody for detecting mouse monocyte chemoattractant protein 1 according to claim 2, It is 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 antibody for detecting mouse monocyte chemoattractant protein 1 according to claim 3, It is characterized in that The light chain constant region of the first antibody and the second antibody is a κ chain, and the heavy chain constant region is an IgG1 type.

5. The antibody for detecting mouse monocyte chemoattractant protein 1 according to claim 1, It is 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 .

6. A nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule or a host cell comprising the nucleic acid molecule, It is characterized in that The nucleic acid molecule encodes the first antibody or the second antibody according to any one of claims 1 to 5.

7. An antibody pair for detecting mouse monocyte chemoattractant protein 1, It is characterized in that The method comprises the first antibody and the second antibody as described in any one of claims 1 to 5.

8. Use of the antibody for detecting mouse monocyte chemoattractant protein 1 according to any one of claims 1 to 5 or the antibody pair for detecting mouse monocyte chemoattractant protein 1 according to claim 7 in the preparation of a mouse monocyte chemoattractant protein 1 detection kit.

9. A detection kit for detecting mouse monocyte chemoattractant protein 1, It is characterized in that The detection kit comprises the antibody for detecting mouse monocyte chemoattractant protein 1 according to any one of claims 1 to 5 or the antibody pair for detecting mouse monocyte chemoattractant protein 1 according to claim 7.

10. The detection kit for detecting mouse monocyte chemoattractant protein 1 according to claim 9, It is 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 is used as a capture antibody, and the second antibody modified with a detection marker is used as a detection antibody.

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