Monoclonal antibody aiming at human KRT19 and application thereof

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to a monoclonal antibody aiming at human KRT19 and application thereof. The amino acid sequences of CDR1-3 on a light chain variable region of the monoclonal 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 monoclonal antibody are respectively shown as SEQ ID NO.8-10. The monoclonal antibody provided by the invention can specifically recognize and accurately target KRT19 in cytoplasm, has good affinity in combination with KRT19, has strong anti-interference ability, effectively ensures that a detection signal is from a target antigen, has a high conformity degree between a detection result and an actual situation, and has good application prospects. A powerful tool is provided for high-sensitivity and high-precision detection of the KRT19 in tissues and / or cells, deep research on functions and regulation mechanisms of the KRT19 in cytoskeleton, epithelial tissue and tumor biology is facilitated, and the kit has important application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and particularly to monoclonal antibodies against human KRT19 and their applications. Background Art

[0002] Keratin (KRT, K), also known as cytokeratin (CK), is a large multi-gene family that constitutes the largest subgroup of intermediate filament proteins. Keratin 19 (CK19, Keratin 19, KRT19) is a member of the type I (acidic) keratin family with a relatively small molecular weight of approximately 40 kDa, which is the smallest known acidic keratin in terms of molecular weight. As a major component of the cytoskeletal intermediate filaments, KRT19 plays an important role in maintaining cell morphology and structural integrity and providing a multi-functional scaffold. It also interacts with a variety of signal transduction molecules, including adapters, effectors, kinases, and receptors, thereby regulating signal pathways and affecting apoptosis, cell cycle arrest, invasion, and metastasis.

[0003] KRT19 is widely distributed in normal epithelial tissues. For example, it is expressed in epithelial cells of various tissues such as human liver bile duct epithelial cells, pancreatic duct epithelial cells, columnar epithelial cells of the gastrointestinal tract, alveolar cells of the lung, and mammary duct epithelial cells. In tumor tissues, the expression of KRT19 is significantly up-regulated, especially in cholangiocarcinoma, ovarian cancer, and thyroid cancer, and it can be used as a diagnostic marker and prognostic indicator for various epithelial tumors. In the field of breast cancer, the role of KRT19 is particularly significant. Studies have shown that KRT19 regulates the NOTCH signaling pathway through direct interaction with the β-catenin / RAC1 complex, thereby affecting the biological characteristics of breast cancer. In addition, KRT19 is also involved in regulating the expression of NUMB and affecting the regulation of breast cancer and cancer stem cell characteristics mediated by the NOTCH pathway. Compared with normal breast tissues, KRT19 is highly expressed in breast cancer, and the expression of KRT19 is closely related to the level of immune infiltration of immune cells. The combination of the two can effectively evaluate the prognosis of breast cancer patients. Generally speaking, cytokeratin 19 is one of the currently very active and relatively mature tumor markers, playing an important role in tumor diagnosis and pathological research.

[0004] In clinical pathological section examination, the expression of KRT19 in cells and / or tissues is often detected by immunohistochemistry (IHC) method, providing more accurate diagnosis and treatment plans for doctors. In the scientific research field, KRT19 is also an important target for Western blot (WB) experiments in scientific research, used to study the cytoskeleton structure and regulatory factors of invasive tumor cells. These applications and detections of KRT19 rely on the development of its high-performance antibodies. Therefore, obtaining monoclonal antibodies with high activity that can specifically and sensitively bind to KRT19 is of great significance for clinical diagnosis and scientific research. Summary of the Invention

[0005] Aiming at the problem of the lack of excellent anti-human KRT19 monoclonal antibodies in the prior art, the present invention provides a rabbit-derived monoclonal antibody against human KRT19, and further provides the application of the monoclonal antibody in the preparation of a human KRT19 immunoassay kit and related immunoassay kits. To achieve the foregoing objectives, the present invention is specifically implemented through the following technical solutions:

[0006] The first aspect of the present invention provides a monoclonal antibody against human KRT19, including 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] Furthermore, 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] Furthermore, the amino acid sequence of the light chain of the monoclonal antibody 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] Furthermore, the monoclonal 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] The second aspect 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 monoclonal antibody against human KRT19 as described above.

[0011] The third aspect of the present invention provides a recombinant vector, and the recombinant vector contains the nucleic acid molecule as described above.

[0012] The fourth aspect of the present invention provides an antibody conjugate, comprising the monoclonal antibody against human KRT19 as described above and a detection label linked to the monoclonal antibody.

[0013] The fifth aspect of the present invention provides the use of the monoclonal antibody against human KRT19 or its antibody conjugate as described above in the preparation of an immunoassay kit for human KRT19.

[0014] Further, the kit is an immunoblot kit or an immunohistochemistry kit.

[0015] The sixth aspect of the present invention provides an immunoassay kit for human KRT19, which comprises the monoclonal antibody against human KRT19 or its antibody conjugate as described above.

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

[0017] Based on the monoclonal antibody provided by the present invention, detection systems such as immunoblotting and immunohistochemistry are constructed. It is observed that the antibody of the present invention can accurately and sensitively detect cytokeratin 19 (KRT19), and only label positive cells expressing KRT19, and the antibody is accurately located in the cytoplasm; it is confirmed that the monoclonal antibody provided by the present invention can specifically recognize and precisely target cytokeratin 19 (KRT19) in the cytoplasm, has good affinity for binding to KRT19, and strong anti-interference ability, effectively ensuring that the detection signal comes from the target protein, and the detection result has a high degree of conformity with the actual situation, providing a powerful tool for the highly sensitive and highly accurate detection of KRT19 in tissues and / or cells, contributing to the in-depth study of the functions and regulatory mechanisms of KRT19 in the cytoskeleton, epithelial tissues and tumor biology, and having important application prospects in the fields of diagnosis and treatment plan formulation of cancerous tumors. BRIEF 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 drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 It is a detection result diagram of the immune serum titer after immunizing New Zealand white rabbits with human KRT19 protein in Example 1 of the present invention;

[0020] Figure 2 It is a vector map for constructing an expression vector of a monoclonal antibody against human KRT19 in Example 1 of the present invention. From left to right, it is the pRB322 vector pre-carried with the constant region of the antibody light chain and the constant region of the heavy chain;

[0021] Figure 3 This is the immunoblot electrophoresis diagram of detecting KRT19 protein in positive cells and negative cells using anti-human KRT19 monoclonal antibody in Example 2 of the present invention;

[0022] Figure 4 This is the immunohistochemical staining diagram of detecting positive tissues and negative tissues using anti-human KRT19 monoclonal antibody in Example 2 of the present invention. Detailed implementation manners

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] Based on the information included 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 schematically illustrating 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.

[0025] In order to better understand the present invention rather than limit the scope of the present invention, 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.

[0026] In addition, it should be noted that, unless otherwise defined, 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.

[0027] The meanings of terms such as "include", "comprise", "contain", "have" and the like are non-restrictive, that is, other steps and other components can be added without affecting the results.

[0028] The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is regarded as including the following situations: (i) A, (ii) B, and (iii) A and B.

[0029] The terms "rabbit monoclonal antibody", "monoclonal antibody", "rabbit-derived antibody", "mAb" and similar terms have the same meaning, and unless otherwise specified, all refer to antibodies that specifically bind to human cytokeratin 19. The terms "KRT19", "CK19", "cytokeratin 19", "keratin 19", "Cytokeratin 19", "Keratin 19", etc. have the same meaning. The modifier "rabbit" indicates that the complementarity-determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences.

[0030] An antibody is an immunoglobulin molecule that can specifically bind to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, as long as they exhibit the desired antigen-binding activity. 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.

[0031] 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 amino acid sequence variations 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 configuration and are connected by three CDRs that form connecting 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.

[0032] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, the cumulative of both the Kabat definition and the Chothia definition, the AbM definition, the contact definition, the IMGT unique numbering definition, and / or the conformational definition, or any CDR determination method well-known in the art. As used in the present invention, they are defined by the Kabat numbering system.

[0033] The light chain constant region (CL) and the heavy chain constant region (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 CLs of different Ig types (κ or λ) are basically the same, but the lengths of CHs 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.

[0034] Full-length antibodies are the most complete antibody molecular structures, with 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.

[0035] Antibody fragments are one or more parts or segments of a full-length antibody, which basically retain the same biological function or activity as the full-length form. Specifically, antibody fragments at least include the same CDR regions as the full-length antibody, and more preferably have the same variable regions, thereby retaining a complete antigen recognition and binding site 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.

[0036] (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, and Fab’ can be obtained. For example, under the action of papain, IgG can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into one F(ab’)2 fragment and one pFc' fragment. The F(ab')2 fragment is further reduced to form two Fab’ fragments. Since Fab has an antigen-binding region and a part of the constant region, it not only has the same antibody-antigen affinity as scFv and excellent tissue penetration, but also has a more stable structure.

[0037] (ii) F(ab)2: A bivalent fragment composed of two Fabs linked by a disulfide bridge in the hinge region.

[0038] (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 (VH-VL dimer) of one VH and one VL. 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.

[0039] (iv) (Fv)2: Composed of two Fv fragments covalently linked together.

[0040] (v) scFv: The single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, which is formed by connecting 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. The linker in the present invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions connected at both ends. 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.

[0041] (vi) The sc(Fv)2 fragment is formed by connecting two heavy-chain variable regions and two light-chain variable regions through a linker or the like.

[0042] 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 a part of the antibody derived from a specific source or species, while the rest is derived from a different source or species. The term "humanized antibody" is a chimeric antibody with the CDR regions of a non-human antibody such as a rabbit antibody and the human FR regions. In some cases, the variable region of the non-human antibody is combined with 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 are combined with 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 this framework sequence is derived from the FR sequences of one or more 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.

[0043] 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.

[0044] The term "specific binding" is well known in the art. A molecule exhibits "specific binding" 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. "Specific binding" or "preferential binding" 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 following detailed description of specific embodiments of the present invention will be given with reference to the accompanying drawings.

[0045] An embodiment of the present invention provides a monoclonal antibody against human KRT19, which includes a light chain variable region and a heavy chain variable region. Both the light chain variable region and the heavy chain variable region include 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.

[0046] The antigen epitope region with specificity on cytokeratin 19 (KRT19) is selected for animal immunization in the present invention, and the produced antibody is more specific and definite in purpose and function. Specifically, based on the antibody provided by the present invention, an immunoblot detection system is constructed, and it is observed that the antibody of the present invention can accurately and sensitively detect a single band of KRT19 in the cell lysate; based on the antibody provided by the present invention, an immunohistochemical detection system is constructed, and it is observed that the antibody of the present invention only labels the cells expressing KRT19, and the antibody is accurately located in the cytoplasm. These results confirm that the monoclonal antibody provided by the present invention can specifically recognize and precisely target cytokeratin 19 (KRT19) in the cytoplasm, has a good affinity for binding to KRT19, has no cross-reaction and specific binding to other irrelevant antigens in the cell, has strong anti-interference ability, effectively ensures that the detection signal comes from the target antigen, and the detection result has a high degree of conformity with the actual situation. It provides a powerful tool for the highly sensitive and highly accurate detection of KRT19 in tissues and / or cells, helps to deeply study the functions and regulatory mechanisms of KRT19 in the cytoskeleton, epithelial tissues and tumor biology, and has important application prospects in the fields of diagnosis and treatment plan formulation of cancerous tumors.

[0047] Optionally, both the light chain variable region and the heavy chain variable region include 4 framework regions (FRs), and the 4 FRs and 3 CDRs are arranged alternately in sequence to form the variable region. The amino acid sequence of the light chain variable region (VL) of the monoclonal antibody of the present invention is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.7.

[0048] Optionally, the monoclonal antibody of the present invention further 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 by public query. For example, through the IMGT online database (www.imgt.org), CH is obtained by searching for rabbit IgG gamma C reign, and CL is obtained by searching for rabbit IgG Kappa C reign.

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

[0050] It should be noted that the monoclonal antibody of the present invention can be a full-length antibody (with 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 regions as described above, and more preferably has the variable regions as described above, thereby retaining the complete antigen recognition and binding sites 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 at least one of Fab, F(ab)2, Fab’, F(ab’)2, Fv, (Fv)2, scFv, and sc(Fv)2. These antigen-binding regions can be obtained by conventional techniques in the art.

[0051] 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 monoclonal antibody against human KRT19 as described above.

[0052] 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.

[0053] The sequence of the nucleic acid molecule can be obtained by conventional means such as codon coding rules based on the antibody AA sequence. The full-length sequence or a fragment of the nucleic acid molecule can usually be obtained by PCR amplification, recombination, or artificial synthesis methods.

[0054] 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 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). Insert the nucleic acid molecule into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule, then introduce it into the host cell and culture it under specific conditions to express and obtain the antibody. This is a well-known technique in the art and will not be described in detail here.

[0055] The nucleic acid molecules encoding the monoclonal antibodies FL and FH of the present invention can be inserted into two 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 appropriate conditions to form an antibody. In other embodiments, the nucleic acid molecules of antibodies FL and FH can also be cloned into one vector, and each nucleic acid sequence is linked 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 expression vector / promoter depends on the type of host cell used to produce the antibody.

[0056] 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, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with the CaCl2 method or MgCl2; 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, etc.

[0057] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present invention include but are not limited to Escherichia coli (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, it is cultured under suitable conditions, and then the antibody can be expressed, and then separated to obtain the purified monoclonal antibody.

[0058] In a typical embodiment, the method for preparing the monoclonal antibody includes: after the heavy chain gene, light chain gene and signal peptide of the monoclonal antibody are concatenated, they are respectively loaded on the expression vector pBR322, co-transfected into human renal epithelial cells (293F), the 293F cells are cultured, and the cell culture supernatant is collected and purified to obtain the target antibody strain. The choice of signal peptide is designed according to the host cell, and the present invention has no special limitation in this regard.

[0059] Another embodiment of the present invention provides an antibody conjugate, comprising the monoclonal antibody against human KRT19 as described above and a detection label linked to the monoclonal antibody.

[0060] The detection label is used to generate a recognizable signal change, so as to identify the antibody of the present invention according to the signal change, and further identify the expression of KRT19 antigen in the sample to be detected through the specific reaction of antigen and antibody. The detection label 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.

[0061] It should be emphasized that the monoclonal antibody 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 monoclonal antibody of the present invention is used as an antigen-binding (or capture) antibody, which specifically recognizes and binds KRT19 in the sample to be detected, and then qualitatively or quantitatively detects the KRT19 protein by analyzing the signal of the detection label linked thereto; in other embodiments, the anti-KRT19 monoclonal 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-KRT19 antibody is a rabbit-derived IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody. Thus, by analyzing the signal change of the detection label generated after the secondary antibody specifically binds to the antibody of the present invention, qualitative or quantitative detection of KRT19 can be achieved, such as the multiple detection systems established in Example 2 below of the present invention.

[0062] Another embodiment of the present invention provides the use of the monoclonal antibody against human KRT19 or its antibody conjugate as described above in the preparation of a human KRT19 immunoassay kit.

[0063] The advantages of the use of the monoclonal antibody against human KRT19 or its antibody conjugate in the preparation of a human KRT19 immunoassay kit are the same as the advantages of the monoclonal antibody against human KRT19 relative to the prior art as described above, and will not be elaborated here.

[0064] Based on the same inventive concept, an embodiment of the present invention also provides a human KRT19 immunoassay kit, which comprises the monoclonal antibody against human KRT19 or its antibody conjugate as described above.

[0065] 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.

[0066] Preferably, the kit is a western blot kit or an immunohistochemistry kit.

[0067] Optionally, the kit further includes a fluorescent secondary antibody against the anti-human KRT19 antibody, and the fluorescent secondary antibody is a fluorescent-conjugated anti-rabbit IgG antibody.

[0068] 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 generally 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 generally according to the conditions recommended by the manufacturer.

[0069] Example 1 Preparation of Rabbit Antibody Against Human KRT19

[0070] 1.1 Animal Immunization: In the present invention, an antigenic epitope region specific to the KRT19 protein is selected to make the purpose and function of the produced antibody more specific and clear. The amino acid (AA) sequence of the KRT19 polypeptide antigen is shown as follows:

[0071] RFVSSSSSGAYGGGYGGVLTASDGLLAGNEKLTMQNLNDRLASYLDKVRALEAANGE LEVKIRDWYQKQGPGPSRDYSHYYTTIQDLRDKILGATIENSRIVLQIDNARLAADDFRTKF ETEQALRMSVEADINGLRRVLDELTLARTDLEMQIEGLKEELAYLKKNH (see SEQ ID NO.11).

[0072] Two New Zealand white rabbits were immunized with human KRT19 protein polypeptide fragments at a dose of 200 μg per rabbit. Before the first immunization, the antigen was mixed with an equal volume of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsifier, which was then injected subcutaneously at multiple points on the abdomen and back of the rabbits. Every three weeks after the first immunization, 100 μg of the immunogen was mixed with an equal volume of incomplete Freund's adjuvant (purchased from Sigma) to prepare an emulsifier, which was injected subcutaneously at multiple points on the abdomen and back of the rabbits for two booster immunizations. After three immunizations, rabbit sera were collected, and the titer against human KRT19 was determined by enzyme-linked immunosorbent assay (ELISA). The rabbit with a higher serum titer was boosted once with 200 μg of the immunogen.

[0073] The steps for determining the titer of immune serum by ELISA are as follows: (1) Coating NeutrAvidin protein: Dilute NeutrAvidin protein with PBS buffer to a final concentration of 2 μg / mL, add 25 μL per well to the enzyme-linked immunosorbent assay plate, and incubate overnight at 4°C; (2) Coating biotinylated KRT19 polypeptide antigen: Dilute the antigen protein with PBS buffer to a final concentration of 1 μg / mL, add 25 μL per well to the enzyme-linked immunosorbent assay plate, and incubate at room temperature for 1 h; (3) Blocking: Wash the plate 5 times by adding 75 μL per well of washing buffer (PBS containing 0.05% (v / v) Tween-20), then add 50 μL per well of blocking buffer (PBS containing 1% BSA, 0.5% gelatin, and 5% sucrose), and incubate at room temperature for 1 h; (4) Serial dilution of the serum to be tested and sample addition: Repeat the plate washing process in (3) to wash the plate, then serially dilute the serum to be tested. Dilute the serum with dilution buffer (PBS containing 1% BSA) starting from 1:1000 and perform three-fold dilutions for a total of 8 gradients; Add 25 μL of the serum dilution per well to the plate and incubate overnight at 4°C; (5) Incubation with secondary antibody: Repeat the plate washing process in (3) to wash the plate, then add 25 μL per well of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (purchased from Jackson ImmunoResearch, catalog number 111-035-045) diluted 1:5000, and incubate at room temperature in the dark for 1 h; (6) Termination of the reaction and color development: Repeat the plate washing process in (3) to wash the plate, then add 100 μL per well of TMB for color development, incubate at 37°C in the dark for 10 min, add 100 μL per well of 0.5 M oxalic acid solution to terminate the reaction, measure the absorbance at 450 nm. Use the pre-immunization rabbit serum as the negative control, and the detection system without adding immune serum as the blank control (NC). A positive immune serum is defined as a ratio of the measured value to the control value ≥ 2.1.

[0074] The preparation method of biotinylated KRT19 polypeptide antigen is as follows: Prepare a KRT19 polypeptide solution, add a labeling reaction solution (50 mM carbonate (CBS) buffer, pH 8.5), mix well, centrifuge at 4°C and 3500 rpm for 12 - 15 min, and discard the filtrate; Repeat the above steps 2 - 3 times. After concentrating to the required volume, transfer the concentrated replacement buffer solution to a 2 mL centrifuge tube, measure the protein concentration after concentration, and ensure that the concentration is not less than 4 mg / mL; Add the required amount of biotin in a ratio of polypeptide:biotin = 1:10 - 100 (molar ratio), mix well, and react at room temperature for 1 h. After the reaction, dialyze and change the solution of the reaction system to obtain the biotinylated KRT19 polypeptide antigen.

[0075] The serum titer detection results are shown in Figure 1 . It can be seen that a strong immune response was generated in the rabbits during the third immunization and the fourth booster immunization, and the sera after the fourth immunization can be used for subsequent monoclonal antibody separation.

[0076] 1.2 Separation of B cells in the spleen and sorting of antigen-specific B cells: For the relevant methods, refer to the published 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] 1.3 Cloning of genes encoding rabbit monoclonal antibodies: Use the antigen-coated ELISA method to detect B cells that can recognize and bind to human KRT19 protein, collect these cells, and after lysis, extract RNA using the Quick-RNA TM Micro Prep kit (purchased from ZYMO, product number R1100 - 250), and reverse transcribe it into cDNA. Using the aforementioned cDNA as a template, adopt the PCR method to amplify the genes of the light chain variable region (VL) and heavy chain variable region (VH) of the naturally paired rabbit-derived antibody. 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 H2O; The PCR amplification program includes: 98°C for 30 s, and then 40 cycles are carried out according to 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 solution is stored at 4°C. The primer sequences (5'-3') for amplifying the VL and VH genes are shown below, where F and R represent the forward and reverse primers respectively.

[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; then the sequence of the constant region was obtained by querying the IMGT online database (www.imgt.org), and antibodies with the complete light chain (FL) shown in SEQ ID NO.1 and the complete heavy chain (FH) shown in SEQ ID NO.6 were obtained. The amino acid (aa) and nucleic acid (DNA) sequences of the antibodies are shown in Table 1. For convenience of description, the light chain complementarity-determining regions CDR1-3 are represented by LCDR1-3 respectively, and the heavy chain complementarity-determining regions CDR1-3 are represented by HCDR1-3 respectively.

[0083] Table 1 Amino acid sequences of rabbit monoclonal antibodies screened in this example

[0084]

[0085]

[0086] 1.4. Expression and large-scale production of antibodies: The obtained heavy chain and light chain genes of the antibody were respectively loaded onto expression vectors. In this example, the light chain constant region (CL) and the heavy chain constant region gene (CH) were pre-inserted into the mammalian expression vector pBR322, and its expression map is shown in Figure 2, 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 chainconstant is the nucleic acid sequence of CL (left figure), and Heavy chain constant is the nucleic acid sequence of CH (right figure). Then, the VL and VH genes with a signal peptide-encoding gene upstream were ligated to the expression vector pBR322 carrying the CL and CH genes, which were linearized with the restriction enzymes XbaI (955 bp) and NheI (949 bp) respectively, by homologous recombination to obtain complete light chain (FL) and heavy chain (FH) gene expression vectors. The successful construction of the vector was verified by sequencing.

[0087] To more conveniently purify the antibody, the secretion expression of the antibody was achieved 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, such as the signal peptide "MDTRAPTQLLGLLLLWLPGATF" upstream of VL and the signal peptide "METGLRWLLLVAVLKGVQC" upstream of VH in the patents "Rabbit monoclonal antibody against human interferon α2 and its application (Publication No.: CN116063487A, Publication Date: May 5, 2023)" and "High-affinity Human IL-5 rabbit monoclonal antibody and its application (Publication No.: CN115819578A, Publication Date: March 21, 2023)". 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 an antibody that recognizes human KRT19 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 SA023100), and the purification protocol was operated according to the instructions of the protein A affinity gel resin. The purity of the antibody was verified to be ≥95% by 12% polyacrylamide gel electrophoresis (SDS-PAGE), and the antibody concentration was 1 mg / mL. The purified antibody was aliquoted and stored at -20 °C for later use.

[0089] Example 2 Evaluation of the reaction specificity and application effect of rabbit monoclonal antibody

[0090] 1. Construction of the detection system by immunoblotting (Western blotting, WB)

[0091] The samples for Western blot contain: human hepatoma cells (Hep G2) and human T lymphocyte leukemia cells (Jurkat). The former is a positive sample with high expression of KRT19 protein, and the latter is a negative sample without KRT19 protein expression. The operations of WB experiment and analysis refer to the Western blot instruction manual, which specifically includes the following steps: (1) Electrophoresis: The cells are lysed to obtain a protein lysate, and conventional electrophoresis solution of Tris-Glycine-SDS-Buffer (purchased from Monad) and 10% SDS-PAGE separating gel are used for electrophoresis; (2) Transfer: According to the conventional method, the gel protein band is transferred to the NC membrane in an electrotransfer system; (3) Blocking: The NC membrane is placed in a TBST blocking solution containing 3% skim milk powder and blocked at room temperature for 30 min; (4) Primary antibody incubation: Add the antibody prepared in Example 1 (the working concentration of the primary antibody is 0.2 μg / mL) and incubate at room temperature for 1 h; (5) Secondary antibody incubation: Wash the membrane 3 - 4 times with TBST, add the secondary antibody working solution (from Abclonal, product number AS014, dilution ratio of the secondary antibody 1:5000), and incubate at room temperature for 1 h; (6) Development: Wash the membrane 3 - 4 times with TBST, add the ECL hypersensitive developing solution for development.

[0092] The results of Western blot are divided into: positive and negative. Among them, a solid band needs to be detected at the expected size position of the antigen protein for the positive signal without any non-specific bands.

[0093] Figure 3 The WB exposure results of human Hep G2 and Jurkat cell samples are shown. It can be seen from the figure that the anti-human KRT19 rabbit monoclonal antibody prepared by the present invention detects a single band of about 40 kDa in the human Hep G2 positive cell sample. The band signal is strong without non-specific bands, which is consistent with the actual size of KRT19, presenting a positive signal. While there is no band in the human Jurkat cell sample, presenting a negative signal. The effect of the WB experiment is consistent with the expectation, indicating that the anti-human KRT19 rabbit monoclonal antibody has high specificity for the KRT19 protein, with good recognition sensitivity and the ability to resist interference from cell components, which is beneficial to improving the accuracy and reliability of detection and reducing false positive and false negative results.

[0094] 2. Construction of the immunohistochemistry (IHC) detection system

[0095] The sliced samples include: human liver tissue sliced samples, human kidney tissue sliced samples, human colon cancer tissue sliced samples, human colon tissue slices and human breast cancer tissue slices. The IHC staining operation is as follows: 1) Sample preparation and baking: Put the paraffin tissue slices into an oven at 56°C for 30 minutes; at the same time, put the dewaxing solution 1 into an oven at 56°C, place the paraffin slices and the slice rack together in the dewaxing solution 1, then take them out of the oven together and place them at room temperature. After 5 minutes, take out the slices and immerse them in the room temperature dewaxing solution 2. Immerse the paraffin slices in the dewaxing solution 2, dewaxing solution 3, absolute ethanol 1, absolute ethanol 2, and absolute ethanol 3 in sequence, place them in the dewaxing solution for 5 minutes, and place them in the absolute ethanol for 3 minutes; then wash the slices with running water for 3 minutes; The dewaxing solutions 1-3 are purchased from Wuxi Jiangyuan Industrial and Trade Co., Ltd.; 2) Antigen repair: High-pressure heat repair with 0.01M Tris-EDTA repair solution (pH 9.0); 3) Inactivation of endogenous peroxidase: Wash 3 times with PBS buffer, 3 minutes each time, to remove the buffer on the slices; then immerse the slices in 3% hydrogen peroxide solution and incubate at room temperature for 10 minutes; 4) Blocking: Wash 3 times with PBS buffer, 3 minutes each time, then remove the buffer, circle the tissue area to be detected on the slide, and drop PBS blocking solution in the circled area and incubate at room temperature for 30 minutes; 5) Primary antibody incubation: Remove the blocking solution, drop the antibody dilution solution prepared in Example 1 (primary antibody dilution ratio 1:1000), and incubate at room temperature for 60 minutes; remove the antibody working solution, quickly rinse once with PBS buffer and soak and wash 3 times, 3 minutes each time; 6) Secondary antibody incubation: Drop the ready-to-use secondary antibody working solution (purchased from DAKO, product number K5007), and incubate at room temperature for 25 minutes; remove the secondary antibody working solution, quickly rinse once with PBS buffer and soak and wash 3 times, 3 minutes each time; 7) Color development: Drop the color development working solution, closely observe the color change under the microscope. After obtaining the appropriate staining intensity, immerse the slices in a large amount of distilled water to terminate the color development, and then wash with running water for 10 minutes; 8) Counterstaining: Immerse the slightly drained tissue slices in Mayer's hematoxylin for counterstaining for 1 minute and wash with running water for 3 minutes; 9) Blueing: Immerse the slightly drained slices in a saturated aqueous solution of lithium carbonate for blueing for 3 seconds and wash with running water for 3 minutes; 10) Dehydration: Immerse the slices in absolute ethanol 2 times, and lift them up and down several times during the immersion. Take them out after 10 seconds; Dry the slices at a high temperature (54-58°C); 11) Sealing: Drop an appropriate amount of neutral gum in the center of the slices and cover with a cover glass. The amount of gum added should be appropriate. After covering the cover glass, it should completely cover the tissue and there should be no gum overflow. Finally, perform slice scanning.

[0096] The results of immunohistochemical staining are divided into: positive and negative. Among them, the positive signal requires brownish-yellow coloring at the expression site of KRT19 antigen in specific tissues and cells with a low background, that is, the cells or tissues that should theoretically show brownish-yellow coloring have medium coloring degree, and the cells and tissues that should not be colored theoretically have no coloring to be regarded as positive. The cell nucleus is counterstained with hematoxylin and appears blue, which is used to judge the subcellular localization of KRT19 antigen.

[0097] Figure 4 The IHC staining results of human liver tissue section samples, human kidney tissue section samples, human colon carcinoma tissue sections, human colon tissue sections, and human breast cancer tissue sections are representatively shown in sequence from left to right and from top to bottom. The 1:200 in the figure indicates the magnification. KRT19 is mainly located in the cytoplasm of epithelial cells. As Figure 4 can be seen, the cytoplasm of human kidney, human colon, and human breast cancer tissue sections shows brownish-yellow coloring, the antibody localization is accurate, the staining is clear without non-specific staining, the background is clean, presenting a positive signal. In human liver tissue, only Kupffer cells are positive and other cells are negative. The immunohistochemical results of this sample are in line with the theoretical coloring and are determined as a negative sample. It can be seen that when detecting KRT19 antigen based on IHC, the antibody of the present invention has good specificity, can efficiently and sensitively recognize the target protein in the cytoplasm, does not cross-react with non-target antigens, has strong anti-cell component interference ability, the detection results are highly consistent with the actual situation, and there are no false positive and false negative results. By detecting pathological tissue samples, it is beneficial to improve the accuracy and reliability of the detection.

[0098] 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. A monoclonal antibody against human KRT19, 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 monoclonal antibody against human KRT19 according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The monoclonal antibody against human KRT19 according to claim 2, characterized in that: The amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The monoclonal antibody against human KRT19 according to claim 1, characterized in that The monoclonal antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody against human KRT19 as described in any one of claims 1 to 4.

6. A recombinant vector, characterized in that: Comprising the nucleic acid molecule as described in claim 5.

7. An antibody conjugate, characterized in that: The method comprises the monoclonal antibody against human KRT19 as claimed in any one of claims 1 to 4 and a detection label connected to the monoclonal antibody.

8. Use of the monoclonal antibody against human KRT19 according to any one of claims 1 to 4 or the antibody conjugate according to claim 7 in the preparation of a human KRT19 immunoassay kit.

9. Use of the monoclonal antibody or antibody conjugate against human KRT19 according to claim 8 in the preparation of a human KRT19 immunoassay kit, characterized in that: The kit is an immunoblotting kit or an immunohistochemistry kit.

10. A human KRT19 immunoassay kit, characterized in that: It comprises the monoclonal antibody against human KRT19 as described in any one of claims 1 to 4 or the antibody conjugate as described in claim 7.

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