Anti-human keratin 8 antibody, antibody conjugate and application thereof

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-human keratin 8 antibody, an antibody conjugate and application thereof. The 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 the 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 monoclonal antibody provided by the invention has strong specificity to CK8 protein in cytoplasm, good binding affinity and high recognition sensitivity, has no cross reaction and non-specific binding to non-target protein in cells, effectively ensures that a detection signal comes from the target protein, is beneficial to reducing background noise and improving the signal-to-noise ratio, and has good application prospects. The accuracy and reliability of the detection result are improved, the detection result is highly consistent with the actual situation, and the method is suitable for construction of the immune biopsy technology of clinical pathological cells or tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and particularly to antibodies against human keratin 8, antibody conjugates and their applications. Background Art

[0002] Keratin 8, also known as Keratin 8, KRT8, Cytokeratin 8, CK8, Type-II keratin, etc., is an intermediate filament protein and belongs to the cytokeratin family. The cytokeratin family contains more than 20 members and is divided into acidic proteins (class I, including CK9-CK20) and basic proteins (class II, including CK1-CK8) according to different isoelectric points. CK8 belongs to class II cytokeratins and has a molecular weight of approximately 40-67 kDa. It consists of a central rod domain (Intermediate filament protein domain), an N-terminal head domain (Keratin type II head domain), and a C-terminal tail domain (HAUS2 domain). The central rod domain is highly conserved and is a common feature of members of the intermediate filament protein family, responsible for forming heterodimers with other keratins (such as CK18). The N-terminal and C-terminal domains are relatively variable and participate in the assembly of keratin filaments and interactions with other cellular structures. Under normal circumstances, CK8 forms heterodimers with corresponding acidic keratins in a 1:1 ratio to construct an intermediate filament network, maintaining the cell morphology and mechanical stability and helping the cell resist external stresses (such as stretching, compression, and shear forces). In addition, CK8 also acts on signal transduction and cell differentiation, interacts with other signal molecules in the cell, participates in intracellular signal transduction, and plays a regulatory role in processes such as cell growth, differentiation, and apoptosis.

[0003] Studies have shown that CK8 is widely expressed in the human body, involving various tissues and organs, such as the glandular epithelium of the colon, prostate, stomach, etc., the renal tubules of the kidney tissue, the lung cells of the lung tissue, and the cytoplasm of structures such as liver cells. These expression locations demonstrate the important role of CK8 in various epithelial cell types. The expression pattern of CK8 also makes it an important biomarker for studying various diseases, especially for differentiating tumors of epithelial origin from those of non-epithelial origin. In the study of lung adenocarcinoma, a group of alveolar intermediate-state cells (KACs) carrying the KRAS oncogene mutation and positive for CK8 expression were found, and these cells can ultimately transform into lung adenocarcinoma cells. In the study of coronary heart disease, the gene mutation frequency of CK8 was significantly higher than that of the healthy population, and its protein expression level was also relatively high. In eye diseases, CK8 is highly expressed in retinal ganglion cells (RGCs), and its expression level increases after induction by acute ocular hypertension (AOH). The lack of CK8 will exacerbate the damage of RGCs under AOH, resulting in the loss of the thickness of the retina and the inner retina, the reduction of RGCs, apoptosis and dysfunction, and the activation of glial cells. At the same time, CK8 and its complementary subunit CK18 are co-expressed in normal glandular epithelium, transitional cell epithelium, and liver cells, but not expressed in squamous epithelium, making CK8 / 18 play a greater role in tumor diagnosis, prognosis, and treatment. For example, in non-cancerous tissues, CK8 and CK18 are usually expressed in normal glandular epithelium, while in cancer tissues, such as human breast cancer or colorectal cancer, the expression of CK8 / 18 decreases, which is related to tumor progression and poor prognosis. On the contrary, in head and neck cancer, oral cancer, and urothelial cancer, the expression of CK8 / 18 is up-regulated and is related to adverse prognosis; in esophageal squamous cell carcinoma, CK8, as a single biomarker, is recognized as an independent prognostic predictor for disease-free survival (DFS) and overall survival (OS), especially in patients with pStage II / III tumors. If the combination of CK8 expression and pathological TNM staging is used at this time, it can be a valuable guidance for the adjuvant treatment decision of OSCC patients. For pStage II / III tumors expressing CK8 and pStage IV tumors regardless of CK8 expression, postoperative chemotherapy may be beneficial, but not for pStage I tumors and pStage II / III tumors negative for CK8.

[0004] Given the diagnostic and labeling value of CK8, the demand for detecting CK8 protein has been steadily increasing. Currently, in clinical practice, the target protein in pathological cells / tissues is often detected by immunofluorescence, flow cytometry, etc. The core lies in developing specific antibodies against the target protein, and the quality of the antibody directly determines the accuracy and reliability of the detection results. However, there is no CK8 antibody with strong comprehensive performance such as specificity and sensitivity on the market. Summary of the Invention

[0005] In view of the problems in the prior art, such as the lack of excellent anti-human CK8 antibodies for cell / tissue immunohistological biopsy, the present invention provides an antibody against human keratin 8 and its antibody conjugate, and further provides the application of the antibody and the antibody conjugate in the preparation of a human keratin 8 immunoassay kit and related immunoassay kits. To achieve the foregoing objectives, the present invention is specifically implemented through the following technical solutions:

[0006] In the first aspect of the present invention, an antibody against human keratin 8 is provided, which 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 respectively as shown in SEQ ID NO.3-5, and the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are respectively as shown in SEQ ID NO.8-10.

[0007] Further, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.7.

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

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

[0010] In the second aspect of the present invention, an antibody conjugate is provided, which includes the antibody against human keratin 8 as described above and a detection label connected to the antibody.

[0011] In the third aspect of the present invention, a nucleic acid molecule, a recombinant vector containing the nucleic acid molecule, or a host cell containing the nucleic acid molecule is provided, and the nucleic acid molecule encodes the antibody against human keratin 8 as described above.

[0012] In the fourth aspect of the present invention, the application of the antibody against human keratin 8 or the antibody conjugate as described above in the preparation of a human keratin 8 immunoassay kit is provided.

[0013] Further, the kit is selected from an immunofluorescence kit or a flow cytometry kit.

[0014] In the fifth aspect of the present invention, a human keratin 8 immunoassay kit is provided, and the kit includes the antibody against human keratin 8 or the antibody conjugate as described above.

[0015] Furthermore, the kit further includes a fluorescent secondary antibody against human keratin 8 antibody, and the fluorescent secondary antibody is a fluorescein-conjugated anti-rabbit IgG antibody.

[0016] The advantages and positive effects of the present invention are as follows:

[0017] The monoclonal antibody strain provided by the present invention can specifically target human keratin 8 (CK8 or KRT8), has strong specificity for CK8 protein in the cytoplasm, good binding affinity, high recognition sensitivity, and no cross-reaction and non-specific binding with non-target proteins in the cell, effectively ensuring that the detection signal comes from the target protein, which is beneficial to reducing background noise, increasing the signal-to-noise ratio, improving the accuracy and reliability of the detection result, and the detection result has a high degree of conformity with the actual situation. It is applicable to the construction of immunobiopsy techniques for clinical pathological cells or tissues and has high practical value in the clinical diagnosis and scientific research detection of human CK8 protein expression levels. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0019] Figure 1 It is a detection result diagram of the immune serum titer after immunizing Japanese white rabbits with human keratin 8 in Example 1 of the present invention;

[0020] Figure 2 It is an immunofluorescence imaging diagram of keratin 8 in a positive cell sample detected by the immune serum after immunizing Japanese white rabbits with human keratin 8 in Example 1 of the present invention;

[0021] Figure 3 It is a vector map of the expression vector pRB322 for constructing an anti-human keratin 8 antibody in Example 1 of the present invention. From left to right, it is a vector pre-carried with the constant region of the antibody light chain and the constant region of the heavy chain;

[0022] Figure 4 It is an immunofluorescence imaging diagram of keratin 8 in positive cells detected by the anti-human keratin 8 antibody in Example 2 of the present invention;

[0023] Figure 5 It is a flow cytometry imaging diagram of keratin 8 in positive cells detected by the anti-human keratin 8 antibody in Example 2 of the present invention. Detailed Embodiments

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

[0025] Based on the information contained in the present invention, those skilled in the art can easily make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for illustrative purposes of specific aspects of the present invention. In fact, various changes that can be made by those skilled in the art or related fields to the embodiments of the present invention are all covered by the scope of the appended claims.

[0026] In order to better understand the present invention rather than limit its scope, all numbers representing amounts, percentages and other numerical values used in the present invention should be understood as being 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 different desired properties. Each numerical parameter should be regarded as being obtained at least according to the reported significant figures and by the conventional rounding method.

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

[0028] The meanings of terms such as "comprising", "including", "containing", "having" and the like are non-restrictive, that is, other steps and other components can be added without affecting the result.

[0029] The term "and / or" should be regarded as a specific disclosure of each of two specified features or components with or without the other. For example, "A and / or B" is regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0030] Terms such as "rabbit monoclonal antibody", "monoclonal antibody", "rabbit-derived antibody" and "monoclonal antibody" have the same meaning. Unless otherwise specified, they all refer to antibodies that specifically bind to human (Human) keratin 8. Terms such as "CK8", "KRT8", "keratin 8", "Cytokeratin 8", "Keratin 8" have the same meaning. The modifier "rabbit" indicates that the complementarity-determining region (CDR) of the antibody is derived from rabbit immunoglobulin sequences.

[0031] An antibody is an immunoglobulin molecule that can specifically bind to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, provided that they exhibit the desired antigen-binding activity. An antibody fragment can be one or more parts or fragments of a full-length antibody that retain the ability of the antibody to specifically bind to the target antigen.

[0032] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). The light chains can be divided into two types, namely κ chains and λ chains; the heavy chains can be classified into five types, namely μ, δ, γ, α, and ε chains, and the antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. The regions with relatively large variations in the amino acid sequences near the N-terminus of the light and heavy chains are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The variable region of the heavy chain (VH) and the variable region of the light chain (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable regions are also called complementarity-determining regions (CDR), which are loop structures. The CDRs of the heavy chain and the CDRs of the light chain are closely juxtaposed and cooperate with each other through the FR regions to jointly form a surface that is complementary to the three-dimensional structure of the target antigen or epitope, determining the specificity of the antibody and being the site where the antibody recognizes and binds to the antigen. The FR regions are the more conserved parts of VH and VL. They generally have a β-sheet conformation and are connected by three CDRs that form linker loops. Each VH and VL usually consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0033] The CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the cumulative of the Kabat definition and the Chothia definition, the AbM definition, the contact definition, the IMGT unique numbering definition, and / or the conformational definition or any CDR determination method well known in the art. As used in the present invention, it is defined by the Kabat numbering system.

[0034] The constant regions of the light chain (CL) and the heavy chain (CH) do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity. The lengths of the CL of different Ig types (κ or λ) are basically the same, but the lengths of the CH of different Ig classes are different. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The amino acid sequences of the constant regions of the antibody heavy and light chains are well known in the art and can be obtained by querying the IMGT database.

[0035] Full-length antibodies are the most complete antibody molecular structures and have a typical Y-shaped molecular structure. Therefore, in the context of the present invention, "full-length antibody", "complete antibody", and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0036] Antibody fragments are one or more parts or fragments of full-length antibodies that basically retain the same biological function or activity as the full-length form. Specifically, antibody fragments include at least the same CDR regions as the full-length antibody, and more preferably have the same variable regions, thereby retaining the complete antigen recognition and binding sites and being able to bind to the same antigen as the full-length antibody, especially to the same epitope. In typical examples, antibody fragments include: Fab, F(ab) 2 、Fab’, F(ab’) 2 、Fv、(Fv) 2 、scFv、sc(Fv) 2 , and these antibody fragments can be obtained by conventional techniques in the art.

[0037] (i) Fab: The antigen-binding fragment (Fab) is a monovalent fragment composed of a complete light chain (variable region and constant region) and a part of the heavy chain (variable region and the first constant region). By protease digestion of the full-length antibody, fragments such as Fab, F(ab’) 2 、Fab’ can be obtained. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab’) 2 fragment and one pFc' fragment. The F(ab') 2 fragment is further reduced to form two Fab’ fragments. Since Fab has an antigen-binding region and a part of the constant region, it not only has the same antibody-antigen affinity and excellent tissue penetration as scFv, etc., but also has a more stable structure.

[0038] (ii) F(ab) 2 : A bivalent fragment containing two Fabs linked by a hinge region disulfide bridge.

[0039] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, contains only the variable regions, and consists of the variable regions of one light chain and one heavy chain. It is a non-covalently bound dimer of a VH and a VL (VH-VL dimer). The three CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind antigens, although the affinity is lower than that of the intact antibody.

[0040] (iv) (Fv) 2 : Consists of two Fv fragments covalently linked together.

[0041] (v) scFv: The single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, formed by linking a heavy-chain variable region (VH) and a light-chain variable region (VL) through a flexible linker (usually composed of 10-25 amino acids). It retains the binding specificity of the original antibody to antigens. In the present invention, the linker as long as it does not interfere with the expression of the antibody variable regions connected at both ends is acceptable and is not particularly limited. Compared with the full-length antibody, scFv has the characteristics of a small molecular weight, so it has higher penetrability and lower immune side reactions.

[0042] (vi) sc(Fv) 2 Fragment, which is formed by connecting two heavy-chain variable regions and two light-chain variable regions through a linker or the like.

[0043] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may contain the 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, such as 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 a part 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 the CDR regions of a non-human antibody such as a rabbit antibody and the FR regions from humans. In some cases, the variable region of the non-human antibody binds to the constant region of the human antibody, such as a human-rabbit chimeric antibody; in other cases, the CDR regions of the non-human antibody bind to the FR regions and constant regions derived from human antibody sequences, that is, the CDR regions of the non-human antibody are grafted onto the human antibody framework (FR) sequences, and such framework sequences are derived from the FR sequences of single or multiple other human antibody variable regions. In the present invention, the CDR regions in the chimeric antibody or humanized antibody are derived from rabbit CDR regions.

[0044] The terms "monoclonal antibody" or "mAb" and similar terms are used interchangeably and refer to a homogeneous population of antibodies, i.e., each antibody constituting the population is identical except for minor mutations and / or post-translational modifications (such as isomerization, amidation) that may occur naturally. "Monoclonal antibodies" are highly specific and exhibit a single binding specificity and affinity for the same or substantially the same epitope on an antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed as limiting the source or method of preparation of the antibody. The antibody can be prepared by a variety of methods, including but not limited to the hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening or single cell sequencing method.

[0045] The term "specifically binds" is a well-known term in the art. A molecule exhibits "specifically binds" if it reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity with a particular target antigen or epitope than with other target antigens or epitopes. "Specifically binds" or "preferentially binds" does not necessarily require (although it can include) exclusive binding. To make the above objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0046] An embodiment of the present invention provides an antibody against human keratin 8, comprising a light chain variable region and a heavy chain variable region. Both the light chain variable region and the heavy chain variable region comprise 3 complementarity determining regions (CDRs), named CDR1, CDR2, and CDR3 respectively. Among them, the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region are shown as SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 respectively; the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 respectively.

[0047] The monoclonal antibody strain provided by the present invention can specifically target human keratin 8 (CK8 or KRT8) and is suitable for the construction of immunobiopsy techniques for clinical pathological cells or tissues, especially for applications such as immunofluorescence and flow cytometry detection. Using the antibody of the present invention as the primary antibody to capture the target antigen and a fluorescein-labeled anti-rabbit IgG secondary antibody for color development to construct an immunofluorescence and flow cytometry detection system, it shows that the antibody of the present invention has strong specificity, good binding affinity, and high recognition sensitivity for the CK8 antigen in the cytoplasm, and has no cross-reaction and non-specific binding to non-target proteins in the cell, effectively ensuring that the detection signal comes from the target protein, which is beneficial to reducing background noise, increasing the signal-to-noise ratio, improving the accuracy and reliability of the detection result, and the detection result is highly consistent with the actual situation, and has high practical value in the clinical diagnosis and scientific research detection of the human CK8 protein expression level.

[0048] Optionally, both the light chain variable region and the heavy chain variable region include four framework regions (FRs), and the four FRs and three CDRs are arranged alternately in sequence to form the variable region. The amino acid sequence of the light chain variable region (VL) of the 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.

[0049] Optionally, the antibody of the present invention further includes a light chain constant region (CL) and a heavy chain constant region (CH). CL and VL form a complete light chain (FL), and CH and VH form a complete heavy chain (FH). The constant region of the antibody can usually be obtained through public queries. For example, through the IMGT online database (www.imgt.org), search for rabbit IgG gamma C reign to obtain CH, and search for rabbit IgGKappa C reign to obtain CL.

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

[0051] It should be noted that the 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 being able to bind to the same antigen as the full-length antibody, especially binding to the same epitope. Optionally, the antigen-binding region is selected from Fab, F(ab) 2 ', Fab', F(ab') 2 ', Fv, (Fv) 2 ', scFv and sc(Fv) 2 ', and at least one of them. These antigen-binding regions can be obtained by conventional techniques in the art.

[0052] Another embodiment of the present invention provides an antibody conjugate, including the anti-human keratin 8 antibody as described above and a detection label linked to the antibody.

[0053] The detection label is used to generate recognizable signal changes to identify the antibody of the present invention according to the signal changes, and further identify the expression of CK8 antigen in the sample to be detected through the specific reaction of antigen and antibody. The detection labels include but are not limited to: biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (such as allophycocyanin, phycoerythrin, PerCP, and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies or combinations thereof.

[0054] It should be emphasized that the antibody of the present invention can be used alone or conjugated with a detection label (covalently or non-covalently) to form an antibody conjugate. In some embodiments, the antibody of the present invention is used as an antigen-binding (or capture) antibody, which specifically recognizes and binds CK8 in the sample to be detected, and then qualitatively or quantitatively detects CK8 protein by analyzing the signal of the detection label conjugated thereto; in other embodiments, the anti-CK8 antibody is not labeled (as a primary antibody or capture antibody), but the detection label is conjugated to a secondary antibody (as a detection antibody) or other molecules that can bind to the primary antibody. For example, if the anti-CK8 antibody is a rabbit-derived IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody. Thus, qualitative or quantitative detection of CK8 is achieved by analyzing the signal changes of the detection label generated after the secondary antibody specifically binds to the antibody of the present invention. For example, multiple detection systems established in Example 2 below of the present invention.

[0055] Another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector containing the nucleic acid molecule, or a host cell containing the nucleic acid molecule, and the nucleic acid molecule encodes the antibody against human keratin 8 as described above.

[0056] The nucleic acid molecule can be in the form of DNA (such as cDNA, genomic DNA, or synthetic DNA) or RNA (such as mRNA or synthetic RNA). The DNA can be single-stranded or double-stranded, and can also be a coding strand or a non-coding strand.

[0057] The sequence of the nucleic acid molecule can be deduced by conventional means such as codon coding rules based on the AA sequence of the antibody. The full-length sequence or fragments of the nucleic acid molecule can usually be obtained by PCR amplification, recombination, or artificial synthesis methods.

[0058] The original vector for constructing the recombinant vector is various conventional 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., used to transfer the nucleic acid molecule into a host and multiply it in large quantities in the host cell) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecule 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 an expression vector carrying the nucleic acid molecule, and then introduced into the host cell and cultured under specific conditions to express and obtain the antibody. This is a well-known technology in the art and will not be described in detail here.

[0059] The nucleic acid molecules encoding the antibody FL and FH of the present invention can be inserted into two vectors respectively, which can be introduced into the same or different host cells. When the heavy chain and the light chain are expressed in different host cells, each chain can be separated from the host cell expressing it, and the separated heavy chain and light chain are mixed and incubated under suitable conditions to form the antibody. In some other embodiments, the nucleic acid molecules of the antibody FL and FH can also be cloned into one vector, and each nucleic acid sequence is ligated downstream of a suitable promoter; for example, each nucleic acid sequence encoding the heavy chain and the light chain can be operably linked to different promoters, or the nucleic acid sequences encoding the heavy chain and the light chain can be operably linked to a single promoter, such that both the heavy chain and the light chain can be expressed by the same promoter. The choice of the expression vector / promoter depends on the type of host cell used for producing the antibody.

[0060] The transfection or transformation of the recombinant vector into the host cell is carried out by conventional techniques. When the host is a prokaryote such as Escherichia coli, the competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with CaCl 2 method or MgCl 2 treatment; it can also be carried out by microinjection, electroporation or liposome packaging, etc. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, microinjection method, electroporation method, liposome packaging or gene gun bombardment and other methods to achieve gene introduction.

[0061] 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 (such as DH5α, JM109, BL21, W3110), Bacillus spp. (such as Bacillus subtilis, Bacillus thuringiensis), Enterobacteriaceae strains (such as Salmonella typhimurium, Serratia marcescens), and Pseudomonas spp. 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 the host cell transfected or transformed with the recombinant vector as described above, culturing under suitable conditions can express the antibody, and then separating it to obtain the purified antibody.

[0062] In a typical embodiment, the method for preparing the antibody includes: after tandemly connecting the heavy chain gene and the light chain gene of the antibody with a signal peptide, respectively loading them on the expression vector pBR322, co-transfecting human renal epithelial cells (293F), culturing the 293F cells, collecting the cell culture supernatant, and purifying to obtain the target antibody strain. The selection of the signal peptide is designed according to the host cell, and the present invention has no special limitation on this.

[0063] Another embodiment of the present invention provides the use of the antibody or antibody conjugate against human keratin 8 as described above in the preparation of a human keratin 8 immunoassay kit.

[0064] The advantages of the use of the antibody or antibody conjugate against human keratin 8 in the preparation of a human keratin 8 immunoassay kit are the same as the advantages of the antibody against human keratin 8 as described above over the prior art, and will not be elaborated here.

[0065] Based on the same inventive concept, an embodiment of the present invention also provides a human keratin 8 immunoassay kit, which includes the antibody or antibody conjugate against human keratin 8 as described above.

[0066] The above-mentioned immunoassay methods include, but are not limited to: Enzyme linked immunosorbent assay (ELISA), Enzyme-linked Immunospot (ELISPOT), Immunohistochemistry (IHC), Immunofluorescence (IF), Western blot (WB), Immunoprecipitation (IP), and Flow Cytometry (FC). The kit can be an enzyme-linked immunosorbent assay kit, an enzyme-linked immunospot kit, an immunohistochemistry kit, an immunofluorescence kit, a Western blot kit, or a flow cytometry kit.

[0067] Preferably, the kit is a flow cytometry kit or an immunofluorescence kit.

[0068] Optionally, the kit further includes a fluorescent secondary antibody against human keratin 8 antibody, and the fluorescent secondary antibody is a fluorescent-conjugated anti-rabbit IgG antibody.

[0069] The present invention will be further described below in conjunction with specific embodiments. For the experimental methods without specific conditions indicated in the following embodiments, they are usually carried out under conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual (Fourth Edition)" published by Cold Spring Harbor Laboratory, or usually according to the conditions recommended by the manufacturer.

[0070] Example 1 Preparation of Rabbit Antibody Against Human Keratin 8 (CK8, KRT8)

[0071] 1.1 Preparation of immunogen: The immunogen used was the 1-483aa protein fragment of human recombinant CK8 protein expressed and purified by 293F. The full-length amino acid sequence of CK8 can be found in Uniprot accession number P05787 or NCBI accession number NP_002264.1, and the gene sequence can be found in NCBI accession number NM_002273.4. The gene sequence corresponding to CK8 1-483aa was constructed into the pBR322 vector, and a recombinant protein with biological activity was expressed in 293F cells. The purity of the recombinant protein was detected to be greater than 90%.

[0072] 1.2. Animal immunization: Immunize 2 Japanese white rabbits with 200 μg of human CK8 protein per rabbit. Before the first immunization, mix the antigen with an equal volume of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsifier, and inject it subcutaneously at multiple points on the abdomen and back of the rabbit; after the first immunization, every 3 weeks, take 100 μg of the immunogen and mix it with an equal volume of incomplete Freund's adjuvant (purchased from Sigma) to prepare an emulsifier, and inject it subcutaneously at multiple points on the abdomen and back of the rabbit for two booster immunizations. After three immunizations, take the serum, dilute it 1:243000, and measure its titer against human CK8 by enzyme-linked immunosorbent assay (ELISA). Take rabbits with OD 450nm greater than 0.2, and boost the immunization once with 200 μg of the immunogen. Three to four days later, take the spleen, dilute the final immune serum, and detect the affinity and specific reaction of the serum with the target antigen in the cell sample to be tested by immunofluorescence (IF) method.

[0073] The steps for measuring the titer of immune serum by ELISA are as follows: 1) Coating: Add 1 μg / mL of human CK8 protein to the enzyme-labeled plate at 25 μL / well and incubate overnight at 4°C; 2) Blocking: Wash 5 times with washing buffer (PBS containing 0.05% (v / v) Tween-20) at 75 μL / well, and then add blocking buffer (PBS containing 1% BSA, 0.5% gelatin, and 5% sucrose) at 50 μL / well and incubate at room temperature for 1 h; 3) Gradient dilution and sample addition of the serum to be tested: Repeat the plate washing process in step 2) to wash the plate, and then perform gradient dilution of the serum to be tested. Dilute the serum with dilution buffer (PBS containing 1% BSA) starting from 1:1000 and perform three-fold dilution for a total of 8 gradients; add the serum dilution to the plate at 25 μL / well and incubate at room temperature for 1 h; 4) Incubation with secondary antibody: Repeat the plate washing process in step 2) to wash the plate, and then add 1:5000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (purchased from ABclonal, catalog number AS014) at 25 μL / well and incubate in the dark at room temperature for 1 h; 5) Termination of reaction and color development: Repeat the plate washing process in step 2) to wash the plate, and then add TMB for color development at 100 μL / well, incubate in the dark at 37°C for 10 min, add 0.5 M oxalic acid solution at 100 μL / well to terminate the reaction, measure the light absorption value at 450 nm, use the pre-immune rabbit serum as a negative control, and the detection system without adding immune serum as a blank control (NC). A positive immune serum is defined as a ratio of the measured value to the control value ≥ 2.1.

[0074] The steps for determining the recognition specificity of immune serum and cell samples (human cervical cancer cells HeLa expressing CK8 protein) by the IF method are as follows: 1) Cell treatment: Prepare adherent HeLa cells, add 4% neutral formaldehyde fixative and fix at room temperature for 10 - 15 min. After fixation, wash 3 times with PBS, 5 min each time; 2) Cell blocking: Add blocking buffer (1×PBS containing 5% BSA) to each well, remove the blocking solution after blocking for 30 - 60 min; 3) Primary antibody incubation: Add the immune serum diluted with PBS (as the primary antibody) at 100 μL / well, incubate overnight at 2 - 8 °C; Take out and warm to room temperature for 15 min, remove the primary antibody working solution, then wash with 1×PBST for 5 min and wash with 1×PBS 2 times, 5 min each time; 4) Secondary antibody incubation: Add the fluorescent secondary antibody diluted with PBS (Cy3-conjugated Goat anti-Rabbit IgG(H+L), from ABclonal, catalog number AS007, secondary antibody dilution ratio 1:500) at 100 μL / well, incubate at room temperature in the dark for 1 h; Remove the secondary antibody working solution, then wash with 1×PBST for 5 min and wash with 1×PBS 2 times, 5 min each time; 5) Nuclear staining: Add DAPI nuclear dye working solution at 100 μL / well, stain for 10 - 30 min, then wash with 1×PBST for 5 min and wash with 1×PBS 2 times, 5 min each time; 6) Immunofluorescence analysis: Observe and photograph and analyze with an ABTK / G2023030034 wide-field fluorescence microscope.

[0075] The detection results of serum titer are shown in Figure 1 , where WA-63743D is the project number, and N16170 and N16171 are rabbit numbers. The detection results of the binding ability of immune serum to the CK8 protein expressed by positive cells HeLa are shown in Figure 2 , where the upper figure is the fluorescence imaging map of using immune serum to bind to HeLa cells. After the antibody in the serum binds to the CK8 protein, it is labeled as Cy3 red by the fluorescent secondary antibody. The lower figure is the comparison map of using DAPI to counterstain the cell nucleus (blue) and merging the red and blue colors. From Figure 1-2 it can be seen that a strong immune response was generated in the rabbit body during the third immunization and the fourth booster immunization, and antibodies that can specifically recognize the CK8 protein were produced in the immune serum, manifested as obvious staining of the filamentous structures in the cytoplasm of HeLa cells, and the antibodies were localized to the intermediate filaments, which was consistent with the expectation. Therefore, the quadruple-immunized serum can be used for subsequent monoclonal antibody separation.

[0076] 1.3. Isolation of B cells in the spleen and sorting of antigen-specific B cells: For related methods, refer to the publicly disclosed patent "Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A, Publication Date: July 16, 2019)" and the patent "An in vitro culture system for B lymphocytes and its application (Publication No.: CN111518765A, Publication Date: August 11, 2020)".

[0077] 1.4. Cloning of genes encoding rabbit monoclonal antibodies: B lymphocytes capable of recognizing and binding to human CK8 protein were detected by antigen-coated ELISA method. The above-mentioned cells were collected, lysed, and RNA was extracted using the Quick-RNA TM Micro Prep kit (purchased from ZYMO Corporation, product number R1100-250), and then reverse-transcribed into cDNA. Using the above cDNA as a template, the naturally paired rabbit antibody light chain variable region (VL) and heavy chain variable region (VH) were amplified by PCR method and sequenced. The PCR reaction system includes: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria HiFi (from ABclonal, product number RK20717) and 6.5 μL H 2 O; The PCR amplification program includes: 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, 72°C for 30 s, and finally 72°C for 5 min. The reaction solution was stored at 4°C. The primer sequences (5'-3') for amplifying VL and VH genes are shown below, where F and R represent the forward primer and the reverse primer respectively.

[0078] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (SEQ ID NO.12);

[0079] VL-R: cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (SEQ ID NO.13);

[0080] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (SEQ ID NO.14);

[0081] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (SEQ ID NO.15).

[0082] The amplified DNA products were sequenced to obtain the VL sequence shown in SEQ ID NO.2 and the VH sequence shown in SEQ ID NO.7. Subsequently, the sequences of the constant regions were obtained by querying the IMGT online database (www.imgt.org), resulting in the antibody 15H6 with the complete light chain (FL) shown in SEQ ID NO.1 and the complete heavy chain (FH) shown in SEQ ID NO.6. The amino acid (aa) and nucleic acid (DNA) sequences of antibody 15H6 are shown in Table 1. For convenience of description, the complementarity-determining regions CDR1-3 of the light chain are denoted as LCDR1-3 respectively, and the complementarity-determining regions CDR1-3 of the heavy chain are denoted as HCDR1-3 respectively. The CDRs are divided based on the Kabat numbering system.

[0083] Table 1 Amino acid sequences of rabbit-derived antibody 15H6 and immunogen in this example

[0084]

[0085]

[0086] 1.5 Expression and large-scale production of antibody 15H6: The obtained heavy and light chain genes of the antibody were respectively loaded onto expression vectors. In this example, the light chain constant region (CL) and heavy chain constant region gene (CH) were pre-inserted into the mammalian expression vector pBR322, and its expression map is shown in Figure 3 , in the figure, pBR322 origin and f1 origin are replication promoters, Ampcillin is a resistance gene, CMV promoter is a transcription promoter, SV40 PA terminator is a polyadenylation signal, Light chain constant is the nucleic acid sequence of CL (left figure), and Heavy chain constant is the nucleic acid sequence of CH (right figure). Then, the above-amplified VL and VH genes were ligated to the expression vector pBR322 carrying the CL and CH genes, which were linearized with the restriction enzymes XbaI (955bp) and NheI (949bp) respectively, by homologous recombination to obtain the expression vectors for the complete light chain (FL) and heavy chain (FH) genes. The successful construction of the vectors was verified by sequencing.

[0087] To more conveniently purify the antibody, the antibody was secreted and expressed by adding a signal peptide upstream of the VL and VH genes. A commonly used antibody expression signal peptide in the art can be used as the signal peptide. For example, in the patent "Rabbit monoclonal antibody against human interferon α2 and its application (Publication No.: CN116063487A, Publication Date: May 5, 2023)" and the patent "High-affinity Human IL-5 rabbit monoclonal antibody and its application (Publication No.: CN115819578A, Publication Date: March 21, 2023)", the signal peptide "MDTRAPTQLLGLLLLWLPGARC" is present upstream of VL, and the signal peptide "METGLRWLLLVAVLKGVQC" is present upstream of VH. 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 in Table 1 of this example.

[0088] The successfully constructed FL and FH expression vectors were co-transfected into 293F cells. After transfection, the cells were cultured for 72 - 96 h to obtain the antibody 15H6 that recognizes human CK8 protein in the culture supernatant. The target antibody was purified from the culture supernatant using protein A affinity gel resin (purchased from Tiandi Renhe, product number SA015100). 12% polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify that the antibody purity was ≥95%, and the antibody concentration was 1.33 mg / mL. The purified antibody was aliquoted and stored at -20 °C for later use.

[0089] Example 2 Establishment of the immunodetection method for antibody 15H6 and evaluation of its application effect

[0090] 1. Establishment of the immunofluorescence (IF) detection method based on antibody 15H6

[0091] The IF detection method was the same as that in Example 1, except that the primary antibody was antibody 15H6 and the working concentration of the primary antibody was 2.66 μg / mL.

[0092] The IF detection results are shown in Figure 4 , in which, the upper figure is the fluorescence imaging diagram of antibody 15H6 binding to CK8 protein in HeLa cells. After antibody 15H6 binds to CK8 protein, it is labeled red by the fluorescent secondary antibody. The lower figure is the imaging diagram showing the nuclear staining in blue and the merging of the red and blue colors. It can be seen from the figure that the intermediate filament fluorescence signal in the cytoplasm of HeLa cells is clear and bright, and the actual color development result is consistent with the theoretical localization of the antibody, indicating that antibody 15H6 can specifically and sensitively bind to the CK8 protein of the intermediate filament.

[0093] 2. Establishment of the flow cytometry (FC) detection method based on antibody 15H6

[0094] FC detection includes the following steps: 1) Disinfect the laminar flow hood by ultraviolet irradiation for 15 - 20 min, turn on the blower for 5 min, and prepare for aseptic work; 2) Collect and wash the cells (HeLa cells with high CK8 expression or human T lymphocyte leukemia cells (Jurkat) with low CK8 expression), measure the total number of cells, and check that the cell viability is between 90% - 95%; 3) Resuspend the cells in 1×PBS solution to about 3×10 6 -5×10 6 cells / mL, dispense the cells into a 96-well V-bottom plate at 100 μL / well, and wash once with 1×PBS; 4) Stain with the Live / Dead staining solution (Zombie NIR, L / D staining solution) of the Zombie NIR Fixable Viability Kit (product number 423105) from Biolegend. Dilute the L / D staining solution with 1×PBS at a ratio of 1:1500, and dispense the diluted L / D staining solution into the well plate at 100 μL / well to resuspend the cells in the wells; 5) Wrap with aluminum foil, gently mix on a microplate shaker for 15 min, then centrifuge at 400 g for 5 min, discard the supernatant, and wash twice with 1×PBS; 6) Dispense 1×IntracelluLar Fixation buffer into the 96-well plate at 100 μL / well to resuspend the cells in each well; 7) Repeat step 5), and wash twice with 1×Permeablization buffer; 8) Dispense the primary antibody 15H6 (final concentration of the primary antibody is 2 μg / mL) diluted with 1×Permbuffer into the well plate at 100 μL / well to resuspend the cells in each well; 9) Dispense the fluorescent secondary antibody (Fluorescein (FITC) AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG, purchased from jackson, product number 111 - 096 - 046) diluted with 1×PBS (dilution ratio 1:200) into the well plate at 100 μL / well to resuspend the cells in the wells; 10) Repeat step 5), then resuspend the cells in each well with 200 μL of 1×PBS and store in the dark; 11) Analyze according to the operation of Beckman cytoflex flow cytometer use and maintenance SOP - 105 - AND - CA - 008.

[0095] The results of FC detection are shown in Figure 5, in which the detection results of Jurkat cells and HeLa cells are shown from left to right. The abscissa in the figure represents the relative fluorescence intensity of the fluorescence signal / scattered light signal of the channel, and the ordinate represents the corresponding number of cells. The red curve is the blank control without adding any antibody, the blue curve is the isotype control (used to eliminate the background staining caused by the non-specific binding of the antibody to the cells), and the yellow curve is antibody 15H6. The results show that antibody 15H6 has specific binding in cell samples with high and low expression of CK8, and there is an obvious difference in the fluorescence signal transition, further confirming that antibody 15H6 can specifically target the CK8 protein expressed in cells, has a high binding sensitivity to the target antigen, has no cross-reaction with non-target antigens, and has good anti-cell component interference ability, which is beneficial to improving the accuracy and reliability of the histopathological biopsy of CK8 protein.

[0096] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-human keratin 8 antibody, characterized in that: It comprises 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 as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively; the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.

2. The anti-human keratin 8 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-human keratin 8 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-human keratin 8 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. An antibody conjugate, characterized in that: The invention comprises the anti-human keratin 8 antibody according to any one of claims 1 to 4 and a detection label connected to the antibody.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-human keratin 8 antibody according to any one of claims 1 to 4.

7. Use of the anti-human keratin 8 antibody according to any one of claims 1 to 4 or the antibody conjugate according to claim 5 in the preparation of a human keratin 8 immunoassay kit.

8. Use of the anti-human keratin 8 antibody or antibody conjugate according to claim 7 in preparing a human keratin 8 immunoassay kit, characterized in that: The kit is selected from an immunofluorescence kit or a flow cytometry kit.

9. A human keratin 8 immunoassay kit, characterized in that: It comprises the anti-human keratin 8 antibody according to any one of claims 1 to 4 or the antibody conjugate according to claim 5.

10. The human keratin 8 immunoassay kit according to claim 9, characterized in that: The invention also includes a fluorescent secondary antibody against human keratin 8 antibody, wherein the fluorescent secondary antibody is an anti-rabbit IgG antibody coupled with fluorescein.

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