Anti-p21 protein monoclonal antibody, antibody conjugate and its application

Abstract

The present invention belongs to the field of antibody technology, and in particular relates to monoclonal antibodies, antibody conjugates and applications thereof against p21 proteins. The amino acid sequences of the complementary determining regions CDR1-3 on the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.3-5, respectively; the amino acid sequences of the complementary determining regions CDR1-3 on the heavy chain variable region are shown as SEQ ID NO.8-10, respectively. The monoclonal antibody provided by the present invention can specifically recognize p21 proteins expressed by human cells and tissues, has good affinity for binding to p21 proteins, high recognition sensitivity, and strong anti-interference ability, is suitable for immunodiagnostic analysis of p21 proteins in tissues / cells, and has broad application prospects and good market prospects in clinical diagnosis and scientific research detection. In addition, the antibody has cross-reactivity to homologous p21 proteins of humans, rats and mice, and has certain applicability to the detection of rat and mouse p21 proteins.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, in particular to monoclonal antibodies against p21 protein, antibody conjugates and applications thereof. Background Art

[0002] The p21 protein, also known as CDKN1, CDKN1A, WAF1, or CIP1, is a member of the cyclin-dependent kinase inhibitor (CDKI) family and a negative regulator of the cell cycle. The eukaryotic cell cycle is divided into interphase (G1, S, and G2) and mitosis (M). In the resting state, p21 binds to cyclin-dependent kinase 2 or cyclin-dependent kinase 4 complexes, such as cyclinD1-CDK4, cyclinE-CDK2, and cyclinA-CDK2, inhibiting the activity of various cyclin-CDK complexes, thereby arresting the cell cycle in the G1 phase and allowing cells ample time to repair damaged DNA or respond to other stimuli. In addition to regulating cell cycle progression through cyclin-CDK, p21 can also interact with the DNA polymerase cofactor proliferating cell nuclear antigen (PCNA), blocking PCNA from activating DNA polymerase activity, thereby inhibiting DNA synthesis and arresting the cell cycle, and playing a regulatory role in S phase DNA replication and DNA damage repair.

[0003] p21 expression is tightly controlled by the tumor suppressor protein p53. Following DNA damage, elevated p53 levels trigger multiple downstream events, including cell cycle arrest, DNA repair and differentiation, and apoptosis. When p53 is inactivated, p21 expression is reduced or eliminated, preventing damaged cells from repairing damaged DNA by arresting in the G1 phase. This leads to DNA malreplication, cellular abnormalities, and malignant transformation. Therefore, p21 plays a crucial role in the development and progression of various tumors. Its abnormal expression is closely correlated with tumor proliferation, differentiation, invasion depth, hyperplasia, and metastasis, and has prognostic value. For example, studies have shown that overexpression of p21 is significantly associated with the transcription of HPV16 / 18E6E7. Tumor suppressor proteins that inhibit p21 expression can overcome cervical damage caused by HPV infection. Low or absent p21 expression is often seen in HPV-negative cervical cancers. This suggests that changes in p21 protein levels may provide insights into the diagnosis and treatment of related diseases.

[0004] With the continuous advancement of immunodiagnostic technology, research on the p21 gene and protein, as well as the preparation of related antibodies, has increased. However, currently available commercially available antibodies do not effectively recognize human p21 protein. While ELISA is commonly used in the medical field to detect p21 expression levels, it is not possible to rapidly diagnose the disease through direct testing of tissues / cells at the site of disease. Because the target antigen p21 is typically present at low levels in cell or tissue testing and is subject to numerous interfering factors, low antibody sensitivity may result in ineffective antigen recognition, resulting in false-negative results. Poor antibody specificity may also lead to cross-reactions with other antigens, resulting in false-positive results. Therefore, the development of highly specific and sensitive anti-p21 antibodies suitable for cell / tissue testing is of great practical significance. Summary of the Invention

[0005] To address the existing problem of a lack of monoclonal antibodies suitable for detecting p21 protein in cells / tissues, the present invention provides an anti-p21 monoclonal antibody that specifically recognizes p21 protein expressed in human cells and tissues. This antibody exhibits good binding affinity, high recognition sensitivity, and strong anti-interference capabilities, making it suitable for immunodiagnostic analysis of p21 protein in tissues / cells. The present invention further provides the use of this monoclonal antibody in the preparation of a p21 protein detection kit, as well as related detection kits.

[0006] To achieve the aforementioned objectives, the present invention is specifically implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides a monoclonal antibody against p21 protein, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of the complementary determining regions CDR1-3 on the light chain variable region are shown as SEQ ID NOs. 3-5, respectively; and the amino acid sequences of the complementary determining regions CDR1-3 on the heavy chain variable region are shown as SEQ ID NOs. 8-10, respectively.

[0008] Furthermore, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7.

[0009] Furthermore, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0010] Furthermore, the monoclonal antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

[0011] The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody against p21 protein.

[0012] The third aspect of the present invention provides an antibody conjugate comprising the above-mentioned monoclonal antibody against p21 protein and a detection label bound to the monoclonal antibody.

[0013] The fourth aspect of the present invention provides the use of the above-mentioned anti-p21 protein monoclonal antibody or antibody conjugate in the preparation of a p21 protein detection kit.

[0014] Furthermore, the p21 protein is human, rat or mouse p21 protein.

[0015] Furthermore, the detection kit is an enzyme-linked immunosorbent assay kit, an enzyme-linked immunosorbent assay kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit, an immunoprecipitation kit or a flow cytometry kit.

[0016] In a fifth aspect, the present invention provides a p21 protein detection kit, which comprises the above-mentioned anti-p21 protein monoclonal antibody or antibody conjugate.

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

[0018] The monoclonal antibody provided by the present invention can specifically recognize p21 protein expressed in human cells and tissues. It has good affinity for binding to p21 protein, high recognition sensitivity, and strong anti-interference ability. When used for detecting p21 protein in cells / tissues, it has good detection sensitivity and specificity, and the detection results are highly consistent with the actual situation. The results are accurate and reliable, and can effectively avoid false positive and false negative results. It is suitable for immunodiagnostic analysis of p21 protein in tissues / cells, especially using western blotting and immunohistochemistry analysis. It has broad application prospects and good market prospects in clinical diagnosis and scientific research. In addition, the antibody has cross-reactivity with human, rat, and mouse homologous p21 proteins and has certain applicability for detecting rat and mouse p21 proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1A map of the mammalian expression vector pRB322 used in constructing an expression vector for a monoclonal antibody against p21 protein in Example 1 of the present invention, including, from left to right, vectors pre-carrying the antibody light chain constant region and heavy chain constant region;

[0021] Figure 2 This is a gel electrophoresis image of p21 expression in positive and negative cells / tissues detected by immunoblotting using a monoclonal antibody against p21 protein in Example 2 of the present invention;

[0022] Figure 3 This is a tissue staining diagram of a human tissue sample detected by immunohistochemistry using a monoclonal antibody against p21 protein in Example 2 of the present invention;

[0023] Figure 4 This is a tissue staining diagram of a mouse tissue sample detected by immunohistochemistry using a monoclonal antibody against p21 protein in Example 2 of the present invention. DETAILED DESCRIPTION

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

[0025] Given the information contained herein, it will be readily apparent to those skilled in the art that various modifications may be made to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the processes, properties, or components defined herein, as these embodiments and other descriptions are intended only to illustrate specific aspects of the present invention. Indeed, various modifications to the embodiments of the present invention that are apparent to those skilled in the art or related fields are intended to be within the scope of the appended claims.

[0026] For a better understanding of the present invention and not to limit the scope of the present invention, all numerals and other numerical values ​​used in the present invention to express amounts, percentages, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant digits and by conventional rounding methods.

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

[0028] The terms "include", "comprising", "containing", "having" and the like are non-limiting in meaning, that is, other steps and other components that do not affect the results may be added.

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

[0030] The terms "rabbit monoclonal antibody," "rabbit antibody," and "rabbit monoclonal antibody" have synonymous meanings and, unless otherwise specified, refer to rabbit antibodies that specifically bind to the p21 protein. The modifier "rabbit" indicates that the complementarity-determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences. The terms "cyclin-dependent kinase inhibitor 1A," "p21," "CDKN1," "CDKN1A," "Waf1 / Cip1," and similar terms have synonymous meanings and can be used interchangeably.

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

[0032] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types: kappa (κ) and lambda (λ); heavy chains can be classified into five types: μ, δ, γ, α, and ε, which define antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of heavy and heavy chains vary greatly, while the remaining amino acid sequences are relatively constant. The regions of the light and heavy chains with the most variable amino acid sequences near the N-terminus are called the variable region (V), while the regions with relatively stable amino acid sequences near the C-terminus are called the constant region (C). The heavy chain variable region (VH) and light chain variable region (VL) are generally the most variable parts of antibodies and contain the antigen recognition site. The VH and VL regions can be further subdivided into hypervariable regions (HVRs) and framework regions (FRs). The HVRs, also known as complementarity-determining regions (CDRs), are circular structures. The heavy and light chain CDRs are closely aligned and interact with each other through the FRs, forming a surface that complements the three-dimensional structure of the target antigen or epitope. This determines the antibody's specificity and is the site of antigen recognition and binding. The FRs are the more conserved portions of the VH and VL sequences. They generally follow a β-pleated sheet configuration and are connected by three CDRs that form a connecting loop. Each VH and VL sequence typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0033] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, a cumulative of the Kabat and Chothia definitions, the AbM definition, the contact definition, the IMGT unique numbering definition and / or the conformational definition, or any CDR determination method known in the art. As used herein, the Kabat numbering system is used to define CDRs.

[0034] The light chain constant region (CL) and heavy chain constant region (CH) are not directly involved in antibody-antigen binding, but they exhibit different effector functions, such as antibody-dependent cytotoxicity. The CL length is essentially the same for different Ig types (κ or λ), but the CH length varies across Ig classes. For example, IgG, IgA, and IgD comprise CH1, CH2, and CH3, while IgM and IgE comprise CH1, CH2, CH3, and CH4. The amino acid sequences of the heavy and light chain constant regions of antibodies are well known in the art and can be obtained by querying the IMGT database.

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

[0036] Antibody fragments are one or more parts or fragments of a full-length antibody that essentially retain the same biological function or activity as the full-length form. Specifically, an antibody fragment includes at least the same CDR regions as the full-length antibody, and more preferably the same variable regions, thereby retaining complete antigen recognition and binding sites and being able to bind to the same antigen as the full-length antibody, particularly to the same epitope. Typical examples include: Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, and sc(Fv)2. These antibody fragments can be obtained using conventional techniques in the art.

[0037] (i) Fab: An antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable and constant regions) and a portion of the heavy chain (variable and first constant regions). By proteolytic cleavage of the full-length antibody, fragments such as Fab, F(ab')2, and Fab' can be obtained. For example, IgG can be degraded into two Fab fragments and an Fc fragment by papain; and into an F(ab')2 fragment and a pFc' fragment by pepsin. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because Fab contains the antigen-binding region and a portion of the constant region, it not only possesses the same antibody-antigen affinity and excellent tissue penetration as scFv, but also has a more stable structure.

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

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

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

[0041] (v) scFv: A single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, consisting of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (typically consisting of 10-25 amino acids). It retains the antigen-binding specificity of the original antibody. The linker in this invention is not particularly limited, as long as it does not hinder the expression of the antibody variable regions connected to it. Compared to full-length antibodies, scFv has a smaller molecular weight, resulting in higher penetration and lower immune side effects.

[0042] (vi) The sc(Fv)2 fragment is composed of two heavy chain variable regions and two light chain variable regions connected by a linker or the like.

[0043] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may comprise a complementary determining region (CDR) and a framework region (FR) from a rabbit immunoglobulin sequence. In other embodiments, the antibody may comprise amino acid residues encoded by non-rabbit immunoglobulin sequences, for example, humanized antibodies, chimeric antibodies, etc., to reduce the body's rejection reaction while maintaining the required specificity and affinity. The term "chimeric antibody" refers to a portion of an antibody derived from a specific source or species, while the rest is derived from a different source or species. The term "humanized antibody" is a chimeric antibody comprising a non-human antibody, such as a rabbit antibody, with a CDR region and a human FR region. In some cases, the variable region of a non-human antibody is combined with a constant region of a human antibody, such as a human-rabbit chimeric antibody; in other cases, the CDR region of a non-human antibody is combined with a FR region and a constant region derived from a human antibody sequence, that is, the CDR region of a non-human antibody is grafted onto a human antibody framework (FR) sequence, which is derived from the FR sequence of a single or multiple other human antibody variable regions. In the present invention, the CDR regions in the chimeric or humanized antibodies are derived from rabbit-derived CDR regions.

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

[0045] The term "specific binding" is a well-known term in the art, and a molecule exhibits "specific binding" if it reacts with a specific target antigen or epitope more frequently, more rapidly, longer-lastingly, and / or with greater affinity than with other target antigens or epitopes. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0046] In order to make the above-mentioned objects and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below.

[0047] An embodiment of the present invention provides an anti-p21 protein monoclonal antibody, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region each comprise three complementarity determining regions (CDRs), respectively designated as CDR1, CDR2, and CDR3, wherein the amino acid (AA) sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively; and the amino acid (AA) sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0048] The monoclonal antibody provided by the present invention can specifically recognize the p21 protein expressed by human cells and tissues, and has good affinity for binding to the p21 protein, high antigen recognition sensitivity, and strong anti-interference ability. It has good detection sensitivity and specificity when used for detecting the p21 protein in cells / tissues. Specifically, by immunoblotting, positive cells or tissues expressing the p21 protein and negative cells or tissues not expressing the p21 protein were detected, and only a single band was detected in the protein lysate of the positive sample, and the size was consistent with the expected size. By immunohistochemistry, positive and negative tissue samples were detected, and the staining in the positive tissue samples was accurately positioned, clear, and with a clean background. In the negative tissue samples, there was no nonspecific staining, and the detection sensitivity and specificity both reached 100%. It can be seen that the detection results of the antibody of the present invention are highly consistent with the actual situation, the results are accurate and reliable, and false positive and false negative results can be effectively avoided. It is suitable for immunodiagnostic analysis of p21 protein in tissues / cells, especially using western blotting and immunohistochemistry analysis and detection, and has broad application prospects and good market prospects in clinical diagnosis and scientific research. Moreover, the antibody has cross-reactivity to homologous p21 proteins of human, rat and mouse, and has certain applicability for the detection of rat and mouse p21 proteins.

[0049] Optionally, the light chain variable region and the heavy chain variable region each include four framework regions (FRs), wherein the four FRs and three CDRs are arranged in a staggered sequence to form a variable region. The amino acid sequence of the light chain variable region (VL) of the monoclonal antibody of the present invention is shown in SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO. 7.

[0050] Optionally, the monoclonal antibody of the present invention further comprises a light chain constant region and a heavy chain constant region, wherein CL and VL constitute the light chain, and CH and VH constitute the heavy chain. The constant region of an antibody is usually obtained by querying the IMGT online database.

[0051] Specifically, the amino acid sequence of the light chain (FL) containing the light chain constant region is shown in SEQ ID NO.1, and the light chain constant region is a κ chain. The amino acid sequence of the heavy chain (FH) containing the heavy chain constant region is shown in SEQ ID NO.6, and the heavy chain is of IgG type.

[0052] It should be noted that the monoclonal antibody of the present invention can be a full-length antibody (having a typical Y-shaped molecular structure) or an antigen-binding region of the full-length antibody; the antigen-binding region refers to a polypeptide that substantially retains the same biological function or activity as the full-length form. Specifically, the antigen-binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining a complete antigen recognition and binding site, and can bind to the same antigen as the full-length antibody, especially to the same epitope. Optionally, the antigen-binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv and sc(Fv)2. These antigen-binding regions can be obtained by conventional techniques in the art.

[0053] Yet another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector comprising the aforementioned nucleic acid molecule, or a host cell comprising the aforementioned nucleic acid molecule, wherein the nucleic acid molecule encodes the aforementioned monoclonal antibody against p21 protein.

[0054] Nucleic acid molecules can be in the form of DNA (e.g., cDNA, genomic DNA, or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can be either a coding strand or a non-coding strand. The sequence of the nucleic acid molecule can be derived from the antibody amino acid sequence by conventional means, such as codon coding rules.

[0055] The full-length sequence of a nucleic acid molecule or a fragment thereof can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0056] The original vector for constructing the recombinant vector is a variety of vectors conventional in the art, as long as it can accommodate the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, phages (such as λgt4λB, λ-Charon, λΔz1 and M13), cosmids and minichromosomes. The vector can be a cloning vector (i.e., for transferring nucleic acid molecules into a host and multiplying them in large quantities in the host cell) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecules inserted into the vector to be expressed in the host cell). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid molecule, and then introduced into a host cell to culture and obtain the antibody. This is a well-known technology in the art and will not be described in detail here.

[0057] The nucleic acid molecules encoding the monoclonal antibodies FL and FH of the present invention can be inserted into two separate vectors and introduced into the same or different host cells. When the heavy chain and light chain are expressed in different host cells, each chain can be isolated from the host cell expressing it, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions to form the antibody. In other embodiments, the nucleic acid molecules of the antibodies FL and FH can also be cloned into a single vector, with each nucleic acid sequence linked downstream to a suitable promoter; for example, each nucleic acid sequence encoding the heavy chain and light chain can be operably linked to a different promoter, or the nucleic acid sequence encoding the heavy chain and light chain can be operably linked to a single promoter so that both the heavy chain and the light chain can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0058] Conventional techniques are used to transfect or transform the recombinant vector into host cells. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with CaCl2 or MgCl2. Alternatively, transfection can be accomplished by microinjection, electroporation, or liposome packaging. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, or particle bombardment to achieve gene introduction.

[0059] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present invention include, but are not limited to, Escherichia coli (e.g., DH5α, JM109, BL21, W3110), Bacillus (e.g., Bacillus subtilis, Bacillus thuringiensis), and Enterobacteriaceae strains (e.g., Salmonella typhimurium, Serratia marcescens), and Pseudomonas. Examples of eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining a host cell transfected or transformed with the recombinant vector described above, the antibody can be expressed by culturing under suitable conditions, and then separated to obtain purified antibodies.

[0060] In the present invention, transfection or transformation of the recombinant vector into host cells is performed using conventional techniques. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2 or MgCl2. If desired, methods such as microinjection, electroporation, or liposome packaging can also be used. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, etc.

[0061] Preferably, the recombinant vector is the expression vector pBR322, and the host cell is a human kidney epithelial (293F) cell.

[0062] Another embodiment of the present invention provides an antibody conjugate comprising the above-mentioned monoclonal antibody against p21 protein and a detection label bound to the monoclonal antibody.

[0063] The detection marker is used to generate a recognizable signal change, so as to identify the monoclonal antibody of the present invention based on the signal change, and then identify the expression of the specific antigen p21 in the sample to be tested through the specific reaction of the antigen and antibody. The detection marker includes, but is not limited to: biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (such as allophycocyanin, phycoerythrin, PerCP and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies or combinations thereof.

[0064] It should be noted that the antibodies of the present invention can be used alone or in conjunction with a detection label (covalently or non-covalently) to form an antibody conjugate. In some embodiments, the antibodies of the present invention are used as antigen-binding antibodies that specifically recognize and bind to the p21 protein in the sample to be tested, and then qualitatively or quantitatively detect p21 by analyzing the detection label signal attached thereto; in other embodiments, the anti-p21 antibody (as a primary antibody or capture antibody) is not labeled, and the detection label is coupled to a secondary antibody (as a detection antibody) or other molecule that can bind to the primary antibody. For example, if the anti-p21 antibody is a rabbit IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody. Thus, by analyzing the change in the detection label signal generated after the secondary antibody specifically binds to the antibody of the present invention, qualitative or quantitative detection of p21 is achieved.

[0065] Yet another embodiment of the present invention provides the use of the above-mentioned anti-p21 protein monoclonal antibody or antibody conjugate in the preparation of a p21 protein detection kit.

[0066] The advantages of using the anti-p21 protein monoclonal antibody or antibody conjugate in preparing a p21 protein detection kit are the same as the advantages of the anti-p21 protein monoclonal antibody or antibody conjugate over the prior art described above, and will not be repeated here.

[0067] The monoclonal antibodies of the present invention have cross-recognition for p21 proteins from multiple species. Specifically, the p21 proteins can be human, rat, or mouse p21 proteins. The amino acid and nucleotide sequences of the p21 proteins can be obtained by conventional techniques. For information on human p21 proteins, see Uniprot No. P38936 or NCBI Accession No. NP_000380.1. For information on mouse p21 proteins, see Uniprot No. P39689 or NCBI Accession No. NP_031695.1. For information on rat p21 proteins, see Uniprot No. Q64315 or NCBI Accession No. NP_542960.1.

[0068] Based on the same inventive concept, an embodiment of the present invention further provides a p21 protein detection kit, which includes the above-mentioned anti-p21 protein monoclonal antibody or antibody conjugate.

[0069] The advantages of the p21 protein detection kit over the prior art are the same as the advantages of the monoclonal antibody against p21 protein over the prior art, which will not be described in detail here.

[0070] The detection method adopts conventional immunoassay, including but not limited to: enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB), immunoprecipitation (IP) and flow cytometry (FC). The detection kit can be an enzyme-linked immunosorbent assay kit, an enzyme-linked immunospot assay kit, an immunohistochemistry assay kit, an immunofluorescence assay kit, an immunoblotting assay kit, an immunoprecipitation assay kit or a flow cytometry assay kit.

[0071] Preferably, the detection kit is an immunoblotting kit or an immunohistochemistry kit.

[0072] The test samples include serum, plasma, urine, cell or tissue samples, among which cells include but are not limited to: human cervical cancer cells, human breast cancer cells, human renal epithelial cells; tissues include but are not limited to: human esophagus, human colon, human liver cancer, human lung, mouse placenta, mouse testicles, rat colon and rat lung.

[0073] The present invention will be further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer.

[0074] Example 1 Preparation of rabbit-derived antibodies against p21 protein

[0075] The immunogen used to prepare rabbit monoclonal antibodies in this example was human p21 protein. Its amino acid sequence is available in Uniprot No. P38936 or NCBI Accession No. NP_000380.1, and its gene sequence is available in NCBI Accession No. NM_000389.5. The full-length gene sequence was constructed into the pBR322 vector and expressed in Escherichia coli Rosetta (DE3) using a prokaryotic expression system to obtain recombinant human p21 protein. The recombinant protein purity was greater than 90%.

[0076] This example is based on B cell labeling and sorting technology. B lymphocytes that can recognize human p21 protein are directly enriched and isolated from the spleen of New Zealand white rabbits after immunization with the immunogen. The isolated B cells are then cultured in the form of single cells to obtain monoclonal antibodies. Finally, through genetic engineering recombinant expression technology, naturally paired antibody heavy chain variable region (VH) and light chain variable region (VL) genes are first obtained by PCR amplification from B lymphocytes secreting monoclonal antibodies, and then recombinant expression is performed to separate and purify the target antibody in large quantities. The antibody sequencing work was completed by Jin Kairui Biotechnology Co., Ltd.; its amino acid (AA) sequence is shown in Table 1. For ease of description, LCDR1-3 in the table represent light chain complementary determining regions CDR1-3, and HCDR1-3 represent heavy chain complementary determining regions CDR1-3, respectively.

[0077] Table 1 Sequence information of the monoclonal antibodies of this example

[0078]

[0079]

[0080] 1. Animal Immunization: Two New Zealand white rabbits were immunized with human p21 protein at a dose of 200 μg per rabbit. Prior to the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to form an emulsion and injected subcutaneously at multiple sites on the rabbit's abdomen and back. Three weeks apart, 100 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to form an emulsion and injected subcutaneously at multiple sites on the rabbit's abdomen and back for two booster immunizations. After the three immunizations, rabbit serum samples were collected and titers against human p21 were determined. Rabbits with high serum titers were given a booster immunization with 200 μg of the immunogen injected subcutaneously at multiple sites. Three days later, the rabbit spleens were harvested.

[0081] The immune serum titer was detected by enzyme-linked immunosorbent assay (ELISA), which includes the following steps: 1) coating with human p21 protein: recombinant p21 protein was diluted with 1× PBS buffer and added to the plate at 25 μL / well, with a coating concentration of 1 μg / mL, and the plate was coated overnight at 4°C; 2) blocking: the plate was removed from the plate after overnight coating, washed five times with 75 μL / well of washing buffer (PBS containing 0.05% (v / v) Tween-20), and then blocked with 50 μL / well of blocking buffer (PBS containing 1% BSA, 0.5% gelatin, and 5% sucrose), and the plate was blocked at 37°C for 2 hours. h; 3) Serial dilution and sample loading of the test serum: Repeat the plate wash process in 2) and perform serial dilution of the test serum. Use dilution buffer (PBS containing 1% BSA) to dilute the serum starting at 1:1000 and perform three-fold dilution for a total of 8 serial dilutions. The dilution ratio can be adjusted according to the actual situation. Add 25 μL / well of the serum dilution solution to the plate and incubate at 37°C for 1 h; 4) Secondary antibody incubation: Repeat the plate wash process in 2) and add 25 μL / well of horseradish peroxidase-labeled goat anti-rabbit IgG (purchased from Jackson) diluted 1:5000 with dilution buffer. ImmunoResearch, Catalog No. AB_2337938), incubate at room temperature in the dark for 1 hour; 5) Termination of reaction and color development: Repeat the washing process in 2) to wash the plate, add 100 μL / well of TMB for color development, incubate at 37°C in the dark for 10 minutes, add 100 μL / well of 0.5 M oxalic acid solution to terminate the reaction, measure the absorbance at 450 nm, use pre-immune mouse serum as a negative control, and determine the titer of the immune serum against human p21 protein if the ratio of the measured value to the control value is ≥2.1.

[0082] 2. Isolate B lymphocytes from the spleen and sort B lymphocytes: Use conventional methods to isolate B lymphocytes from the spleen, and sort out antigen-specific B lymphocytes. For related methods, please refer to the patents "Method for Efficiently Isolating Single Antigen-Specific B Lymphocytes from Spleen Cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)" and "A B Lymphocyte In Vitro Culture System and Application (Publication No.: CN111518765A, Publication Date: 2020-08-11)".

[0083] 3. Cloning of rabbit monoclonal antibody gene: The supernatant of cultured B lymphocytes was used to identify positive clones by immunogen-coated ELISA. The positive clones of single B lymphocytes were collected and lysed and then analyzed by Quick-RNA. TMRNA was extracted using a MicroPrep kit (purchased from ZYMO, catalog number R1051) and reverse transcribed into cDNA. Using cDNA as a template, naturally paired rabbit monoclonal antibody VL and VH genes were amplified by PCR. The PCR reaction system consisted of 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2× Gloria HiFi (from ABclonal, catalog number RK20717), and 6.5 μL H2O. The amplification procedure was 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s, followed by a final incubation at 72°C for 5 min. The resulting reaction solution was stored at 4°C. The primer sequences (5'-3') for amplifying the VL and VH genes are shown below, where F and R represent the forward and reverse primers, respectively:

[0084] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (SEQ ID NO. 11);

[0085] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (SEQ ID NO. 12);

[0086] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (SEQ ID NO. 13);

[0087] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (SEQ ID NO. 14).

[0088] 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 IMGT online database (www.imgt.org) was queried to obtain the sequence of the constant region, and 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.

[0089] 4. Production and purification of rabbit monoclonal antibodies: The heavy chain and light chain genes of the selected rabbit monoclonal antibodies were loaded onto expression vectors and co-transfected into host cells. In this example, the light chain constant region (CL) and heavy chain constant region genes (CH) were pre-inserted into the mammalian expression vector pBR322. The expression profile is shown in FIG. Figure 1In the figure, pBR322 origin and f1origin are replication promoters, Ampcillin is a resistance gene, CMV promoter is a transcription promoter, SV40 PAterminator is a tailing signal, the light chain constant is the nucleic acid sequence of CL (left), and the heavy chain constant is the nucleic acid sequence of CH (right). The amplified VL and VH genes were then ligated via homologous recombination with the expression vector pBR322 carrying the CL and CH genes, which had been linearized with the restriction enzymes XbaI (955 bp) and NheI (949 bp), respectively, to obtain complete light chain (FL) and heavy chain (FH) gene expression vectors. Sequencing verified the successful construction of the vectors.

[0090] Typically, to achieve secretory expression of antibodies, signal peptides can be added to the front ends of the VL and VH genes. Signal peptides commonly used in the art for antibody expression can be used. For example, the patent "Anti-human interferon α2 rabbit monoclonal antibody and its application (Publication No.: CN116063487A, Publication Date: 2023-05-05)" and the patent "High-affinity human IL-5 rabbit monoclonal antibody and its application (Publication No.: CN115819578A, Publication Date: 2023-03-21)" have a signal peptide "MDTRAPTQLLGLLLLWLPGARC" upstream of the VL and a signal peptide "METGLRWLLLVAVLKGVQC" upstream of the 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 of Table 1 of this Example.

[0091] The constructed FL and FH expression vectors were co-transfected into 293F cells and cultured for 72-96 hours. Rabbit monoclonal antibodies recognizing human p21 protein were obtained in the culture supernatant. The target antibodies were purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, Catalog No. SA023100). Antibody purity was verified by 12% polyacrylamide gel electrophoresis (SDS-PAGE) to be ≥95%. The purified antibodies were aliquoted and stored at -20°C until further use.

[0092] Example 2 Anti-p21 protein antibody for cell / tissue detection and analysis

[0093] 1. Western blot analysis (WB) of monoclonal antibodies

[0094] Positive cells / tissue samples expressing p21 and negative cells / tissue samples not expressing p21 were lysed to obtain protein lysates. 10% SDS-PAGE electrophoresis was performed, and the gel protein bands were transferred to a NC membrane using a conventional electrotransfer system. The membrane was incubated in TBST blocking buffer containing 3% skim milk powder for 1 hour at room temperature. The anti-p21 protein antibody prepared in Example 1 (primary antibody dilution ratio 1:1000, final concentration 1 μg / mL) was added and incubated for 1 hour at room temperature. The membrane was then washed 3-4 times with TBST, and HRP-conjugated goat anti-rabbit IgG (H+L) (purchased from Jackson, Catalog No. AS014, secondary antibody dilution ratio 1:5000) was added and incubated for 1 hour at room temperature. The membrane was washed again with TBST 3-4 times, and finally, ECL ultrasensitive colorimetric solution was added for development. β-actin protein was used as an internal control.

[0095] Figure 2 Shown are the Western Blot results of lysates from human cervical cancer cells (HeLa), human breast cancer cells (MCF7), human renal epithelial cells (293T), and 293T cells homozygous for CDKN1A knockout (293T-CDKN1A-1 (homozygous sample), as well as mouse large intestine, mouse testis, mouse spleen, rat lung, and rat heart tissue samples. HeLa and MCF7 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the 293T cell line was purchased from Beina Biotechnology. 293T-CDKN1A-1 (homozygous) was prepared by homozygous knockout of the CDKN1A gene in the 293T cell line, and tissue samples were prepared from the corresponding animals. Of all the samples, only 293T-CDKN1A-1 (homozygous) was negative; the rest were positive.

[0096] Western blotting analysis revealed that the antibody of the present invention specifically binds to the human p21 protein. Only a single clear band was detected in HeLa, MCF7, and 293T cells, with the band size consistent with the theoretical localization. No band was detected in CDKN1A knockout homozygotes, demonstrating the antibody's excellent specificity, effectively avoiding false-positive results and ensuring high confidence in the test results. Furthermore, the antibody of the present invention simultaneously recognized p21 proteins in some mouse and rat samples, demonstrating its applicability to p21 proteins from different species.

[0097] 2. Immunohistochemistry (IHC) analysis using monoclonal antibodies

[0098] The tissue section samples used in this embodiment include: 1) human samples: human esophagus, human lung, human colon, human liver cancer, and human liver; 2) rat samples: rat colon; and 3) mouse samples: mouse placenta.

[0099] The IHC staining procedure is as follows: 1) Sample preparation and baking: paraffin sections were placed in a 56°C constant temperature baking oven for 30 minutes; at the same time, dewaxing solution 1 was placed in a 56°C constant temperature box, and the paraffin sections and the section rack were placed in dewaxing solution 1. Then, they were taken out of the constant temperature box and placed at room temperature. After 5 minutes, the sections were taken out and immersed in dewaxing solution 2 at room temperature. The paraffin sections were immersed in dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3 in this order. The sections were placed in dewaxing solution for 5 minutes and in anhydrous ethanol for 3 minutes. The sections were then washed with running water for 3 minutes. Dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.; 2) Antigen retrieval: 0.01M Tris-EDTA retrieval solution (pH 7.0) was used. 9.0) High-pressure heat repair; 3) Inactivation of endogenous peroxidase: Wash with PBS buffer three times for 1 min each time, remove the buffer on the slice; then immerse the slice in 3% hydrogen peroxide solution and incubate at room temperature for 10 min; 4) Blocking: Wash with PBS buffer three times for 3 min each time, remove the buffer on the slice; circle the tissue area to be tested on the slide with an immunohistochemistry pen, and add PBS blocking solution in the circled area; place the slice horizontally in an incubation humidified box with water at the bottom, start the time from the addition of blocking solution, and incubate at room temperature for 30 min; 5) Primary antibody incubation: Remove the blocking solution, add anti-p21 protein antibody dilution solution (primary antibody dilution ratio 1:200, final concentration 5μg / mL) to the tissue slice, and incubate at room temperature for 60 min; remove the antibody working solution, quickly rinse with PBS buffer once, and soak and wash three times for 3 min each time; 6) Secondary antibody incubation: Add ready-to-use secondary antibody working solution (purchased from DAKO, product number K5007) to the tissue slice ) and incubate at room temperature for 25 minutes; remove the secondary antibody working solution from the slices, quickly rinse once with PBS buffer, and soak and wash three times, each time for 3 minutes; 7) Color development: add color development solution to the slices, closely observe the color change under a microscope, and after obtaining the appropriate staining intensity, immerse the slices in a large amount of distilled water to stop the color development, and then rinse in running water for 10 minutes; 8) Counterstaining: Immerse the slightly drained tissue slices in Mayer's hematoxylin for counterstaining for 1 minute, and rinse with running water for 3 minutes. min; 9) Bluing: Immerse the slightly drained sections in a saturated aqueous solution of lithium carbonate for 3 seconds, then rinse with running water for 3 minutes; 10) Dehydration: Immerse the washed sections in anhydrous ethanol once, lifting them up and down several times during the soaking period, and remove them after 10 seconds; Dry the sections at high temperature (54-58°C); 11) Sealing: Add an appropriate amount of neutral gum to the center of the section and cover with a coverslip. The amount of gum added should be appropriate. After sealing with the coverslip, the tissue should be completely covered without any gum overflow. Finally, scan the sections.

[0100] Immunohistochemical staining results are divided into: positive and negative, wherein positive expression must have brown staining and low background or no background in the specific cell antigen site of the corresponding tissue to be considered positive, and negative refers to the absence of brown staining in the cells of the specific tissue. In the present embodiment, human esophagus (Human esophagus), human colon (Human colon), human hepatocellular carcinoma (Human hepatocellular carcinoma), human lung (Human lung), mouse placenta (Mouse placenta) and rat colon (Rat colon) are positive tissue samples, and the positive cells are colon and tonsil cells. The p21 protein is subcellularly localized in the cell nucleus. Therefore, a positive result is shown as a moderate to strong nuclear staining reaction in the aforementioned cells. Normal human liver tissue is a negative sample and has no staining. Figure 3 From left to right and from top to bottom, representative IHC staining results of human esophagus, human lung, human colon, human hepatocarcinoma, and human liver are shown. Figure 4 Representative IHC staining results of mouse placenta and rat colon are shown from left to right. In the figure, 1:200 represents the dilution ratio of the anti-p21 protein monoclonal antibody of the present invention, and 40×lens represents the lens magnification.

[0101] The above results show that the IHC detection results of the antibody of the present invention for 6 positive tissue samples are consistent with the actual expression and localization of p21 in the corresponding samples. The positive staining is localized in the cell nucleus, the staining is clear, and the background is clean. There is no nonspecific staining in the 1 negative sample. This shows that the antibody has high specificity, good antigen recognition sensitivity, strong anti-interference ability, good detection sensitivity and specificity, and no false positive or false negative results occur. The results are highly accurate and reliable.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A monoclonal antibody against p21 protein, characterized in that: It includes a light chain variable region and a heavy chain variable region, and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The anti-p21 protein monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The monoclonal antibody against p21 protein according to claim 2, characterized in that The amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The monoclonal antibody against p21 protein according to claim 1, characterized in that The monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody against p21 protein according to any one of claims 1 to 4.

6. An antibody conjugate, characterized in that The invention comprises the anti-p21 protein monoclonal antibody according to any one of claims 1 to 4 and a detection label combined with the monoclonal antibody.

7. Use of the anti-p21 protein monoclonal antibody according to any one of claims 1 to 4 or the antibody conjugate according to claim 6 in the preparation of a p21 protein detection kit.

8. Use of the anti-p21 protein monoclonal antibody according to claim 7 in preparing a p21 protein detection kit, characterized in that: The p21 protein is human, rat or mouse p21 protein.

9. Use of the anti-p21 protein monoclonal antibody according to claim 7 in preparing a p21 protein detection kit, characterized in that: The detection kit is an enzyme-linked immunosorbent assay kit, an enzyme-linked immunospot assay kit, an immunohistochemistry kit, an immunofluorescence assay kit, an immunoblotting assay kit, an immunoprecipitation assay kit or a flow cytometry assay kit.

10. A p21 protein detection kit, characterized in that: The detection kit comprises the anti-p21 protein monoclonal antibody according to any one of claims 1 to 4 or the antibody conjugate according to claim 6.

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