Monoclonal antibodies against Ki-67 protein and their applications

Abstract

The present invention belongs to the technical field of antibody preparation, and in particular to monoclonal antibodies against Ki-67 protein and their applications. The amino acid sequences of the complementary determining regions CDR1-3 on the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.3-5, respectively; the amino acid sequences of the complementary determining regions CDR1-3 on the heavy chain variable region are shown in SEQ ID NO.8-10, respectively. The monoclonal antibody provided by the present invention can effectively identify Ki-67 protein expressed by cells / tissues, has good antigen binding specificity, high affinity and excellent anti-interference ability, is conducive to greatly improving the accuracy, sensitivity and credibility of detecting whether tissues / cells express Ki-67, and the antibody has cross-reactivity to human and mouse (including rats and mice) homologous Ki-67, and has wide applicability to the detection of Ki-67 proteins of different species.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and in particular to monoclonal antibodies against Ki-67 protein and applications thereof. Background Art

[0002] Ki-67 is a specific nuclear protein closely associated with cell proliferation. Under normal physiological conditions, Ki-67 expression levels gradually increase from the G1 phase of mitosis to the S and G2 phases, then rapidly decrease after the M phase, and finally cease expression in the G0 phase (quiescence phase). Therefore, Ki-67 is an important cell proliferation marker, and its expression level reflects the rate and activity of cell proliferation.

[0003] Ki-67 has important clinical pathological diagnostic value and has become one of the most commonly used clinical pathological targets, widely used in oncology research. In tumor tissue, high Ki-67 expression often indicates high tumor cell proliferation activity, and the Ki-67 proliferation index is closely correlated with tumor differentiation, infiltration and metastasis, and prognosis. Therefore, Ki-67 can, in certain circumstances, be used as an independent marker to study tumor biological behavior, assess tumor progression and prognosis, and predict the risk of recurrence and metastasis. For example, in solid tumors such as breast cancer, prostate cancer, and gastrointestinal tumors, a high Ki-67 index is often associated with higher tumor grade, lymph node metastasis, and poorer survival. Clinically, Ki-67 is also used to guide treatment decisions. In certain situations, high Ki-67 levels may indicate the need for more aggressive treatment strategies, while patients with low Ki-67 expression may be more suitable for more moderate treatment approaches such as endocrine therapy. This personalized treatment strategy based on Ki-67 levels can help optimize treatment efficacy and reduce unnecessary treatment burden. Ki-67 expression levels can also be used to predict treatment response. If the expression level of Ki-67 in tumor tissue is detected to be significantly decreased, it usually indicates a better treatment effect; if the expression level of Ki-67 is significantly increased, the treatment plan may need to be adjusted.

[0004] Given the guiding role of Ki-67 in assessing cell proliferation activity, assisting in tumor diagnosis, determining tumor prognosis, predicting the risk of recurrence and metastasis, monitoring treatment efficacy, and guiding personalized treatment, detecting Ki-67 in pathological cells or tissues can provide an important reference for the diagnosis and treatment of related tumors. With the widespread use of immunohistochemistry in clinical pathology, the development of Ki-67 immunohistochemistry techniques is imperative. Key to this is the specificity and sensitivity of the antibody. In cell or tissue biopsies, the target antigen Ki-67 is often present at low levels or when the epitope is partially masked. Low antibody sensitivity can result in ineffective antigen recognition, leading to false-negative results. Furthermore, due to the numerous interfering factors of cellular components, poor antibody specificity can lead to cross-reactions with other antigens, resulting in nonspecific staining and increased background staining, leading to false-positive results and impairing the accurate identification of Ki-67-positive cells. This presents a challenge for detecting tumors with low proliferation activity or those whose proliferation is inhibited after treatment. Therefore, the development of anti-Ki-67 antibodies with high specificity and sensitivity for cell / tissue detection is of great value. Summary of the Invention

[0005] To address the existing problem of a lack of monoclonal antibodies suitable for detecting Ki-67 in cells / tissues, the present invention provides a monoclonal antibody against the Ki-67 protein. This antibody can specifically recognize and bind to Ki-67 on the surface of cells / tissues, exhibiting good antigen-binding specificity, high affinity, and excellent anti-interference capabilities. The present invention further provides the use of this monoclonal antibody in the preparation of a Ki-67 protein detection kit, as well as related detection kits. To achieve the aforementioned objectives, the present invention is specifically implemented through the following technical solutions:

[0006] In a first aspect, the present invention provides a monoclonal antibody against Ki-67 protein, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of the complementarity determining regions CDR1-3 on the light chain variable region are shown as SEQ ID NOs. 3-5, respectively; and the amino acid sequences of the complementarity determining regions CDR1-3 on the heavy chain variable region are shown as SEQ ID NOs. 8-10, respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region 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.

[0008] Furthermore, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0009] Furthermore, the monoclonal antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

[0010] The second aspect of the present invention provides a nucleic acid molecule encoding the monoclonal antibody against Ki-67 protein as described above.

[0011] The third aspect of the present invention provides a recombinant vector or a host cell, wherein the recombinant vector or the host cell comprises the nucleic acid molecule as described above.

[0012] A fourth aspect of the present invention provides use of the above-mentioned monoclonal antibody against Ki-67 protein in the preparation of a Ki-67 protein detection kit.

[0013] Furthermore, the Ki-67 protein is human, rat or mouse Ki-67 protein.

[0014] Furthermore, the detection kit is an enzyme-linked immunosorbent assay kit, an enzyme-linked immunosorbent assay kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit or a flow cytometry kit.

[0015] In a fifth aspect, the present invention provides a Ki-67 protein detection kit, which comprises the anti-Ki-67 protein monoclonal antibody or its antibody conjugate as described above, wherein the antibody conjugate is formed by linking the monoclonal antibody to a detection label.

[0016] The advantages and positive effects of the present invention include: the monoclonal antibody provided herein can effectively recognize Ki-67 proteins expressed in cells / tissues, exhibiting good antigen-binding specificity, high affinity, and excellent anti-interference capabilities. This significantly improves the accuracy, sensitivity, and reliability of detecting whether tissues / cells express Ki-67, effectively avoiding false-positive and false-negative results. The antibody is particularly suitable for immunoblotting, immunofluorescence, and immunohistochemistry analysis, and has broad application prospects and promising market prospects in clinical diagnosis and scientific research. Furthermore, the antibody exhibits cross-reactivity with homologous Ki-67 from humans and mice (including rats and mice), making it widely applicable for detecting Ki-67 proteins from different species. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 1 A map of the mammalian expression vector pRB322 used to construct the anti-human Ki-67 protein antibody expression vector in Example 1 of the present invention, including, from left to right, vectors pre-carrying the antibody light chain constant region and heavy chain constant region;

[0019] Figure 2 This is an electrophoresis gel image of the Ki-67 expression in positive and negative cells / tissues detected by immunoblotting using an anti-human Ki-67 protein antibody in Example 2 of the present invention;

[0020] Figure 3 This is a fluorescence staining image of the Ki-67 expression in positive cells / tissue samples detected by immunofluorescence using an anti-human Ki-67 protein antibody according to Example 2 of the present invention;

[0021] Figure 4 This is a tissue staining diagram of Example 2 of the present invention, in which anti-human Ki-67 protein antibody is used to detect the expression of Ki-67 in positive and negative tissues by immunohistochemistry. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The terms "include", "comprising", "containing", "having" and the like are non-limiting in meaning, that is, other steps and other components that do not affect the results may be added.

[0027] 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" includes the following situations: (i) A, (ii) B, and (iii) A and B.

[0028] The terms "rabbit monoclonal antibody," "rabbit antibody," and "rabbit monoclonal antibody" have synonymous meanings and, unless otherwise specified, refer to rabbit antibodies that specifically bind to the Ki-67 protein. The modifier "rabbit" indicates that the complementarity determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences.

[0029] 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 encompasses various 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. Antibody fragments can be one or more portions or fragments of a full-length antibody that retain the antibody's ability to specifically bind to the target antigen.

[0030] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types: kappa (κ) and lambda (λ); heavy chains can be classified into five types: μ, δ, γ, α, and ε, which define antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of heavy and heavy chains vary greatly, while the remaining amino acid sequences are relatively constant. The regions of the light and heavy chains with the most variable amino acid sequences near the N-terminus are called the variable region (V), while the regions with relatively stable amino acid sequences near the C-terminus are called the constant region (C). The heavy chain variable region (VH) and light chain variable region (VL) are generally the most variable parts of antibodies and contain the antigen recognition site. The VH and VL regions can be further subdivided into hypervariable regions (HVRs) and framework regions (FRs). The HVRs, also known as complementarity-determining regions (CDRs), are circular structures. The heavy and light chain CDRs are closely aligned and interact with each other through the FRs, forming a surface that complements the three-dimensional structure of the target antigen or epitope. This determines the antibody's specificity and is the site of antigen recognition and binding. The FRs are the more conserved portions of the VH and VL sequences. They generally follow a β-pleated sheet configuration and are connected by three CDRs that form a connecting loop. Each VH and VL sequence typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0031] 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.

[0032] 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 length is essentially the same for different Ig types (κ or λ), but the CH length varies across Ig classes. 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 and can be obtained by querying the IMGT database.

[0033] 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.

[0034] 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.

[0035] (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.

[0036] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0037] (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.

[0038] (iv) (Fv)2: Consists of two covalently linked Fv fragments.

[0039] (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.

[0040] (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.

[0041] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may comprise a complementary 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 non-rabbit immunoglobulin sequences, for example, humanized 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 "humanized antibody" is a chimeric antibody comprising a non-human antibody, such as a rabbit antibody, with a CDR region and a human FR region. In some cases, the variable region of a non-human antibody is combined with a constant region of a human antibody, such as a human-rabbit chimeric antibody; in other cases, the CDR region of a non-human antibody is combined with a FR region and a constant region derived from a human antibody sequence, that is, the CDR region of a non-human antibody is grafted onto a human antibody framework (FR) sequence, which is derived from the FR sequence of a single or multiple other human antibody variable regions. In the present invention, the CDR regions in the chimeric or humanized antibodies are derived from rabbit-derived CDR regions.

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

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

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

[0045] An embodiment of the present invention provides an anti-Ki-67 protein monoclonal antibody, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region each include three complementarity determining regions (CDRs), respectively designated as CDR1, CDR2, and CDR3, wherein the amino acid (AA) sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively; and the amino acid (AA) sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10, respectively.

[0046] The antibodies provided by the present invention are suitable for a variety of immunological detection methods. Specifically, by immunoblotting, positive cells or tissues expressing Ki-67 protein and negative cells or tissues not expressing Ki-67 protein were detected. Bands were detected only in the protein lysate of the positive samples and were consistent with expectations, indicating that the antibody has good specificity and good reactivity to Ki-67 proteins of different species. By immunofluorescence, multiple positive samples such as Hela cell line, A431 cell line, mouse spleen, C2C12 cell line, rat spleen and PC-12 cell line were detected. Similarly, obvious specific positive staining was shown in the positive samples, and the localization of the antigen Ki-67 was consistent with the theory. By immunohistochemistry, positive and negative tissue samples were detected. No staining was found in the negative tissue samples, while the staining location was accurate in the positive tissue samples, there was no nonspecific staining, and the positive staining was clear and bright with a clean background. The detection sensitivity and specificity could reach 100%. Combined with the above results, it can be seen that the antibody of the present invention can effectively recognize Ki-67 protein expressed in cells / tissues, with good antigen-binding specificity, high affinity, and excellent anti-interference ability. The detection results are highly consistent with the actual situation. This greatly improves the accuracy, sensitivity, and reliability of immunoassays for direct detection of Ki-67 expression in tissues / cells, effectively avoiding false positive and false negative results. It is particularly suitable for immunoblotting, immunofluorescence, and immunohistochemistry analysis, and has broad application prospects and promising market prospects in clinical diagnosis and scientific research. In addition, this antibody has cross-reactivity with human and mouse (including rat and mouse) homologous Ki-67, making it widely applicable to the detection of Ki-67 proteins from different species.

[0047] Optionally, the light chain variable region and the heavy chain variable region each include four framework regions (FRs), wherein the four FRs and three CDRs are arranged in a staggered sequence to form a variable region. The amino acid sequence of the light chain variable region (VL) of the monoclonal antibody of the present invention 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.

[0048] Optionally, the monoclonal antibody of the present invention further comprises a light chain constant region and a heavy chain constant region, wherein CL and VL constitute the light chain, and CH and VH constitute the heavy chain. The constant region of an antibody is usually obtained by querying the IMGT online database.

[0049] Specifically, the amino acid sequence of the monoclonal antibody light chain (FL) is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain (FH) is shown in SEQ ID NO.6, and the heavy chain is of IgG type.

[0050] It should be noted that the monoclonal antibody of the present invention can be 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 retains the same biological function or activity as the full-length form. 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.

[0051] Yet another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector comprising the aforementioned nucleic acid molecule, or a host cell comprising the aforementioned nucleic acid molecule, wherein the nucleic acid molecule encodes the monoclonal antibody against Ki-67 protein as described above.

[0052] Nucleic acid molecules can be in the form of DNA (e.g., cDNA, genomic DNA, or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can be either a coding strand or a non-coding strand. The sequence of the nucleic acid molecule can be derived from the antibody amino acid sequence by conventional means, such as codon coding rules.

[0053] The full-length sequence of a nucleic acid molecule or a fragment thereof can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0054] The original vector for constructing the recombinant vector is a variety of vectors conventional in the art, as long as it can accommodate the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, phages (such as λgt4λB, λ-Charon, λΔz1 and M13), cosmids and minichromosomes. The vector can be a cloning vector (i.e., for transferring nucleic acid molecules into a host and multiplying them in large quantities in the host cell) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecules inserted into the vector to be expressed in the host cell). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid molecule, and then introduced into a host cell to culture and obtain the antibody. This is a well-known technology in the art and will not be described in detail here.

[0055] The nucleic acid molecules encoding the monoclonal antibodies FL and FH of the present invention can be inserted into two separate vectors and 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 downstream to a suitable promoter; 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.

[0056] 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.

[0057] 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.

[0058] In the present invention, transfection or transformation of the recombinant vector into host cells is performed using conventional techniques. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2 or MgCl2. If desired, methods such as microinjection, electroporation, or liposome packaging can also be used. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, etc.

[0059] Preferably, the recombinant vector is the expression vector pBR322, and the host cell is a human kidney epithelial (293F) cell.

[0060] Yet another embodiment of the present invention provides the use of the above-mentioned monoclonal antibody against Ki-67 protein in the preparation of a Ki-67 protein detection kit.

[0061] The advantages of using the anti-Ki-67 protein monoclonal antibody in preparing a Ki-67 protein detection kit are the same as the advantages of the anti-Ki-67 protein monoclonal antibody over the prior art as described above, and will not be repeated here.

[0062] The monoclonal antibodies of the present invention have cross-recognition for Ki-67 proteins from multiple species. Specifically, the Ki-67 protein can be human, rat, or mouse Ki-67 protein. The amino acid and nucleotide sequences of the Ki-67 protein can be obtained by conventional techniques. For information on human Ki-67 protein, see Uniprot Protein Accession Number: P46013, GeneID Number: 4288; or, NCBI Protein Accession Number: NP_002408.3, Gene Accession Number: NM_002417.5. For information on mouse Ki-67 protein, see Uniprot Protein Accession Number: E9PVX6, GeneID Number: 17345; or, NCBI Protein Accession Number: NP_001074586.2, NCBI Gene Accession Number: NM_001081117.2. For information on rat Ki-67 protein, please refer to Uniprot protein accession number: Q5RJMO, GeneID number: 246042; or, NCBI protein accession number: NP_631925.2, NCBI gene accession number: NM_139186.2.

[0063] Based on the same inventive concept, an embodiment of the present invention further provides a Ki-67 protein detection kit, which includes the anti-Ki-67 protein monoclonal antibody or antibody conjugate as described above, wherein the antibody conjugate is formed by linking the monoclonal antibody to a detection label.

[0064] The advantages of the Ki-67 protein detection kit over the prior art are the same as those of the monoclonal antibody against Ki-67 protein over the prior art as described above, and will not be repeated here.

[0065] It should be noted that the antibodies of the present invention can be used alone or in combination with a detection label to form an antibody conjugate. In some embodiments, the antibodies of the present invention are used as antigen-binding antibodies that specifically recognize and bind to Ki-67 in the sample to be tested, and then qualitative or quantitative detection of Ki-67 is achieved by analyzing the signal of the detection label attached thereto. In other embodiments, the anti-Ki-67 antibody (as a primary antibody or capture antibody) is not labeled, 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-Ki-67 antibody is a rabbit IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody. Thus, qualitative or quantitative detection of Ki-67 is achieved by analyzing the change in the detection label signal generated after the secondary antibody specifically binds to the antibody of the present invention.

[0066] The detection label is used to generate a recognizable signal change to identify the sample to be tested based on the signal change. Detection labels include, but are not limited to, biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, and 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, and avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.

[0067] Immunoassay methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB), immunoprecipitation (IP), and flow cytometry (FC). The detection kit can be an enzyme-linked immunosorbent assay (ELISA), an ELISPOT assay, an immunohistochemistry assay, an immunofluorescence assay, an immunoblotting assay, or a flow cytometry assay.

[0068] Preferably, the detection kit is an immunoblotting kit, an immunohistochemistry kit or an immunofluorescence kit.

[0069] The test samples include serum, plasma, urine, cell or tissue samples, wherein cells include but are not limited to: human Hela cell line, human A431 cell line, mouse C2C12 cell line or rat PC-12 cell line; tissues include but are not limited to: human spleen, human colon, human colon cancer, human tonsil, human breast cancer, human invasive ductal carcinoma of the breast, human brain, human appendix, human kidney, human lung cancer, mouse colon, mouse spleen, mouse testicle, rat colon, rat spleen or rat testicle.

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

[0071] Example 1 Preparation of rabbit-derived antibodies against Ki-67 protein

[0072] The immunogen used to prepare the rabbit monoclonal antibody in this example is a 42-amino acid polypeptide fragment from position 2295 to 2336 of the human Ki-67 protein. The polypeptide was synthesized by Suzhou Modif Biotechnology Co., Ltd. and the initial AA residue C was added. The sequence of the immunogen is: C-NLPGSKRWPQTPKEKAQALEDLAGFKELFQTPGTDKPTTDEK (see SEQ ID NO. 11).

[0073] In this example, B cell labeling and sorting techniques were used to enrich and isolate B lymphocytes capable of recognizing the human Ki-67 protein directly from the spleens of New Zealand white rabbits immunized with the immunogen. These isolated B lymphocytes were then cultured as single cells to produce the monoclonal antibody. For large-scale production, naturally paired antibody heavy chain variable region (VH) and light chain variable region (VL) genes were amplified by PCR from monoclonal antibody-secreting B lymphocytes and recombinantly expressed. Finally, the target antibody 4E5 was isolated and purified in large quantities from the host cell culture supernatant.

[0074] The amino acid (AA) sequence of antibody 4E5 is shown in Table 1. For ease of description, LCDR1-3 in the table represent light chain complementary determining regions CDR1-3, and HCDR1-3 represent heavy chain complementary determining regions CDR1-3.

[0075] Table 1 Sequence information of monoclonal antibody 4E5 in this example

[0076]

[0077] 1. Animal Immunization: Four Japanese large-eared white rabbits were immunized with the above-mentioned immunogen; each rabbit received 300 μg of the immunogen. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to form an emulsion and injected subcutaneously at multiple sites on the abdomen and back of the rabbit. Two weeks after the first immunization, 150 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to form an emulsion and injected subcutaneously at multiple sites on the abdomen and back of the rabbit. Serum samples were collected after three immunizations and titers against human Ki-67 protein were determined by ELISA. Antibodies were purified from the serum after the final immunization and tested for specificity and sensitivity against endogenous detection samples using Western blotting, immunofluorescence, and immunohistochemistry. Rabbits with high serum titers and the best endogenous detection performance were given a booster immunization with 300 μg of the immunogen injected subcutaneously at multiple sites. Three days later, the animals were sacrificed and their spleens were harvested.

[0078] 2. Isolate B lymphocytes from the spleen and sort B lymphocytes: Use conventional methods to isolate B lymphocytes from the spleen, and sort out antigen-specific B lymphocytes. For related methods, please refer to the patents "Method for Efficiently Isolating Single Antigen-Specific B Lymphocytes from Spleen Cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)" and "A B Lymphocyte In Vitro Culture System and Application (Publication No.: CN111518765A, Publication Date: 2020-08-11)".

[0079] 3. Cloning of the rabbit monoclonal antibody gene: The supernatant of cultured B lymphocytes was used to identify positive clones by ELISA coated with biotinylated screening peptide (sequence: NLPGSKRWPQTPKEKAQALEDLAGFKELFQTPGTDKPTTDEK-Biotin, synthesized by Suzhou Modifu Biotechnology Co., Ltd.). The screening criteria was OD 450 The value is greater than 0.7.

[0080] The positive clones of single B lymphocytes were collected and lysed and then analyzed by Quick-RNA TMRNA was extracted using a MicroPrep kit (ZYMO, Cat. No. R1051) and reverse transcribed into cDNA. Using cDNA as a template, naturally paired rabbit monoclonal antibody light chain variable regions (VL) and heavy chain variable regions (VH) were amplified from cDNA of corresponding positive clones using PCR. The PCR reaction system consisted of 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2× Gloria HiFi (ABclonal, Cat. No. RK20717), and 6.5 μL 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, followed by a final hold at 72°C for 5 min. The resulting reaction solution was stored at 4°C. The primer sequences (5'-3') for amplifying the VL and VH genes are shown below, where F and R represent the forward and reverse primers, respectively:

[0081] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (SEQ ID NO. 12);

[0082] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (SEQ ID NO. 13);

[0083] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (SEQ ID NO. 14);

[0084] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (SEQ ID NO. 15).

[0085] The amplified DNA product was sequenced by Jinkairui Biotechnology Co., Ltd., obtaining the VL amino acid sequence shown in SEQ ID NO. 2 and the VH amino acid sequence shown in SEQ ID NO. 7. The IMGT online database (www.imgt.org) was then queried to obtain the constant region sequence, resulting in the complete light chain (FL) amino acid sequence shown in SEQ ID NO. 1 and the complete heavy chain (FH) amino acid sequence shown in SEQ ID NO. 6. This resulted in a rabbit monoclonal antibody against Ki-67 protein, designated 4E5.

[0086] 4. Production and purification of rabbit monoclonal antibodies: To obtain multiple antibodies that recognize human Ki-67, the heavy and light chain genes of the selected antibody 4E5 were loaded onto expression vectors, and the plasmids were transfected into 293F cells. The mammalian expression vector pBR322 used pre-carries the light chain constant region (CL) and heavy chain constant region (CH) genes, and its expression profile is shown in Figure 1 In the figure, the pBR322 origin and f1 origin are replication promoters, Ampcillin is the resistance gene, CMV promoter is the transcription promoter, SV40 PAterminator is the tailing signal, the light chain constant is the nucleic acid sequence of CL (left figure), and the heavy chain constant is the nucleic acid sequence of CH (right figure). Then, the VL and VH genes are inserted upstream of the CL and CH genes of the pBR322 vector, respectively. The construction process of the light chain and heavy chain gene expression vectors is as follows: the mammalian cell expression vector pBR322 containing rabbit monoclonal antibodies CH and CL is conventionally linearized with NheI and XbaI restriction endonucleases, respectively. After the PCR products amplified above are purified, the VL and VH genes with signal peptide encoding genes are constructed into the expression vector pBR322 by homologous recombination. The successful construction of the vectors was verified by sequencing.

[0087] Typically, to achieve secretory expression of an antibody, a signal peptide sequence is added to the front end of its VL and VH genes. Signal peptides can be commonly used antibody expression signal peptides in the art, such as the signal peptides "MDTRAPTQLLGLLLLWLPGARC" upstream of the VL and "METGLRWLLLVAVLKGVQC" upstream of the VH in the patent "Anti-human interferon α2 rabbit monoclonal antibody and its application (Publication No.: CN116063487A, Publication Date: 2023-05-05)" and "High-affinity human IL-5 rabbit monoclonal antibody and its application (Publication No.: CN115819578A, Publication Date: 2023-03-21)." Of course, those skilled in the art can also replace other signal peptides for antibody expression after obtaining the antibody sequence of the present invention. Therefore, the signal peptide sequence is not shown in the antibody sequence of Table 1 of this example.

[0088] The constructed FL and FH expression vectors were co-transfected into 293F cells and cultured for 72-96 hours. The culture supernatant contained recombinant rabbit monoclonal antibodies that recognized human Ki-67. The target antibodies were purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, Catalog No. SA023100). Electrophoresis on a 12% SDS-PAGE gel revealed an antibody concentration of 0.55 mg / mL and a purity greater than 90%.

[0089] Example 2 Reaction Specificity and Application Analysis of Monoclonal Antibodies Against Ki-67 Protein

[0090] 1. Western blot analysis of monoclonal antibody 4E5 (WB)

[0091] Positive cell / tissue samples with high Ki-67 expression and negative cell / tissue samples without Ki-67 expression were lysed separately to obtain protein extracts, which were subjected to 10% polyacrylamide gel electrophoresis. The gel protein bands were transferred to a PVDF membrane using an electrotransfer system according to conventional methods. The membrane was placed in TBST blocking solution containing 5% skim milk powder and incubated at room temperature for 1 hour. Antibody 4E5 (primary antibody dilution ratio 1:1000, working concentration 0.55 μg / mL) was added and incubated at room temperature for 1 hour. The membrane was then washed with TBST, and goat anti-rabbit secondary antibody (purchased from Jackson, product number AS014, secondary antibody dilution ratio 1:10000) was added and incubated at room temperature for 1 hour. The membrane was washed with TBST again. Finally, ECL supersensitive color development solution was added for development.

[0092] The positive cell / tissue samples in this embodiment include: HeLa cell line, and the negative cell / tissue samples include: MKI67 (Ki67 encoding gene) homozygous knockout HeLa cell line (HeLa-MKI67-1E6), mouse embryonic fibroblast cell line NIH / 3T3, mouse liver tissue and rat liver tissue. The aforementioned Hela cell line is derived from the cell bank of the Chinese Academy of Sciences in Shanghai. The Hela cell line was starved and stimulated with nocodazole before detection. The starvation group was serum starved for 18 hours, and the nocadazole stimulation group was stimulated with 1μg / mL nocadazole for 16 hours. Nocadazole is a fast and reversible microtubule inhibitor that binds to β-tubulin and disrupts microtubule assembly / disassembly dynamics, thereby arresting the cells in the G2 / M phase. Ki-67 is most highly expressed in the G2 phase, so the expression of Ki-67 in cells treated with nocadazole is enhanced. The test results of the corresponding samples are shown in Figure 2 .

[0093] As can be seen in the figure, a distinct Ki-67 protein band appears in the positive samples. Because Ki-67 is rapidly degraded upon expression, the detected bands are diffuse and tailing, which is normal. The bands are all located below 359 kDa, consistent with the diffuse distribution characteristic of Ki-67 protein. Furthermore, the figure shows that expression is significantly enhanced in the nocadazole-stimulated group compared to the starvation-treated group, consistent with the expected results. Furthermore, no bands are observed in homozygous Ki-67 knockout cells, regardless of stimulation or in the absence of any bands. Therefore, the tailing bands in the positive samples are distinct degradation bands of the target protein. No bands were detected in the remaining negative samples, demonstrating that antibody 4E5 uniquely recognizes Ki-67 protein in cell lysates. The antibody has high specificity and good binding affinity for Ki-67, resulting in high detection accuracy and effective avoidance of false-positive results. Furthermore, the results demonstrate that this antibody can be used for the specific detection of Ki-67 protein expressed in multiple species, including human, mouse, and rat.

[0094] 2. Immunofluorescence analysis (IF) of monoclonal antibody 4E5

[0095] Positive samples expressing Ki-67 protein were taken, and the aforementioned cell / tissue samples were completely covered with 5% blank goat serum and incubated in a 37°C constant temperature and humidity incubator for 30 minutes. The blocking solution was removed, and then the antibody 4E5 working solution prepared in TBS buffer (primary antibody dilution ratio 1:200, working concentration 2.75 μg / mL) was added dropwise and incubated at 4°C overnight; the sample was taken out and rewarmed at room temperature for 15 minutes, the primary antibody working solution was removed, and the sample was washed with TBST buffer. Then, goat anti-rabbit secondary antibody working solution (secondary antibody dilution ratio 1:10000, from ABclonal, product number AS007) was added dropwise, incubated at 37°C for 1 hour in the dark, the secondary antibody working solution was removed, and the sample was washed with TBST buffer. DAPI working solution was added to the sample and incubated for 10 minutes; the DAPI working solution was removed, and anti-fluorescence attenuation mounting medium was added. The sample was observed under a fluorescence microscope and images were collected. α-Tubulin scaffold protein was used as a reference. The primary and secondary antibodies were both from ABclonal, with catalog numbers AC012 (dilution ratio 1:200) and AS076 (dilution ratio 1:500), respectively.

[0096] The positive cell / tissue samples in this example include: human cervical cancer cell line (Hela) and human epidermal cancer cell line (A431), mouse spleen tissue and mouse myoblast cell line (C2C12), rat spleen tissue sample and rat adrenal pheochromocytoma cell line (PC-12). The corresponding sample results are shown in Figure 3Among them, the upper figure from left to right is the fluorescence imaging of the human Hela cell line, human A431 cell line, mouse spleen tissue, mouse C2C12 cell line, rat spleen tissue and rat PC-12 cell line samples using antibody 4E5. After the antibody 4E5 binds to the Ki-67 protein, it is labeled with red by the fluorescent secondary antibody. The lower figure from left to right is the comparison of the corresponding samples using DAPI nuclear counterstaining and displaying the red, blue and green three-color merge. DAPI counterstaining cell nuclei is blue, and green is the reference for α-Tubulin skeletal protein.

[0097] As can be seen from the figures, the nuclear staining of human Hela and A431 cell lines, mouse C2C12 cell lines, and rat PC-12 cell lines shows clear, bright, and strong positive signals, indicating accurate antibody localization. Mouse and rat spleen tissue samples show positive staining of proliferating cell nuclei and negative staining of other cellular components, demonstrating acceptable specificity. This demonstrates that the antibody 4E5 of the present invention exhibits high specificity for the Ki-67 protein, with actual IF detection results consistent with theoretical localization. The results are highly reliable and demonstrate good applicability for Ki-67 proteins from diverse species.

[0098] 3. Immunohistochemistry (IHC) analysis of monoclonal antibody 4E5

[0099] Ki-67 protein is expressed in proliferating cells in all active cell cycle phases (late G1, S, G2, and M phases), and therefore is expressed to varying degrees in most normal and tumor tissues, with subcellular localization in the nucleus. The tissue samples used in this example include: 1) human samples: spleen, liver, hepatocellular carcinoma, tonsil, lung, lung cancer, colon, colon cancer, kidney, thyroid cancer, breast, breast cancer, esophagus, and pancreas; 2) rat samples: heart, liver, spleen, lung, kidney, brain, colon, and testicles; and 3) mouse samples: heart, liver, spleen, lung, kidney, brain, colon, and testicles, for a total of 30 samples. Tissue samples with high Ki-67 expression include: human tonsil, human colon, human lung cancer, rat colon, rat spleen, rat testicle, mouse colon, mouse spleen, and mouse testicles; rat liver, mouse liver, and human liver show little expression, while other tissues express Ki-67 to varying degrees.

[0100] IHC staining was performed as follows: 1) Sample preparation and baking: paraffin sections were placed on a slice rack in the same orientation and placed in a 56°C constant temperature incubator for 30 min; at the same time, dewaxing solution 1 was placed in a 56°C constant temperature incubator; paraffin sections and slice racks were placed in dewaxing solution 1, and then removed from the constant temperature incubator and placed at room temperature. After 5 min, the sections were taken out and immersed in room temperature dewaxing solution 2, and paraffin sections were placed in the dewaxing solution reagent tank in the order of dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3. The sections were placed in each tank for 5 min when placed in the dewaxing solution reagent tank, and in each tank for 3 min when placed in the anhydrous ethanol reagent tank; the sections were washed with running water for 3 min; dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.; 2) Antigen retrieval: 0.01 M High-pressure heat repair was performed using Tris-EDTA repair solution (pH 9.0). 3) Endogenous peroxidase inactivation: The sections were rinsed three times with PBS buffer for 1 minute each time, and the buffer was removed. The sections were completely immersed in 3% hydrogen peroxide solution and incubated at room temperature for 10 minutes. 4) Blocking: The sections were rinsed three times with PBS buffer for 3 minutes each time, and the buffer was removed. The tissue area to be examined was circled on the slide using an immunohistochemistry pen, and PBS blocking solution was added dropwise to the circled area. The sections were placed horizontally in a humidified incubation chamber with water at the bottom. The timer was started from the addition of blocking solution, and the sections were incubated at room temperature for 30 minutes. 5) Primary antibody incubation: The blocking solution was removed, and anti-Ki-67 protein antibody 4E5 diluted solution was added dropwise to the tissue sections (primary antibody dilution ratio 1:1000, working concentration 0.55μg / mL), placed horizontally in an incubation humidified box, and incubated at room temperature for 60min; remove the antibody working solution, rinse quickly with PBS buffer once, and soak and wash three times with PBS buffer, each time for 3min; 6) Secondary antibody incubation: add ready-to-use secondary antibody working solution (from ABclonal ready-to-use immunohistochemistry pika and rabbit universal secondary antibody kit, product number RK50015) to the tissue section, then place horizontally in an incubation humidified box and incubate at room temperature for 25min; remove the secondary antibody working solution on the section, rinse quickly with PBS buffer once, and soak and wash three times with PBS buffer, each time for 3min; 7) Color development: add the color development solution of ABclonal ready-to-use immunohistochemistry pika and rabbit universal secondary antibody kit to the section, and incubate at room temperature for 5min Afterwards, immerse the sections in a large amount of distilled water to terminate color development. After terminating color development, rinse in running water for 10 minutes. 8) Counterstaining: Immerse the slightly drained tissue sections in Mayer's hematoxylin for counterstaining for 1 minute and rinse in running water for 3 minutes. 9) Blueing: Immerse the slightly drained sections in a saturated aqueous solution of lithium carbonate for blueing for 3 seconds and rinse in running water for 3 minutes. 10) Dehydration: Soak the washed sections once in anhydrous ethanol, lifting them up and down several times during the soaking period, counting for 10 seconds before removing them. Place them in a constant temperature forced air drying oven at high temperature (54-58°C) to completely dry. 11) Mounting: Add an appropriate amount of neutral gum to the center of the section and cover with a coverslip. The amount of gum added should be appropriate, ensuring that the tissue is completely covered after the coverslip is applied, and no gum overflows. Finally, scan the sections.

[0101] Immunohistochemical staining results are categorized as either positive or negative. Positive expression requires brown staining at the specific antigenic site of the corresponding cells and tissues against a low or absent background. Negative expression refers to the absence of brown staining in the specific tissues and cells. For example, a human tonsil sample can serve as both a positive and negative control. Nearly all B cells in the dark zone of the germinal center of a human tonsil must show moderate to strong nuclear staining, while at least weak to moderate staining must be seen in the majority of B cells in the light zone, with the vast majority of mantle zone B cells showing no staining. Additionally, liver or pancreas can be used primarily as supplementary negative tissue controls. In these cases, <1% of hepatocytes and exocrine pancreatic epithelial cells should be positive (inflammation may induce Ki-67 elevation). Figure 4Representative IHC staining results for human colon, human tonsil, human breast cancer, mouse colon, mouse spleen, mouse testis, rat spleen, and rat testis are shown from left to right and from top to bottom. Antibody 4E5 is accurately localized within proliferating cell nuclei, with clear staining, no nonspecific staining, and a clean background, demonstrating high antibody specificity. Furthermore, the IHC detection results for 30 tissue samples from the present invention are consistent with the actual expression and localization of Ki-67 in the corresponding tissue samples and theoretical expectations, further demonstrating the high specificity, strong anti-interference ability, good detection accuracy and reliability, excellent detection sensitivity and specificity, and the absence of false positive or false negative results.

[0102] 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. A monoclonal antibody against Ki-67 protein, characterized in that: It includes a light chain variable region and a heavy chain variable region, and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the light chain variable region 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.

2. The anti-Ki-67 protein monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region 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.

3. The monoclonal antibody against Ki-67 protein according to claim 2, characterized in that The amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The monoclonal antibody against Ki-67 protein according to claim 1, characterized in that The monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody against Ki-67 protein according to any one of claims 1 to 4.

6. A recombinant vector or host cell, characterized in that: The recombinant vector or the host cell comprises the nucleic acid molecule according to claim 5.

7. Use of the anti-Ki-67 protein monoclonal antibody according to any one of claims 1 to 4 in the preparation of a Ki-67 protein detection kit.

8. Use of the anti-Ki-67 protein monoclonal antibody according to claim 7 in preparing a Ki-67 protein detection kit, characterized in that: The Ki-67 protein is human, rat or mouse Ki-67 protein.

9. Use of the anti-Ki-67 protein monoclonal antibody according to claim 7 in preparing a Ki-67 protein detection kit, characterized in that: The detection kit is an enzyme-linked immunosorbent assay kit, an enzyme-linked immunosorbent assay kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit or a flow cytometry kit.

10. A Ki-67 protein detection kit, characterized in that: The detection kit comprises the anti-Ki-67 protein monoclonal antibody or its antibody conjugate according to any one of claims 1 to 4, wherein the antibody conjugate is formed by linking the monoclonal antibody to a detection label.

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