Anti-EpCAM protein antibody and application thereof in immunodetection

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-EpCAM protein antibody and application thereof in immunodetection. Amino acid sequences of CDR1-3 on a light chain variable region of the antibody are respectively shown as SEQ ID NO.3-5, and amino acid sequences of CDR1-3 on a heavy chain variable region of the antibody are respectively shown as SEQ ID NO.8-10. The antibody can specifically recognize EpCAM protein naturally expressed in cells and tissues, and has good applicability in a plurality of immunodetection systems, especially immunoblotting, immunofluorescence, immunohistochemistry, flow cytometry and the like. Moreover, the immunodetection kit has the advantages of high specificity, high affinity and / or high sensitivity, cross-species scene applicability and the like, not only can the accuracy of clinical diagnosis be improved, but also a key tool can be provided for basic research and targeted therapy, and the immunodetection kit has important scientific significance and clinical value.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, in particular to an antibody against EpCAM protein and an application thereof in immunoassay. Background Art

[0002] Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein with a molecular weight of approximately 40 kDa. Also known as CD326, ESA, KSA, DIAR5, KS1 / 4, TROP1, TACSTD1, and GA733-2, it is expressed to varying degrees in all normal epithelial cells except squamous cells, primarily located at tight junctions. The EpCAM protein consists of 314 amino acids and has a large extracellular domain, a transmembrane region, and a short (26 amino acid) cytoplasmic tail. The extracellular domain includes an epidermal growth factor-like domain and a putative thyroglobulin domain, mediating homotypic epithelial cell adhesion. Under physiological conditions, EpCAM not only regulates cell adhesion but also participates in a variety of biological processes, including cell-matrix interactions, cell migration, proliferation and differentiation, cell cycle regulation, and signal transduction.

[0003] In recent years, EpCAM has attracted much attention as a target for tumor diagnosis and treatment. Evidence from multiple studies has shown that EpCAM molecules are expressed in the vast majority of epithelial tumor tissues. Among common tumors, the expression rate of esophageal cancer, gastric cancer, colon cancer, prostate cancer, lung cancer, and ovarian cancer reaches or approaches 100%. Therefore, EpCAM is often used as an epithelial tumor marker. Pathologically, EpCAM can be used to distinguish epithelial tumors from tumors of non-epithelial tissue origin, such as lung adenocarcinoma, ovarian cancer, and EpCAM-negative mesothelioma (except for epithelioid mesothelioma that may express EpCAM). EpCAM expression levels are also associated with tumor progression and prognosis, and are closely related to the length of patient survival. Studies have found that prostate cancer patients with high EpCAM expression have significantly lower overall survival and metastasis-free survival than those with low EpCAM expression. In addition, the widespread expression of EpCAM and its involvement in cell proliferation also make EpCAM a therapeutic target for immunotherapy strategies.

[0004] Immunological detection techniques such as immunoblotting, immunohistochemistry, immunofluorescence, and flow cytometry have been widely used in the diagnosis of pathological samples such as tumor tissues and cells, leading to a steadily increasing demand for EpCAM antibodies. However, there are few anti-EpCAM antibodies on the market, and even fewer antibodies that can be used in immunological detection. Most antibodies also suffer from poor specificity and low antigen binding affinity, resulting in less than ideal EpCAM detection results. For example, some antibodies can cross-react with non-target proteins such as cell adhesion molecules, leading to false-positive results. Low-affinity antibodies cannot effectively recognize tumor cells with low EpCAM expression, resulting in false-negative results. Furthermore, antibodies only recognize human EpCAM and are unable to bind to homologous proteins in model animals such as mice and rats, limiting the verification of EpCAM function in preclinical animal studies. Therefore, the development of novel antibodies with high selectivity, high sensitivity, and / or broad-spectrum EpCAM recognition is an urgent need and has important implications for the early diagnosis, treatment, and prognosis of tumors. Summary of the Invention

[0005] To address the aforementioned issues in the prior art, the present invention provides a novel anti-EpCAM protein antibody with high specificity, high affinity, and / or high sensitivity, and cross-species applicability, providing a reliable antibody tool for clinical diagnosis and basic research. Further provided are the use of this antibody or its antibody conjugate in the preparation of an EpCAM protein immunoassay kit, as well as related immunoassay 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 an anti-EpCAM protein antibody, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NOs. 3-5, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in 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 antibody light chain 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 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 or a recombinant vector, wherein the recombinant vector comprises the nucleic acid molecule, and the nucleic acid molecule comprises a gene sequence encoding the above-mentioned anti-EpCAM protein antibody.

[0011] Optionally, the nucleic acid molecule further comprises a gene sequence encoding a signal peptide.

[0012] The third aspect of the present invention provides the use of the above-mentioned anti-EpCAM protein antibody or its antibody conjugate in the preparation of an EpCAM protein immunoassay kit, wherein the antibody conjugate comprises the antibody and a detection label connected to the antibody.

[0013] Furthermore, the EpCAM protein is human, mouse or rat EpCAM protein.

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

[0015] Furthermore, the immunoassay kit is an immunoblotting kit, an immunohistochemistry kit, an immunofluorescence kit or a flow cytometry kit.

[0016] In a fourth aspect, the present invention provides an EpCAM protein immunoassay kit, comprising the above-described anti-EpCAM protein antibody or an antibody conjugate thereof, wherein the antibody conjugate comprises the antibody and a detection label linked to the antibody.

[0017] The advantages and positive effects of the present invention are:

[0018] The antibodies of the present invention can specifically recognize EpCAM proteins naturally expressed in cells and tissues and have excellent applicability in multiple immunoassay systems, particularly immunoblotting, immunofluorescence, immunohistochemistry, and flow cytometry. Furthermore, they exhibit high specificity, high affinity, and / or high sensitivity in immunoassays, as well as cross-species applicability. They not only enhance the accuracy of clinical diagnoses but also provide key tools for basic research and targeted therapies, possessing significant scientific significance and clinical value. 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 This is a graph showing the immune serum titer test results after rabbits were immunized with human EpCAM protein in Example 1 of the present invention;

[0021] Figure 2 This is a map of the expression vectors used to construct the anti-EpCAM protein antibody in Example 1 of the present invention, from left to right are the pBR322 vectors pre-carrying the antibody light chain constant region and heavy chain constant region;

[0022] Figure 3 This is an immunohistochemical staining diagram of human brain, human kidney, human colon and human small intestine tissue sections detected using anti-EpCAM protein antibodies in Example 2 of the present invention;

[0023] Figure 4 This is an immunofluorescence staining image of mouse colon, mouse small intestine, and rat small intestine tissue section samples detected using an anti-EpCAM protein antibody in Example 2 of the present invention;

[0024] Figure 5 This is an immunoblot image of human colon cancer cells, mouse myoblasts, and mouse colon tissue samples detected using an anti-EpCAM protein antibody according to Example 2 of the present invention;

[0025] Figure 6 This is a flow cytometry fluorescence intensity graph of 293T cells overexpressing the human EpCAM gene and wild-type 293T cells detected using an anti-EpCAM protein antibody in Example 2 of the present invention. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those skilled in the art.

[0030] 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.

[0031] 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 situations: (i) A, (ii) B, and (iii) A and B.

[0032] The terms "rabbit monoclonal antibody," "monoclonal antibody," "rabbit-derived antibody," and "rabbit monoclonal antibody" have synonymous meanings and, unless otherwise specified, refer to antibodies that specifically bind to epithelial cell adhesion molecule (EpCAM). The terms "EpCAM," "EpCAM protein," "CD326," and "CD326 / EpCAM" have synonymous meanings. The modifier "rabbit" indicates that the complementarity determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences.

[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 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.

[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 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.

[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 cell-mediated cytotoxicity (ADCC). The CL length is generally consistent across different Ig types (κ or λ), but the CH length varies across different 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.

[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 both 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 include CDR regions and FR regions from rabbit immunoglobulin sequences. In other embodiments, the antibody may contain 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 an antibody in which part of the antibody is 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 with a CDR region of a non-human antibody such as a rabbit antibody and a FR region from a human. In some cases, the variable region of a non-human antibody is combined with the 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 the FR region and 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, and this framework sequence 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.

[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 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.

[0049] One embodiment of the present invention provides an anti-EpCAM protein 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 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; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0050] The present invention uses commercial recombinant human EpCAM (CD326 / EpCAM) protein as an immunogen for immunization, and then obtains rabbit monoclonal antibodies against EpCAM protein based on the monoclonal antibody development technology of single B lymphocyte screening and culture. The antibody of the present invention can specifically recognize EpCAM protein naturally expressed in cells and tissues, and has good applicability in multiple immunoassay systems, especially immunoblotting, immunofluorescence, immunohistochemistry, flow cytometry, etc. During immunoassay, it can effectively and accurately distinguish positive and negative samples. The actual detection results in positive samples are highly consistent with theoretical expectations, and there is no specific binding in negative samples. The detection sensitivity and specificity are high, indicating that the antibody of the present invention can effectively resist the interference of non-target proteins, has good specificity and high affinity, and can sensitively identify cell tissues with low expression of EpCAM, which is beneficial to improving the accuracy of early clinical pathological diagnosis and reducing the false negative rate in pathological diagnosis. The high specificity is also beneficial to reducing the false positive rate in pathological diagnosis. In addition, the antibodies of the present invention can recognize homologous EpCAM proteins in humans, mice, and rats. Their cross-species reaction characteristics can support the dynamic monitoring of EpCAM in mouse and rat tumor models, accelerate the functional verification of clinical drugs targeting EpCAM proteins, and greatly expand the application scenarios of antibodies.

[0051] 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.

[0052] Optionally, the monoclonal antibody of the present invention further comprises a light chain constant region (CL) and a heavy chain constant region (CH), wherein CL and VL constitute a complete light chain (FL), and CH and VH constitute a complete heavy chain (FH). Antibody constant regions are typically obtained by querying the IMGT online database, for example, by searching the IMGT online database (www.imgt.org) for rabbit IgG gamma C reign to obtain CH, and for rabbit IgG kappa C reign to obtain CL.

[0053] Specifically, the amino acid sequence of the antibody light chain is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 6. Correspondingly, CL is of κ type, and CH is of IgG type.

[0054] 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.

[0055] Yet another embodiment 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 comprises a gene sequence encoding the above-mentioned anti-EpCAM protein antibody.

[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 by conventional means such as codon coding rules. The full-length sequence of the nucleic acid molecule or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0058] In order to achieve secretory expression of the antibody, the nucleic acid molecule may optionally further include a gene sequence encoding a signal peptide, wherein the signal peptide is located upstream of the antibody gene sequence.

[0059] The original vector for constructing the recombinant vector is a conventional various vectors 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, which is then introduced into the host cell and cultured under specific conditions to express the antibody. This is a well-known technology in the art and will not be described in detail here.

[0060] The nucleic acid molecules encoding the monoclonal 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 chain and light chain are mixed and incubated under appropriate conditions to form antibodies. In other embodiments, the nucleic acid molecules of the monoclonal 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.

[0061] Conventional techniques are used to transfect or transform recombinant vectors 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 gene gun bombardment to achieve gene introduction.

[0062] 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, CHOS, COS7, 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.

[0063] In a preferred embodiment, the above-mentioned recombinant vector is a mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell (293F cell).

[0064] In a typical embodiment, a method for preparing a monoclonal antibody comprises: concatenating the heavy and light chain genes of the antibody with a signal peptide, loading the genes separately into the expression vector pBR322, co-transfecting 293F cells, culturing and collecting the cell culture supernatant, and purifying the target antibody. The choice of signal peptide is designed based on the host cell and is not particularly limited in the present invention.

[0065] Another embodiment of the present invention provides the use of the above-mentioned anti-EpCAM protein antibody or its antibody conjugate in preparing an EpCAM protein immunoassay kit, wherein the antibody conjugate comprises the antibody and a detection label connected to the antibody.

[0066] The advantages of using the anti-EpCAM protein antibody or its antibody conjugate in preparing an EpCAM protein immunoassay kit are the same as the advantages of the anti-EpCAM protein antibody over the prior art as described above, and will not be repeated here.

[0067] It should be emphasized that the antibodies of the present invention can be used alone or linked (covalently or non-covalently) to a detection label to form an antibody conjugate. In some embodiments, the antibodies of the present invention are used as antigen-binding (or capture) antibodies that specifically recognize and bind to EpCAM protein in the sample to be tested, and then qualitatively or quantitatively detect EpCAM by analyzing the detection label signal linked thereto; in other embodiments, the anti-EpCAM protein antibody is not labeled (as a primary antibody or capture antibody), and the detection label is linked to a secondary antibody (as a detection antibody) or other molecule. For example, if the anti-EpCAM antibody is a rabbit IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody, thereby qualitatively or quantitatively detecting EpCAM by analyzing the changes in the detection label signal generated after the secondary antibody specifically binds to the antibody of the present invention, such as the multiple detection systems established in Example 2 below.

[0068] The detection marker is used to generate a recognizable signal change, so as to identify the antibody of the present invention or its secondary antibody according to the signal change, and then identify the expression of EpCAM protein in the sample to be tested through the specific reaction of the antigen and antibody. The detection marker includes, but is 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.

[0069] The above-mentioned 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).

[0070] During drug development, it is often necessary to evaluate the activity of EpCAM proteins in humans and animal models, and the cross-reactivity to cross-species homologous proteins determines the applicability of the antibody in these models. The monoclonal antibodies of the present invention can bind to EpCAM proteins of humans and commonly used experimental animals (mice, rats), have multi-species cross-reactivity, and can be used across species. Therefore, in a preferred embodiment of the present invention, the EpCAM protein can be a human, mouse or rat EpCAM protein. The amino acid and nucleotide sequences of the EpCAM protein can be obtained by conventional techniques. Among them, for information on human EpCAM protein, see Uniprot No. P16422, for information on mouse EpCAM protein, see Uniprot No. Q99JW5, and for information on rat EpCAM protein, see Uniprot No. O55159.

[0071] Based on the same inventive concept, an embodiment of the present invention further provides an EpCAM protein immunoassay kit, which comprises the above-mentioned anti-EpCAM protein antibody or antibody conjugate thereof.

[0072] Optionally, the immunoassay kit is an enzyme-linked immunosorbent assay kit, an enzyme-linked immunospot assay kit, an immunohistochemistry kit, an immunofluorescence assay kit, an immunoblotting kit, an immunoprecipitation kit, or a flow cytometry kit.

[0073] Preferably, the immunoassay kit is an immunoblotting kit, an immunohistochemistry kit, an immunofluorescence kit or a flow cytometry kit.

[0074] Preferably, the immunoassay kit further comprises a fluorescein-coupled anti-rabbit IgG secondary antibody or a horseradish peroxidase (HRP)-coupled anti-rabbit IgG secondary antibody.

[0075] 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.

[0076] Example 1 Preparation of rabbit monoclonal antibodies against EpCAM protein

[0077] 1.1. Animal Immunization: Two New Zealand white rabbits were immunized with commercially available recombinant human CD326 / EpCAM protein (from ABclonal, Catalog No. RP00073) as the immunogen at a dose of 200 μg / rabbit. 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. Every three weeks 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. Two booster immunizations were performed. After the three immunizations, rabbit serum samples were collected and the titer against human CD326 / EpCAM was determined by enzyme-linked immunosorbent assay (ELISA). Rabbits with high serum titers were boosted once with 200 μg of the immunogen at multiple points subcutaneously. The spleens were harvested three days later.

[0078] The results of ELISA determination of immune serum titer are shown in Figure 1 , where the coating was originally recombinant human CD326 / EpCAM protein, the final coating concentration was 1μg / mL, the serum to be tested was the primary antibody, the serum was initially diluted with 1×PBS buffer at 1:1000, and then diluted in a 1:3 ratio, with a total of 8 gradients, horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG (from ABclonal, product number AS014) was the secondary antibody, the secondary antibody dilution ratio was 1:5000, pre-immune rabbit serum was used as the negative control, and the detection system without immune serum was the blank control (NC), N22693 and N22694 are the rabbit numbers. Figure 1 It can be seen that after the third to fourth immunizations, a strong immune response was produced in the rabbit body, the titer of the neutralizing antibody was high, and the specific antibody activity was good, which can be used to separate antibodies.

[0079] 1.2. Isolation of B lymphocytes in the spleen and sorting of antigen-specific B lymphocytes: For related methods, please refer to the published patents "Method for Efficient Isolation of 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)".

[0080] 1.3 Cloning of the rabbit monoclonal antibody gene: The cultured B lymphocyte supernatant was used to identify antigen-specific B lymphocytes using ELISA coated with recombinant human CD326 / EpCAM protein. TMRNA was extracted using a Micro Prep kit (ZYMO, Cat. No. R1100-250) and reverse transcribed into cDNA. Using the cDNA as a template, naturally paired rabbit antibody light chain variable region (VL) and heavy chain variable region (VH) genes were amplified by PCR and sequenced. The PCR 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 protocol consisted of 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 72°C for 5 min. The reaction mixture 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 forward and reverse primers, respectively.

[0081] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO. 11);

[0082] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 12);

[0083] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 13);

[0084] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 14).

[0085] The amplified DNA product was sequenced to obtain the VL sequence shown in SEQ ID NO.2 and the VH sequence shown in SEQ ID NO.7; then, the IMGT online database (www.imgt.org) was queried to obtain the sequence of the constant region, and an antibody with a complete light chain (FL) shown in SEQ ID NO.1 and a complete heavy chain (FH) shown in SEQ ID NO.6 was obtained.

[0086] The antibody sequencing was completed by Jinkairui Biotechnology Co., Ltd. The amino acid sequence of the antibody is shown in Table 1, where LCDR1-3 represent the complementarity determining regions CDR1-3 of the light chain variable region, and HCDR1-3 represent the complementarity determining regions CDR1-3 of the heavy chain variable region, respectively. The variable region numbering system is the Kabat numbering system.

[0087] Table 1 Sequence information of rabbit monoclonal antibodies in this example

[0088]

[0089]

[0090] 1.4. Antibody expression and large-scale production: The amplified VL and VH genes are inserted into the expression vector in series with the light chain constant region (CL) and the heavy chain constant region (CH), and the monoclonal antibody is produced in large quantities by recombinantly expressing the antibody genes. In this example, the CL and CH genes are pre-inserted into the mammalian expression vector pBR322, and their expression patterns are shown in FIG. Figure 2 In the figure, 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), and the heavy chain constant is the nucleic acid sequence of CH (right). The VL and VH genes were then ligated via homologous recombination with the pBR322 expression vector carrying the CL and CH genes, which had been linearized with the XbaI (955 bp) and NheI (949 bp) restriction enzymes, respectively, to generate the FL and FH gene expression vectors. Sequencing verified the successful construction of the vectors.

[0091] To facilitate antibody purification, signal peptides are added upstream of the VL and VH genes to achieve secretory expression of the antibodies. Signal peptides commonly used in the art can be used for antibody expression. For example, the patent "Anti-human interferon α2 rabbit monoclonal antibody and its application (Publication No.: CN116063487A, Publication Date: 2023-05-05)" and the patent "High-affinity human IL-5 rabbit monoclonal antibody and its application (Publication No.: CN115819578A, Publication Date: 2023-03-21)" have a signal peptide "MDTRAPTQLLGLLLLWLPGARC" upstream of the VL gene and a signal peptide "METGLRWLLLVAVLKGVQC" upstream of the VH gene. 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 sequences are not shown in the antibody sequences in Table 1 of this example.

[0092] 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 the CD326 / EpCAM protein. The target antibodies were purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, Catalog No. SA023100). Antibody purity was verified by 12% polyacrylamide gel electrophoresis (SDS-PAGE) to be ≥95% at a concentration of 1 mg / mL. The purified antibodies were aliquoted and stored at -20°C until needed.

[0093] Example 2 Establishment and Effect Evaluation of Rabbit Monoclonal Antibody Immunoassay Method

[0094] 2.1 Immunohistochemistry (IHC) analysis using rabbit monoclonal antibodies

[0095] In normal tissues, CD326 / EpCAM is primarily distributed at the tight junctions or basolateral surfaces of epithelial cells. In connective tissue, hematopoietic cells, brain tissue, and vascular endothelial cells, CD326 / EpCAM expression is essentially absent. In pathological conditions, the average EpCAM positivity rate in malignant epithelial tumor cells exceeds 90%. The tissue sections selected for immunohistochemistry in this example included human brain, human kidney, human colon, and human small intestine, with human brain tissue being the negative sample and the remaining samples being the positive samples.

[0096] IHC staining and analysis include the following steps: (1) baking and dewaxing: immerse the paraffin sections of tissue that have been baked at a constant temperature of 56°C for 30 minutes in dewaxing solution 1. After 5 minutes, take out the sections and immerse the sections in the order of dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3. Place the sections in the dewaxing solution for 5 minutes and in the anhydrous ethanol for 3 minutes. Then, wash the sections with running water for 3 minutes. Dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.; (2) antigen repair: 0.01M Tris-EDTA repair solution (pH 9.0) High-pressure heat repair; (3) Inactivation of endogenous peroxidase: Immerse the slides in PBS buffer for 3 times, 1 min each time, remove the buffer on the slides; then immerse the slides in 3% hydrogen peroxide solution and incubate at room temperature for 10 min; (4) Blocking: Immerse the slides in PBS buffer for 3 times, 3 min each time, then remove the buffer, circle the area to be examined on the slide, add PBS blocking solution in the area to be examined, and incubate at room temperature for 30 min; (5) Primary antibody incubation: Remove the blocking solution, add antibody dilution solution (primary antibody dilution ratio 1:900), incubate at room temperature for 60 min; remove the antibody working solution, quickly rinse once with PBS buffer, and soak and wash three times, 3 min each time; (6) Secondary antibody incubation: Add ready-to-use secondary antibody working solution (purchased from Dako, product number K5007), incubate at room temperature for 25 min; remove the secondary antibody working solution, quickly Rinse once and soak and wash three times, each time for 3 minutes; (7) Color development: add color development working solution, observe the color change closely under a microscope, and after obtaining the appropriate staining intensity, immerse the slice in a large amount of distilled water to stop color development, and then rinse in running water for 10 minutes; (8) Restaining: immerse the slightly drained tissue slice in Mayer's hematoxylin for restaining for 1 minute, and rinse in running water for 3 minutes; (9) Rebluing: immerse the slightly drained slice in a saturated aqueous solution of lithium carbonate for blueing for 3 seconds, and rinse in running water for 3 minutes; (10) Dehydration: immerse the slice in anhydrous ethanol twice, lift it up and down several times during the immersion period, and take it out after 10 seconds; dry the slice at high temperature (54-58℃); (11) Sealing: add an appropriate amount of neutral gum to the center of the slice and cover it with a cover glass. The amount of glue added should be appropriate. After sealing the cover glass, the tissue should be completely covered and no glue should overflow. Finally, scan the slice.

[0097] Immunohistochemical staining results are divided into: positive and negative. Positive signals require brown staining and low background in the antigen-expressing areas of specific tissues and cells, while the rest are negative. Figure 3Shown from left to right and top to bottom are IHC staining results for tissue sections from human brain, human kidney, human colon, and human small intestine, magnified at 1:1000. Blue staining indicates nuclear localization, while tan staining indicates subcellular localization of CD326 / EpCAM protein expression. The results demonstrate accurate localization of CD326 / EpCAM staining in positive tissues, including kidney, colon, and small intestine, with a subtle or absent background and no nonspecific staining. CD326 / EpCAM is expressed on the membranes of most normal epithelial cells, with strong staining intensity at the basolateral level and at tight junctions. For example, in the kidney, nearly all epithelial cells lining the collecting tubules exhibit moderate to strong membranous staining, while a predominantly basolateral staining signal is observed in most epithelial cells of the proximal tubules and scattered epithelial cells lining the Bowman's capsule. However, negative tissues such as the human brain showed no specific staining, exhibiting a negative signal, consistent with expectations. This demonstrates that the antibodies of the present invention can be used for labeling CD326 / EpCAM-positive cells and immunohistochemical analysis. The antibodies exhibit advantages such as good specificity, high sensitivity in recognizing the target protein, strong resistance to interference from cellular components, and binding to CD326 / EpCAM proteins that are unaffected by cell / tissue complexity, thereby improving the accuracy and reliability of detection.

[0098] 2.2 Establishment of Immunofluorescence (IF) Detection System

[0099] In this example, positive tissues such as mouse colon, mouse small intestine, and rat small intestine tissues were selected for fluorescent staining.

[0100] The IF detection method is as follows: (1) Tissue section preparation: Take positive tissue and prepare paraffin sections; (2) Slice baking and dewaxing: The operation is the same as IHC staining; (3) Antigen repair: 0.01M Tris-EDTA repair solution (pH9.0) high-pressure heat repair: The operation is the same as IHC staining; (4) Blocking: The operation is the same as IHC staining; (5) Primary antibody incubation: Add the antibody prepared in Example 1 (primary antibody working concentration is 6.5μg / mL), incubate at 4℃ overnight; take out and warm to room temperature for 15min, remove the primary antibody working solution, and then wash once with PBST and twice with PBS, each washing for 5min; (6) Secondary antibody incubation: Add fluorescent secondary antibody solution (Cy3 Goat Anti-Rabbit IgG (H+L), from ABclonal, catalog number AS007, secondary antibody dilution ratio 1:500), incubated at 37℃ in the dark for 1 hour; remove the secondary antibody working solution, then wash once with PBST and twice with TBS, each for 5 minutes; (7) Nuclear staining: add 1μg / mL DAPI nuclear dye working solution, stain at room temperature for 30 minutes, remove the DAPI working solution, then wash once with PBST and twice with PBS, each for 5 minutes; (8) Color development: add anti-fluorescence attenuation mounting medium, observe and collect images under a fluorescence microscope.

[0101] See the results Figure 4 , where the left picture shows confocal imaging of mouse colon, mouse small intestine, and rat small intestine tissue using CD326 / EpCAM rabbit monoclonal antibody. After the antibody binds to CD326 / EpCAM protein, it is labeled red with a fluorescent secondary antibody. The right picture shows the localization information of CD326 / EpCAM using DAPI nuclear staining (blue) and merging red and blue. Through immunofluorescence detection, it can be seen that the rabbit monoclonal antibody of the present invention can specifically bind to CD326 / EpCAM in mouse colon, mouse small intestine, and rat small intestine tissue. The test results are consistent with its theoretical positioning, further proving that the rabbit monoclonal antibody prepared in this application has good recognition specificity and high affinity for human CD326 / EpCAM.

[0102] 2.3 Establishment of Western blot (WB) detection system

[0103] In this embodiment, the samples include human colon cancer cells (HCT116), mouse myoblasts (C2C12), and mouse colon. Mouse C2C12 cells are negative samples, and the others are positive samples.

[0104] The WB experimental analysis operation is as follows: (1) protein sample preparation: the above cells or tissues are lysed separately to obtain protein lysate; (2) electrophoresis: select 10% separation gel and perform polyacrylamide gel electrophoresis (SDS-PAGE); (3) membrane transfer: transfer the gel protein band to the NC membrane in the electrotransfer system according to the conventional method; (4) blocking: place the NC membrane in TBST buffer containing 3% skim milk powder and block it at room temperature for 1 hour; (5) primary antibody incubation: add the antibody prepared in Example 1 (primary antibody concentration is 0.39 μg / mL) and incubate at 4°C overnight; (6) secondary antibody incubation: wash the membrane with TBST 3-4 times, add HRP-conjugated goat anti-rabbit IgG secondary antibody (HRP-conjugated Goat anti-Rabbit IgG secondary antibody) IgG (H+L), from Abclonal, catalog number AS014, secondary antibody dilution ratio 1:5000), incubate at room temperature for 1 hour; (7) Color development: wash the membrane 3-4 times with TBST, add ECL ultrasensitive color development solution (from ABclonal, catalog number RM00020) and develop.

[0105] See the results Figure 5 , the left picture is the result of WB detection of human HCT116 cells, and the right picture is the result of WB detection of mouse C2C12 cells and colon tissue. It can be seen that the rabbit monoclonal antibody of the present invention can specifically bind to CD326 / EpCAM in HCT116 cells and mouse colon tissue, while there is no specific band in C2C12 cells, showing a negative signal. The result is consistent with expectations, indicating that the rabbit monoclonal antibody prepared in this application has good recognition specificity for human and mouse CD326 / EpCAM, as well as the advantage of cross-reaction with human and mouse homologous proteins. The 35-40kDa single or double band of CD326 / EpCAM is the result of the combined action of post-translational modification (especially glycosylation) and splice variants. The double band phenomenon usually reflects the cell state (such as cancer) or modification heterogeneity, while the single band suggests a more uniform expression form.

[0106] 2.4 Establishment of Flow Cytometry (FC) Detection System

[0107] The samples in this example were 293T cells overexpressing the human EpCAM gene (sequence: NCBI gene ID: 4072) (293T-EpCAM) and wild-type 293T cells, the latter serving as a negative sample. The 293T-EpCAM cell preparation process was briefly as follows: pcDNA3.1(+) vector containing the EpCAM gene was transfected into 293T cells using High Gene transfection reagent (ABclonal, Cat. No. RM09014). Cell lines stably expressing the EpCAM protein were obtained by screening for G418 or Puromycin resistance and gene expression assays.

[0108] The FC assay method is as follows: (1) sterilize the clean bench by ultraviolet irradiation for 15-20 minutes, turn on the fan for 5 minutes, and prepare for sterile work; (2) collect and wash the cells to be tested, determine the total number of cells, and check that the cell viability is 90%-95%; (3) resuspend the cells in PBS buffer to approximately 3×10 6 -5×10 6 cells / mL, dispense the cells into a 96-well V-shaped plate at 100 μL / well, and wash once with 1× PBS; (4) dilute the L / D staining solution (Live / Dead staining solution) of the Zombie NIR Fixable Viability Kit (purchased from Biolegend, product number 423105) at a ratio of 1:1500, and dispense the diluted staining solution into the well plate at 100 μL / well, resuspend the cells in the well, and react at room temperature for 15 minutes; (5) centrifuge at 400 g for 5 minutes, discard the supernatant, and wash twice with FACS buffer; (6) dispense 100 μL / well 1× Intracellular Fixation buffer into a 96-well V-shaped plate, resuspend the cells in the well, and react at room temperature for 30 minutes; (7) centrifuge at 400 g for 5 minutes, discard the supernatant, and wash twice with 1× Permeablization buffer; (8) centrifuge at 100 μL / well with 1× Perm The primary antibody diluted with buffer (the antibody prepared in Example 1, the antibody concentration is 2 μg / mL) was dispensed into the well plate, the cells in each well were resuspended, and the reaction was carried out at room temperature for 30 minutes; (9) centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were washed twice with 1× Permeablization buffer; (10) 100 μL / well of fluorescent secondary antibody (Fluorescein (FITC) AffiniPure F(ab')2Fragment Goat Anti-Rabbit IgG, purchased from Jackson, product number 111-096-046) diluted with 1× Permeablization buffer (dilution ratio 1:200) was dispensed into the well plate, the cells in the well were resuspended, and the reaction was carried out at room temperature for 30 minutes; (11) centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were washed twice with 1× Permeablization buffer; (12) The cells in each well were resuspended with 200 μL FACS buffer and stored in the dark; (13) The cells were detected by Beckman Analyses were performed using the Cytoflex flow cytometer according to SOP-105-AND-CA-008. Rabbit IgG isotype control (ABclonal, Cat. No. AC042) was used as an isotype control.

[0109] The results of flow cytometry were as follows Figure 6 As shown, from left to right are wild-type 293T cells and 293T-EpCAM cells, the horizontal axis represents the relative intensity of the fluorescence signal / scattered light signal of the channel, the vertical axis represents the corresponding number of cells, the red curve is the blank control without adding any antibody, the blue curve is the isotype control, and the yellow curve is the antibody prepared by the present invention. It can be seen that there are two cell populations on the 293T-EpCAM cells (of which the high-expressing cell population has a signal transition of 3.0 orders of magnitude). Since the present invention uses a CD326 / EpCAM transient overexpression cell line, which contains cell populations with different efficiencies such as high expression, medium expression, and low expression, the aforementioned two transition signals are generated during the detection process to indicate cell populations with different expression efficiencies. It can be seen that the antibody of the present invention can recognize CD326 / EpCAM proteins with different expression levels and has higher antigen recognition and binding sensitivity. However, there was no signal transition change in the negative sample 293T cells, blank controls, and isotype controls, which proves that the antibody prepared by the present invention has good specificity and is used for FC detection without non-specific signals.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-EpCAM protein antibody, characterized in that It includes a light chain variable region and a heavy chain variable region, the amino acid sequences of 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, 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.

2. The anti-EpCAM protein 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 anti-EpCAM protein antibody according to claim 2, characterized in that The amino acid sequence of the antibody light chain 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 anti-EpCAM protein antibody according to claim 1, characterized in that The 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.

5. A nucleic acid molecule or recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule, and the nucleic acid molecule comprises a gene sequence encoding the anti-EpCAM protein antibody according to any one of claims 1 to 4.

6. The nucleic acid molecule or recombinant vector according to claim 5, characterized in that The nucleic acid molecule also includes a gene sequence encoding a signal peptide.

7. Use of the anti-EpCAM protein antibody or its antibody conjugate according to any one of claims 1 to 4 in the preparation of an EpCAM protein immunoassay kit, characterized in that: The antibody conjugate comprises the antibody and a detection label linked to the antibody.

8. Use of the anti-EpCAM protein antibody or its antibody conjugate according to claim 7 in preparing an EpCAM protein immunoassay kit, characterized in that: The EpCAM protein is human, mouse or rat EpCAM protein.

9. Use of the anti-EpCAM protein antibody or its antibody conjugate according to claim 7 in preparing an EpCAM protein immunoassay kit, characterized in that: The immunoassay kit is an immunoblotting kit, an immunohistochemistry kit, an immunofluorescence kit or a flow cytometry kit.

10. An EpCAM protein immunoassay kit, characterized in that: The invention comprises the anti-EpCAM protein antibody or its antibody conjugate according to any one of claims 1 to 4, wherein the antibody conjugate comprises the antibody and a detection label connected to the antibody.

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