Antibodies and antibody pairs against mouse TNF-α protein and their applications

Abstract

The present invention belongs to the field of immunological detection technology, and more particularly relates to antibodies and antibody pairs against mouse TNF-α proteins 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 two antibodies provided by the present invention have high affinity and high specificity, and are used to develop a mouse TNF-α protein immunoassay system with the advantages of high sensitivity, high specificity, good stability, reliable results, a wide range of detection concentrations, a small amount of sample required, simple and easy steps, and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, in particular to an antibody and an antibody pair against mouse TNF-alpha protein and applications thereof. Background Art

[0002] Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine that connects inflammation and the immune system. It is a ligand of the TNF superfamily and is primarily secreted by macrophages, monocytes, neutrophils, CD4+ T cells, and NK cells. It plays a central role in inflammation, apoptosis, and immune system development, and can also coordinate the production of other cytokines, cell survival, and death to coordinate tissue homeostasis. TNF-α is a proinflammatory cytokine. In normal immune responses, TNF-α activates the immune system for immunomodulation, exerting anti-infection and anti-injury effects. However, excessive production or abnormal release of TNF-α can trigger a cytokine storm and play a key role in a variety of autoimmune and inflammatory diseases, including rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, and ulcerative colitis. Therefore, TNF-α is a marker representing the degree of systemic inflammation. Numerous studies have found that TNF-α levels are significantly elevated in the serum, synovium, and synovial fluid of patients with autoimmune diseases such as rheumatoid arthritis. TNF-α can even trigger the release of other inflammatory factors, such as IL-1, IL-6, IL-8, and TGF-β, exacerbating tissue damage. TNF-α can also be produced by malignant and immune cells within the tumor-associated microenvironment. As an endogenous tumor promoter, it promotes the progression of malignant diseases by generating an inflammatory ecosystem. Furthermore, clinical studies have shown that TNF-α can be used as an important indicator for screening for recurrent miscarriage. TNF-α levels in the peripheral blood of patients with recurrent miscarriage and preeclampsia are significantly elevated compared to those in normal pregnant women. At high concentrations, TNF-α can cause miscarriage through various pathways, and high plasma levels of TNF-α are thought to increase the risk of miscarriage in women with recurrent pregnancy loss. Therefore, detecting TNF-α is crucial for the diagnosis of many pathological conditions.

[0003] Over the past decade, the research and application of immunoassay technology has developed rapidly and has been widely used in various fields of basic biomedical research and clinical disease diagnosis. Methods for detecting TNF-α primarily include enzyme-linked immunosorbent assay (ELISA), which relies on specific anti-TNF-α antibodies and uses antigen-antibody immune reactions to establish qualitative and quantitative analysis methods. However, the anti-TNF-α antibodies currently prepared in the prior art suffer from low antibody specificity and poor affinity for the antigen, resulting in poor sensitivity and reliability in TNF-α protein detection. Summary of the Invention

[0004] In response to the problems of low antibody specificity and / or poor affinity for antigens in the prior art, the present invention provides two rabbit-derived antibodies with high affinity and specificity against mouse TNF-α and an antibody pair composed of the same, which are used to develop a mouse TNF-α protein immunoassay system with advantages such as high sensitivity, high specificity, good stability, reliable results, a wide detection concentration range, a small sample size, simple and easy operation steps, etc. The present invention further provides the use of the aforementioned antibodies and antibody pairs in the preparation of a mouse TNF-α detection kit, and provides a detection kit containing the aforementioned antibodies and antibody pairs. The present invention is specifically implemented through the following technical solutions:

[0005] In a first aspect, the present invention provides an anti-mouse TNF-α protein 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 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.

[0010] Furthermore, the nucleotide sequence of the light chain variable region of the first antibody is shown as SEQ ID NO.22, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.24; the nucleotide sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.26, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.28.

[0011] Furthermore, the nucleotide sequence of the light chain of the first antibody is shown as SEQ ID NO.21, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO.23; the nucleotide sequence of the light chain of the second antibody is shown as SEQ ID NO.25, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO.27.

[0012] The third aspect of the present invention provides an anti-mouse TNF-α protein antibody pair consisting of the first antibody and the second antibody as described above.

[0013] A fourth aspect of the present invention provides use of the above-mentioned anti-mouse TNF-α protein antibody or antibody pair in the preparation of a mouse TNF-α protein detection kit.

[0014] A fifth aspect of the present invention provides a detection kit for detecting mouse TNF-α protein, wherein the detection kit comprises the above-mentioned anti-mouse TNF-α protein antibody or antibody pair.

[0015] 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 modified with a detection label.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are:

[0017] 1. The two monoclonal antibodies provided by the present invention can bind to mouse tumor necrosis factor α (TNF-α) protein with high affinity, and the affinity constant K D As low as 0.1nM level, and the antibody only weakly cross-reacts with rat TNF-α protein with extremely high homology, and has no reaction with other cytokines. It has high sensitivity and high specificity to mouse TNF-α, providing excellent antibody material for qualitative or quantitative detection of mouse TNF-α protein.

[0018] 2. The two antibodies provided by the present invention recognize different antigenic epitopes on the surface of mouse TNF-α protein and can form a double-antibody sandwich enzyme-linked immunosorbent assay system. When used for the quantitative detection of TNF-α content in biological samples, it has the advantages of high sensitivity, high specificity, good stability, reliable results, a wide detection concentration range, small sample volume required, simple and easy operation steps, etc. The detection sensitivity can be as low as 1.004 pg / mL, which is suitable for high-specificity and high-sensitivity detection of low-concentration TNF-α. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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 paying any creative work.

[0020] 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;

[0021] Figure 2 This is an affinity curve of the rabbit monoclonal antibody 7C7 binding to mouse TNF-α in Example 2 of the present invention;

[0022] Figure 3 This is an affinity curve of the rabbit monoclonal antibody 5E12 binding to mouse TNF-α in Example 2 of the present invention;

[0023] Figure 4 This is a graph showing the recognition of TNF-α antigen epitopes by rabbit monoclonal antibodies 7C7 and 5E12 according to Example 2 of the present invention;

[0024] Figure 5 This is the standard curve for detecting mouse TNF-α using a double-antibody sandwich ELISA system established based on rabbit monoclonal antibodies 7C7 and 5E12 in Example 3 of the present invention;

[0025] Figure 6 This is a graph showing the specificity determination results of the double-antibody sandwich enzyme-linked immunosorbent assay system established based on rabbit monoclonal antibodies 7C7 and 5E12 in Example 4 of the present invention;

[0026] Figure 7 This is a graph showing the thermal stability test results of the double-antibody sandwich ELISA system established based on rabbit monoclonal antibodies 7C7 and 5E12 in Example 5 of the present invention. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The terms "rabbit monoclonal antibody," "monoclonal antibody," "rabbit-derived antibody," and similar expressions have synonymous meanings and, unless otherwise specified, refer to antibodies that specifically bind to mouse TNF-α protein. The modifier "rabbit" indicates that the complementarity-determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. The terms "mouse TNF-α protein," "Mouse TNF-α," "mouse TNF-α," and similar expressions have synonymous meanings.

[0033] 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.

[0034] 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 forming 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] (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.

[0040] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0041] (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.

[0042] (iv) (Fv)2: Consists of two covalently linked Fv fragments.

[0043] (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.

[0044] (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.

[0045] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may comprise a complementarity determining region (CDR) and a framework region (FR) from a rabbit immunoglobulin sequence. In other embodiments, the antibody may comprise amino acid residues encoded by a non-rabbit immunoglobulin sequence, for example, murinized antibodies, chimeric antibodies, etc., to reduce the body's rejection reaction while maintaining the required specificity and affinity. The term "chimeric antibody" refers to an antibody in which a portion is derived from a specific source or species, while the remaining portion is derived from a different source or species. The term "murinized antibody" is a chimeric antibody comprising a non-murine antibody, such as a rabbit antibody, with a CDR region and a mouse FR region. In some cases, the variable region of a non-murine antibody is combined with a constant region of a mouse antibody, such as a mouse-rabbit chimeric antibody; in other cases, the CDR region of a non-murine 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-murine antibody is grafted onto a mouse antibody framework (FR) sequence, which is derived from a single or multiple other mouse antibody variable region framework sequences. In the present invention, the CDR regions in the chimeric or murinized antibodies are derived from rabbit CDR regions.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] An embodiment of the present invention provides an anti-mouse TNF-α protein antibody, 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 complementarity determining regions (CDRs), respectively named CDR1, CDR2, and CDR3; wherein: the amino acid (AA) 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.

[0050] The monoclonal antibody provided by the present invention can bind to mouse tumor necrosis factor α (TNF-α) protein with high affinity, wherein the affinity constants K of the first antibody and the second antibody to mouse TNF-α areD The specificity of the antibody is 0.441nM and 0.738nM respectively. Moreover, in the antibody specificity identification, the antibody of the present invention only weakly cross-reacts with the rat TNF-α protein with extremely high homology, and does not react with human TNF-α and other mouse cytokines. It has high sensitivity and high specificity for mouse TNF-α, providing an antibody material with excellent performance for qualitative or quantitative detection of mouse TNF-α protein. Moreover, the two antibodies of the present invention recognize and bind to different antigenic epitopes of mouse TNF-α protein, and can be used as paired antibodies to construct a double antibody sandwich enzyme-linked immunosorbent assay system. Taking the first antibody as the capture antibody and the second antibody as the detection antibody as an example, when used for quantitative detection of TNF-α content in biological samples, it has the advantages of high sensitivity, high specificity, good stability, reliable results, wide detection concentration range, small sample size, simple and easy steps, etc., and effectively improves the minimum detection limit of TNF-α, and the detection sensitivity can be as low as 1.004pg / mL, which is suitable for high specificity and high sensitivity detection of low concentration TNF-α in biological samples.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The sequence of the nucleic acid molecule can be derived from the antibody 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.

[0058] Specifically, the nucleotide sequence of the light chain variable region of the first antibody is shown as SEQ ID NO.22, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.24; the nucleotide sequence of the light chain variable region of the second antibody is shown as SEQ ID NO.26, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.28.

[0059] Specifically, the nucleotide sequence of the light chain of the first antibody is shown as SEQ ID NO.21, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO.23; the nucleotide sequence of the light chain of the second antibody is shown as SEQ ID NO.25, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO.27.

[0060] The original vector for constructing the recombinant vector is a variety of 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Preferably, the recombinant vector is the expression vector pBR322, and the host cell is a human kidney epithelial (293F) cell.

[0065] Another embodiment of the present invention provides an anti-mouse TNF-α protein antibody pair, consisting of the first antibody and the second antibody as described above.

[0066] The first antibody and the second antibody provided by the present invention recognize and bind to different epitopes of the mouse TNF-α protein, and are used to develop a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) system for the detection of mouse TNF-α protein, which has the advantages of high specificity, high sensitivity, low detection limit, and reliable results.

[0067] Yet another embodiment of the present invention provides the use of the above-mentioned anti-mouse TNF-α protein antibody or antibody pair in the preparation of a mouse TNF-α protein detection kit.

[0068] The advantages of using the anti-mouse TNF-α protein antibody or antibody pair in preparing a mouse TNF-α protein detection kit are the same as the advantages of the anti-mouse TNF-α protein antibody or antibody pair over the prior art as described above, and will not be repeated here.

[0069] Based on the same inventive concept as above, an embodiment of the present invention further provides a detection kit for detecting mouse TNF-α protein, wherein the detection kit comprises the first antibody and / or the second antibody as described above.

[0070] 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 are used 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 coupled to a detection label, and qualitative or quantitative detection of TNF-α is achieved by analyzing the changes in the identifiable signal generated by the detection label. In other embodiments, the first antibody and / or the second antibody against mouse TNF-α is not labeled (as a primary antibody or capture antibody), and the detection label is coupled to a secondary antibody (as a detection antibody) or other molecule that can bind to the primary antibody. For example, if the anti-mouse TNF-α 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 to the detection label. When used in pairs, one of the first antibody and the second antibody is used as a primary antibody or capture antibody, and the other is used as a secondary antibody or detection antibody.

[0071] The above-mentioned detection methods utilize conventional immunological methods, including but not limited to enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT), immunohistochemistry (IHC), immunofluorescence assay (IF), immunoblotting (WB), flow cytometry (FC), and the like. Detection targets include recombinantly expressed TNF-α, naturally secreted or expressed TNF-α, and test samples include but are not limited to TNF-α in serum, plasma, urine, cell culture fluid, tissue homogenate, and the like.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Example 1 Preparation of anti-TNF-α rabbit monoclonal antibody

[0076] This example provides a method for preparing a rabbit monoclonal antibody against mouse TNF-α. The immunogen used to prepare the mouse TNF-α rabbit monoclonal antibody is derived from a high-quality recombinant mouse TNF-α mature protein with biological activity expressed in a mammalian expression system (purchased from Sino-Bio, Catalog No. 50349-MNAE). The preparation method is based on a monoclonal antibody development technology based on single B lymphocyte screening and culture, and specifically includes the following steps:

[0077] 1.1. Animal immunization: Two New Zealand white rabbits were immunized with recombinant Mouse TNF-α protein as the immunogen; each rabbit was immunized with 200 μg of the immunogen. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma) 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 (purchased from Sigma) 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 the three immunizations, rabbit serum samples were collected and diluted 1:243,000 to determine the titer against Mouse TNF-α using the ELISA method. The OD value was taken. 450nm Rabbits with a IgG level exceeding 0.2 were boosted once with 200 μg of immunogen injected subcutaneously at multiple points. Three days later, the animals were sacrificed and the spleens were removed.

[0078] 1.2. Isolate spleen cells: Aseptically remove a culture dish in a safety cabinet, add 30-40 mL of basal culture medium, place a cell sieve, remove the spleen and place it in the cell sieve, trim excess connective tissue and fat from the rabbit spleen tissue, chop the spleen tissue into pieces and place it in the cell sieve, use a clean grinding rod to crush and grind the tissue to slowly free the cells in the membrane, pass through the cell sieve, and 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.

[0079] 1.3. B lymphocyte sorting and culture: Refer to the patent "Method for Efficient Isolation of 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)" to obtain antigen-specific B lymphocytes.

[0080] 1.4 Cloning of rabbit monoclonal antibody gene: The supernatant of cultured B lymphocytes was used to identify positive clones by antigen-coated ELISA. The cells of positive clones were collected and lysed, and then the positive clones were identified by Quick-RNA TM RNA was extracted using a Micro Prep Kit (purchased from ZYMO, Cat. No. R1051) and reverse transcribed into cDNA. Using cDNA as a template, naturally paired rabbit monoclonal antibody light chain variable region (VL) and heavy chain variable region (VH) genes were amplified from the cDNA of corresponding positive clones using PCR. Several clones were selected for sequencing. The primer sequences (5'-3') for amplifying the VL and VH genes are as follows, where F and R represent the forward and reverse primers, respectively:

[0081] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO. 29);

[0082] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 30);

[0083] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 31);

[0084] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 32).

[0085] 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, product number RK20717), and 6.5 μL H2O. The PCR amplification procedure included: initial denaturation at 98°C 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 maintaining at 72°C for 5 min. The resulting reaction solution was stored at 4°C.

[0086] 1.5. Monoclonal antibody preparation and purification: In order to produce anti-Mouse TNF-α antibodies on a large scale, the mammalian expression vector pBR322 carrying the light chain constant region (CL) and heavy chain constant region (CH) genes was linearized with XbaI and NheI restriction enzymes respectively. The VL and VH genes containing the signal peptides amplified by PCR were purified and then constructed into the aforementioned expression vectors by homologous recombination to obtain light chain gene and heavy chain gene expression vectors. The expression vectors were successfully constructed by sequencing. The expression map of the vector used is 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 PA terminator 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 reign (CH) and rabbit IgG kappa C reign (CL).

[0087] The signal peptide of this embodiment can adopt the antibody expression signal peptide 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)", which has a signal peptide "MDTRAPTQLLGLLLLWLPGATF" or "MDTRAPTQLLGLLLLWLPGARC" upstream of VL and a signal peptide "METGLRWLLLVAVLKGVQC" upstream of VH.

[0088] The constructed expression vectors containing the light chain (FL) and heavy chain (FH) genes were transfected into 293F cells and cultured for 72-96 hours. The culture supernatant contained recombinant rabbit monoclonal antibodies that recognized mouse TNF-α. The target antibodies were purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, Catalog No. SA023100) to a purity greater than 95%. The purified antibodies were aliquoted and stored at -20°C until use.

[0089] 1.6 Monoclonal Antibody Screening and Identification: After obtaining multiple recombinantly expressed Mouse TNF-α antibodies, the antibodies were identified for affinity and antigen recognition epitopes. The specific methods are as follows:

[0090] 1.6.1 Monoclonal Antibody Screening: The affinity of the obtained rabbit monoclonal antibodies was preliminarily determined using the Probe Life Gator Biomolecular Interaction Analyzer. The screening reagent was recombinant Mouse TNF-α protein (purchased from Sino-Bio, Cat. No. 50349-MNAE) at a concentration of 3 μg / mL, and the rabbit monoclonal antibody was at a concentration of 2 μg / mL. By comparing the affinities of the various antibodies, the affinity constants of ≤1×10 -9 of antibodies.

[0091] 1.6.2 Identification of Antigen Recognition Epitopes: The obtained rabbit monoclonal antibodies were paired using the Probe Life Gator Biomolecular Interaction Analyzer to test their recognition of epitope determinants. The screening reagent was recombinant Mouse TNF-α protein (purchased from Sino-Bio, Catalog No. 50349-MNAE) at a concentration of 3 μg / mL. The concentrations of the first rabbit monoclonal antibody and the second rabbit monoclonal antibody were 3 μg / mL. By analyzing the pairing data between the two antibodies, two antibodies were selected that recognized different epitope determinants and were designated as antibodies 7C7 and 5E12, respectively.

[0092] The affinity of rabbit monoclonal antibodies 7C7 and 5E12 was determined, and the affinity curves are shown in Figure 2. 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 1, and the dissociation coefficient K off The constant that characterizes the dissociation rate between antibody and antigen, the binding coefficient K on A constant that characterizes how quickly an antibody binds to its target, the affinity constant K D K off / K on The ratio of , characterizes the equilibrium dissociation constant between antibody and antigen. Figure 2-3 As shown in Table 1, the affinity constants K of antibodies 7C7 and 5E12 for Mouse TNF-α protein are D The affinity of the antibody to mouse TNF-α protein was 0.441 nM and 0.738 nM, respectively.

[0093] Table 1 Results of affinity-related parameters determination of rabbit monoclonal antibodies

[0094] Antibody strain <![CDATA[K off (1 / s)]]> <![CDATA[K on (1 / Ms)]]> <![CDATA[K D (M)]]> 7C7 <![CDATA[8.91×10 -4 ]]> <![CDATA[2.02×10 6 ]]> <![CDATA[4.41×10 -10 ]]> 5E12 <![CDATA[7.62×10 -4 ]]> <![CDATA[1.03×10 6 ]]> <![CDATA[7.38×10 -10 ]]>

[0095] The results of antigenic determinant determination of rabbit monoclonal antibodies 7C7 and 5E12 are shown in Figure 4 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. In the figure, 7C7 represents the rabbit monoclonal antibody 7C7, and 5E12 represents the rabbit monoclonal antibody 5E12. As can be seen, antibodies 7C7 and 5E12 recognize different epitopes on the TNF-α protein, and their recognition and binding sites do not interfere with each other. Therefore, they can be used as paired antibodies in a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) system.

[0096] The screened antibodies were sequenced by Jinkairui Biotechnology Co., Ltd. The amino acid (AA) and nucleotide (DNA) sequences of antibodies 7C7 and 5E12 are shown in Tables 2-3, respectively. LCDR1-3 denotes the light chain complementary determining regions (CDR1-3), and HCDR1-3 denotes the heavy chain complementary determining regions (CDR1-3). The sequences of the light chain variable regions (VL) and heavy chain variable regions (VH) of antibodies 7C7 and 5E12 exhibited 75.45% and 66.38% identity, respectively.

[0097] Table 2 Sequence information of rabbit monoclonal antibody 7C7 in this example

[0098]

[0099]

[0100] Table 3 Sequence information of rabbit monoclonal antibody 5E12 in this example

[0101]

[0102]

[0103] Example 2 Establishment of a double-antibody sandwich ELISA system based on antibodies 7C7 and 5E12 and its sensitivity test

[0104] Biotinylation of Antibody 5E12: Antibody 5E12 was prepared into a 1 mg / mL solution, and NHS-LC-biotin (N-succinimidyl 6-biotin aminocaproic acid, purchased from Thermo Fisher Scientific) was prepared into a 60 mg / mL solution using dimethyl sulfoxide (DMSO). To 200 μL of the 1 mg / mL antibody 5E12 solution, 10 μL of the 60 mg / mL NHS-LC-biotin solution was added. The mixture was mixed and allowed to stand at room temperature for 30 minutes. The reaction was terminated by adding 50 μg of 500 mM Tris-HCl solution (pH 9.0). Finally, 4 mL of 1× PBS buffer (pH 7.4) was added, and the mixture was centrifuged using a spin column with a 30 kDa exclusion limit to remove excess biotin molecules and equilibrate the buffer system to obtain biotin-labeled antibody 5E12 (5E12-biotin).

[0105] A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established using antibody 7C7 as the capture antibody and biotin-labeled antibody 5E12-biotin as the detection antibody. The steps are as follows:

[0106] 2.1. Coating with capture antibody 7C7: Dilute antibody 7C7 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 in a 4°C refrigerator for 16-20 hours.

[0107] 2.2. Washing: After incubation, discard the liquid in the wells, wash the plate once with 1×PBST, add 300μL of sample, let it stand for 40s, then discard the liquid in the wells and pat dry on flat paper;

[0108] 2.3. 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 of the plate. 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.

[0109] 2.4. Adding antigen protein TNF-α: TNF-α protein was serially diluted in diluent (1× PBS containing 2% BSA, 0.05% Tween 20, 0.1% proclin 300, pH 7.2) to the following concentrations: 150 pg / mL, 75 pg / mL, 37.5 pg / mL, 18.75 pg / mL, 9.37 pg / mL, 4.68 pg / mL, and 2.34 pg / mL. Different concentrations were then added to the ELISA plate at 100 μL / well, covered with a cover film, and incubated at 37°C for 2 h. The diluent without protein was used as a blank control.

[0110] 2.5. Wash the plate: Same as step 2.2;

[0111] 2.6. Add detection antibody 5E12: Dilute 5E12-biotin to 0.08 μg / mL and add 100 μL / well to the ELISA plate. Cover with film and incubate at 37°C for 1 hour.

[0112] 2.7, plate washing: same as step 2.2;

[0113] 2.8. Add 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.

[0114] 2.9, plate washing: same as step 2.2;

[0115] 2.10. Add TMB colorimetric solution: Add 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution to the ELISA plate, cover with film, and incubate at 37°C for 15 min.

[0116] 2.11. Reading: After incubation, remove the ELISA plate, add 50 μL of stop solution (1 mol / L hydrochloric acid) to each well, and immediately read the plate at 450 nm using a ELISA reader.

[0117] The concentration of mouse TNF-α protein was used as the horizontal axis, and the absorbance value OD 450nm As the vertical axis, the standard curve was obtained by fitting (see Figure 5 ), with the sensitivity of the double-antibody sandwich ELISA assay defined as the concentration at which the average absorbance value was greater than three times the average absorbance value of the blank control. The results demonstrated excellent linearity in the standard curve of the double-antibody sandwich ELISA assay established with antibodies 7C7 and 5E12, with a sensitivity as low as 1.004 pg / mL, enabling highly sensitive and reliable detection of extremely trace levels of TNF-α protein in biological samples.

[0118] Example 3 Specificity test of double antibody sandwich enzyme-linked immunosorbent assay system based on antibodies 7C7 and 5E12

[0119] Thirteen proteins similar to Mouse TNF-α were used to cross-react to determine the antigen recognition specificity of the double antibody sandwich ELISA detection system. The cross-reactive test proteins were: mouse IL-1α (purchased from ABclonal, product number RM00437), mouse IL-1β (purchased from ABclonal), mouse IL-2 (purchased from RD, product number 840139), mouse IL-4 (purchased from RD, product number 840142), mouse IL-5 (purchased from RD, product number 840479), mouse IL-6 (purchased from ABclonal, product number RP01321), mouse IL-10 ( 417-ML-005 / CF), mouse IL-12 / IL-23p40 (purchased from ABclonal, catalog number RM00093), mouse IFN-β (purchased from RD, catalog number 844567), mouse IFN-γ (purchased from RD, catalog number 485-MI-100 / CF), mouse EGF (purchased from ABclonal, catalog number RM01481), human TNF-α (purchased from RD, catalog number 840121), rat TNF-α (purchased from ABclonal, catalog number RM00141). The double antibody sandwich ELISA detection system in Example 2 was used for detection, and the results are shown in FIG. Figure 6 .

[0120] The results showed that antibodies 7C7 and 5E12 had no cross-reactivity with other proteins except for mouse and rat TNF-α (TNF-alpha). The cross-reactivity with rat TNF-α is due to the high homology of rat and mouse TNF-α antigens and the extremely similar protein structures. Overall, the antibodies provided by the present invention are highly specific for the mouse TNF-α protein.

[0121] Example 5 Thermal stability test of a double antibody sandwich ELISA system based on antibodies 7C7 and 5E12

[0122] The ELISA plate coated with the capture antibody, freeze-dried Mouse TNF-α protein, and 100× concentrated biotinylated detection antibody were sealed and stored at -20°C, 4°C, and 37°C for 7 days, then taken out and tested according to the double antibody sandwich ELISA detection system of Example 2. The standard curves established by the antibody samples treated at different temperatures were compared to analyze the thermal stability of the double antibody sandwich ELISA system. The results are shown in Figure 2. Figure 7 .

[0123] from Figure 7 As can be seen in the figure, the OD values ​​of rabbit monoclonal antibodies 7C7 and 5E12 at -20℃, 4℃ and 37℃ are 450nm The values ​​were not much different, and different storage temperatures had little effect on the detection sensitivity and linear range, indicating that the Mouse TNF-α antibody prepared by the present invention has a high degree of thermal stability.

[0124] 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 antibody against mouse TNF-α protein, characterized in that is a primary antibody or a secondary 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 in SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively; the amino acid sequences of the complementary determining regions 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 the complementary determining regions 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 the complementary determining regions 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-mouse TNF-α protein 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-mouse TNF-α protein 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-mouse TNF-α protein 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. 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 first antibody or the second antibody as described in any one of claims 1 to 4.

6. The nucleic acid molecule, the recombinant vector comprising the nucleic acid molecule or the host cell comprising the nucleic acid molecule according to claim 5, characterized in that: The nucleotide sequence of the light chain variable region of the first antibody is shown in SEQ ID NO. 22, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 24; The nucleotide sequence of the light chain variable region of the second antibody is shown in SEQ ID NO.26, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

28.

7. The nucleic acid molecule, the recombinant vector comprising the nucleic acid molecule or the host cell comprising the nucleic acid molecule according to claim 6, characterized in that: The nucleotide sequence of the light chain of the first antibody is shown in SEQ ID NO.21, and the nucleotide sequence of the heavy chain is shown in SEQ ID NO.23; The nucleotide sequence of the light chain of the second antibody is shown in SEQ ID NO. 25, and the nucleotide sequence of the heavy chain is shown in SEQ ID NO.

27.

8. An anti-mouse TNF-α protein 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.

9. Use of the anti-mouse TNF-α protein antibody according to any one of claims 1 to 4 or the anti-mouse TNF-α protein antibody pair according to claim 8 in preparing a mouse TNF-α protein detection kit.

10. A detection kit for detecting mouse TNF-α protein, characterized in that: The detection kit comprises the anti-mouse TNF-α protein antibody according to any one of claims 1 to 4 or the anti-mouse TNF-α protein antibody pair according to claim 8.

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