Monoclonal antibody against p21 protein, antibody conjugate and application thereof

Abstract

The invention belongs to the technical field of antibodies, and particularly relates to an anti-p21 protein monoclonal antibody, an antibody conjugate and application thereof. Amino acid sequences of complementary determining regions 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 the complementary determining regions CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NO.8-10. The monoclonal antibody provided by the invention can specifically recognize the p21 protein expressed by human cells and tissues, has good affinity of binding with the p21 protein, high recognition sensitivity and strong anti-interference capability, is suitable for immunodiagnosis analysis of the p21 protein in tissues / cells, and has wide application prospects and good market prospects in clinical diagnosis and scientific research detection. The antibody has cross reactivity to human, rat and mouse homologous p21 proteins, and has certain applicability to detection of the rat and mouse p21 proteins.

Description

Technical Field

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

[0002] The full name of p21 protein is cyclin-dependent kinase inhibitor 1A, also known as CDKN1, CDKN1A, WAF1 or CIP1. It belongs to the family of cyclin-dependent kinase inhibitors (CDKIs) and is a negative regulator of the cell cycle. The eukaryotic cell cycle is divided into interphase (G1, S and G2 phases) and mitosis (M) phase. In the resting state of cells, p21 protein binds to cyclin-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, ensuring that cells have sufficient time to repair damaged DNA or respond to other stimuli. In addition to regulating the cell cycle process 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] The expression of p21 is strictly controlled by the tumor suppressor protein p53. After DNA damage, the increase in p53 protein levels can lead to a variety of downstream events, including cell cycle arrest, DNA repair or differentiation, and apoptosis. When p53 is inactivated, the expression of the p21 gene decreases or disappears, and the damaged cells cannot rely on blocking the G1 phase to repair the damaged DNA, which leads to incorrect replication of cell DNA, cell alienation, or malignant transformation. Therefore, p21 plays an important role in the occurrence and development of various tumors. Its abnormal expression is closely related to tumor proliferation, differentiation, infiltration depth, hyperplasia, and metastasis, and has the value of judging prognosis. For example, studies have shown that: p21 protein overexpression is significantly related to the transcription of HPV16 / 18E6E7, and tumor suppressor proteins with the function of inhibiting p21 protein expression can overcome the damage to the cervix after HPV infection; low or no expression of p21 protein is more common in HPV-negative cervical cancer; this suggests that changes in p21 protein levels can provide a reference for the diagnosis and treatment of related diseases.

[0004] With the continuous development of immunodiagnostic technology, more and more studies have been conducted on p21 genes and proteins, as well as the preparation of related antibodies. However, currently available commercial antibodies cannot recognize human p21 proteins very well. In addition, ELISA is commonly used in the medical field to detect the expression level of p21, but it is not possible to quickly quantify the expression through direct detection of tissues / cells at the lesion site. Since the content of the target antigen p21 is usually low and there are many interfering factors in cell or tissue detection, if the antibody sensitivity is low, it may not be able to effectively recognize the antigen, resulting in a false negative result; if the antibody specificity is poor, it may cross-react with other antigens, resulting in a false positive result. Therefore, it is of great practical significance to develop anti-p21 antibodies with good specificity and high sensitivity suitable for cell / tissue detection. Summary of the invention

[0005] In view of the problem that the prior art lacks monoclonal antibodies suitable for detecting p21 protein in cells / tissues, the present invention provides a monoclonal antibody against p21 protein, which can specifically recognize p21 protein expressed by human cells and tissues, has good affinity for binding to p21 protein, high recognition sensitivity, and strong anti-interference ability, and is suitable for immunodiagnosis analysis of p21 protein in tissues / cells. The present invention further provides the use of the monoclonal antibody in preparing a p21 protein detection kit and a related detection kit.

[0006] To achieve the above-mentioned purpose, the present invention is specifically implemented through the following technical solutions:

[0007] The first aspect of 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 respectively shown as SEQ ID NOs.3-5; and the amino acid sequences of the complementary determining regions CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NOs.8-10.

[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, which encodes the monoclonal antibody against p21 protein as described above.

[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 combined with 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 immunospot assay kit, an immunohistochemistry kit, an immunofluorescence assay kit, an immunoblotting kit, an immunoprecipitation kit or a flow cytometry kit.

[0016] The fifth aspect of 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 the p21 protein expressed by human cells and tissues, has good affinity for binding to the p21 protein, high recognition sensitivity, and strong anti-interference ability, has good detection sensitivity and specificity when used for detecting the p21 protein in cells / tissues, has a high degree of conformity between the detection result and the actual situation, has good accuracy and credibility, can effectively avoid false positive and false negative results, is suitable for immunodiagnosis analysis of p21 protein in tissues / cells, especially using protein immunoblotting and immunohistochemistry analysis and detection, 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. 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 creative work.

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

[0021] Figure 2 This is a gel electrophoresis gel image of the 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 solution and advantages of the present invention more clearly understood, the present invention is 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.

[0025] Based on the information contained in the present invention, it is easy for those skilled in the art to 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 the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0026] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, 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 according to the reported significant figures 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 of ordinary skill in the art.

[0028] The terms "include", "comprising", "containing", "having" and the like are not 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" and other similar terms have the same meaning and, unless otherwise specified, refer to rabbit antibodies that specifically bind to p21 protein. The modifier "rabbit" indicates that the complementarity determining region (CDR) of the antibody is derived from a rabbit immunoglobulin sequence. The terms "cyclin-dependent kinase inhibitor 1A", "p21", "CDKN1", "CDKN1A", "Waf1 / Cip1" and other similar terms have the same meaning and can be used interchangeably.

[0031] An antibody is an immunoglobulin molecule that is able to 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 a 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, namely κ chain and λ chain; heavy chains can be classified into five types, namely μ, δ, γ, α and ε chains, and 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 near the N-terminus of the light and heavy chains where the amino acid sequences vary greatly are called variable regions (V), and the regions near the C-terminus where the amino acid sequences are relatively stable are called constant regions (C). The heavy chain variable region (VH) and the light chain variable region (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 (CDRs), which are ring-shaped structures. The heavy chain CDR and the light chain CDR are closely together and cooperate with each other through the FR region, forming a surface that can complement the three-dimensional structure of the target antigen or epitope, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. The FR region is the more conservative part of VH and VL. They are generally in a β-folded configuration and are connected by three CDRs that form a connecting loop. Each VH and VL is usually composed of three CDRs and four FRs, arranged from the amino terminus to the 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, the accumulation 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 known in the art. As used herein, the Kabat numbering system is used to define.

[0034] The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity of antibodies. The length of CL of different types of Ig (κ or λ) is basically the same, but the length of CH of different classes of Ig is different, such as 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 heavy chain 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", "complete antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

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

[0037] (i) Fab: Antigen-binding fragment (Fab) is a monovalent fragment consisting 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 proteolytic cleavage 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 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: Single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, which is composed of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (generally composed of 10-25 amino acids), which retains the binding specificity of the original antibody to the antigen. The linker in the present invention is not particularly limited as long as it does not hinder the expression of the antibody variable regions connected to its two ends. Compared with full-length antibodies, scFv has the characteristics of small molecular weight, so it has 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 via a linker or the like.

[0043] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include a complementary determining region (CDR) and a framework region (FR) from a rabbit immunoglobulin sequence. In other embodiments, the antibody may include 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 of a non-human antibody such as a rabbit antibody's 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, and this framework sequence is derived from a single or multiple FR sequences of other human antibody variable regions. In the present invention, the CDR region in the chimeric antibody or humanized antibody is derived from a rabbit-derived CDR region.

[0044] The terms "monoclonal antibody" or "single antibody" and the like are used interchangeably and refer to a homogeneous antibody population, i.e., the individual antibodies constituting 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 epitopes on an 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 understandable, the specific implementation methods of the present invention are described in detail below.

[0047] The embodiment of the present invention provides a monoclonal antibody against p21 protein, 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 complementary determining regions (CDRs), which are named CDR1, CDR2 and CDR3, respectively, wherein the amino acid (AA) 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; and the amino acid (AA) 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.

[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, strong anti-interference ability, and has good detection sensitivity and specificity when used for the detection of p21 protein in cells / tissues. Specifically, by immunoblotting, positive cells or tissues expressing p21 protein and negative cells or tissues not expressing p21 protein are detected, and only a unique band is detected in the protein lysate of the positive sample, and the size is consistent with the expectation; by immunohistochemistry, positive and negative tissue samples are detected, and the staining positioning in the positive tissue sample is accurate, the staining is clear, and the background is clean, and there is no non-specific staining in the negative tissue sample, and the detection sensitivity and specificity can reach 100%. It can be seen that the detection result of the antibody of the present invention is highly consistent with the actual situation, the result accuracy and credibility are good, and false positive and false negative results can be effectively avoided. It is suitable for immunodiagnosis analysis of p21 protein in tissues / cells, especially using protein immunoblotting and immunohistochemistry analysis and detection, and has broad application prospects and good market prospects in clinical diagnosis and scientific research detection. Moreover, the antibody has cross-reactivity to homologous p21 proteins of human, rat and mouse, and has certain applicability to 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), and the four FRs and three CDRs are arranged alternately in 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, CL and VL constitute the light chain, and CH and VH constitute the heavy chain. The constant region of the 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 as 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 as 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 maintains 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] 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 above-mentioned monoclonal antibody against p21 protein.

[0054] The nucleic acid molecule can be in the form of DNA (such as cDNA or genomic DNA or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can also be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule can be derived based on 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 any conventional vector in the art, as long as it can hold the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, bacteriophages (such as λgt4λB, λ-Charon, λΔz1 and M13), cosmids and mini-chromosomes. The vector can be a cloning vector (i.e., used to transfer nucleic acid molecules to a host and multiply in a 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 vectors respectively and introduced into the same or different host cells. When the heavy chain and the light chain are expressed in different host cells, each chain can be separated from the host cell expressing it, and the separated heavy chain and light chain are mixed and incubated under suitable conditions to form antibodies. In other embodiments, the nucleic acid molecules of antibodies FL and FH can also be cloned into a vector, and each nucleic acid sequence is connected to a suitable promoter downstream; for example, each nucleic acid sequence encoding the heavy chain and the light chain can be operably connected to different promoters, or the nucleic acid sequence encoding the heavy chain and the light chain can be operably connected 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] The recombinant vector is transfected or transformed into the host cell using conventional techniques. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA are harvested after the exponential growth period and treated with CaCl2 or MgCl2; they can also be transfected 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 a recombinant vector as described above, the antibody can be expressed by culturing under suitable conditions, and then separated to obtain a purified antibody.

[0060] In the present invention, the recombinant vector is transfected or transformed into the host cell using conventional techniques. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl2 or MgCl2. If necessary, microinjection, electroporation or liposome packaging can also be used. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, as well as 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 combined with 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 according to 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 antibody of the present invention can be used alone or combined with a detection label (covalently or non-covalently) to form an antibody conjugate. In some embodiments, the antibody of the present invention is used as an antigen-binding antibody, which specifically recognizes and binds to the p21 protein in the sample to be tested, and then qualitatively or quantitatively detects p21 by analyzing the detection label signal connected 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 molecules that can bind to the primary antibody. For example, if the anti-p21 antibody is a rabbit IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody, thereby qualitatively or quantitatively detecting p21 by analyzing the changes in the detection label signal generated after the secondary antibody specifically binds to the antibody of the present invention.

[0065] 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 as described above, and will not be repeated here.

[0067] The monoclonal antibody of the present invention has cross-recognition effect on multi-species p21 protein, specifically, the p21 protein can be human, rat or mouse p21 protein. The amino acid and nucleotide sequences of p21 protein can be obtained by conventional techniques. Among them, the information of human p21 protein is referred to Uniprot number P38936 or NCBI accession number NP_000380.1. The information of mouse p21 protein is referred to Uniprot number P39689 or NCBI accession number NP_031695.1. The information of rat p21 protein is referred to Uniprot number Q64315 or NCBI accession number 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 as described above, and will not be elaborated here.

[0070] The detection method adopts conventional immunoassay, including but not limited to: enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (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, wherein 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 is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in the Molecular Cloning Laboratory Guide (Fourth Edition) published by Cold Spring Harbor Laboratory, or usually under the conditions recommended by the manufacturer.

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

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

[0076] This embodiment is based on B cell labeling and sorting technology, and directly enriches and separates B lymphocytes that can recognize human p21 protein from the spleen of New Zealand white rabbits immunized with immunogens, and then culture the separated B cells in the form of single cells to obtain monoclonal antibodies. Finally, through genetic engineering recombinant expression technology, the 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 in large quantities to obtain the target antibody. 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 respectively represent light chain complementary determining regions CDR1-3, and HCDR1-3 respectively represent heavy chain complementary determining regions CDR1-3.

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

[0078]

[0079]

[0080] 1. Animal immunization: 2 New Zealand white rabbits were immunized with human p21 protein at 200μg / rabbit. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant to make an emulsifier, and injected subcutaneously at multiple points on the rabbit's abdomen and back. After 3 weeks, 100μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant to make an emulsifier, and injected subcutaneously at multiple points on the rabbit's abdomen and back for two booster immunizations. After three immunizations, rabbit serum samples were collected to determine the serum titer against human p21. Rabbits with high serum titers were selected and boosted with 200μg of the immunogen at multiple points subcutaneously. The spleen of the rabbit was taken three days later.

[0081] The immune serum titer was detected by enzyme-linked immunosorbent assay (ELISA), which includes the following steps: 1) coating human p21 protein: dilute the recombinant p21 protein with 1× PBS buffer, add 25 μL / well to the plate, the coating concentration is 1 μg / mL, and coat overnight at 4°C; 2) blocking: take out the plate coated overnight, add washing buffer (PBS containing 0.05% (v / v) Tween-20) at 75 μL / well, wash 5 times, then add blocking buffer (PBS containing 1% BSA, 0.5% gelatin and 5% sucrose) at 50 μL / well, and block at 37°C for 2 hours. h; 3) Serial dilution and sample loading of the serum to be tested: Repeat the process of 2) washing the plate to clean the well plate, perform serial dilution of the serum to be tested, and use dilution buffer (PBS containing 1% BSA) to dilute the serum starting from 1:1000 and perform three-fold dilution, with a total of 8 serial dilutions. The dilution ratio can be adjusted according to the actual situation; add the serum dilution solution to the well plate at 25 μL / well and incubate at 37°C for 1h; 4) Secondary antibody incubation: Repeat the process of 2) washing the plate to clean the well plate, and add 25 μL / well of horseradish peroxidase-labeled goat anti-rabbit IgG (purchased from Jackson Biotechnology Co., Ltd.) diluted 1:5000 with dilution buffer. ImmunoResearch, catalog number AB_2337938), incubate at room temperature in the dark for 1 hour; 5) terminate the reaction and develop color: repeat 2) plate washing process to clean the well plate, add TMB at 100 μL / well for color development, incubate at 37°C in the dark for 10 minutes, add 0.5 M oxalic acid solution at 100 μL / well to terminate the reaction, measure the light absorption value at 450 nm, use the pre-immune mouse serum as the negative control, and the ratio of the measured value to the control value ≥ 2.1 is positive to judge the titer of the immune serum against human p21 protein.

[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 separating single antigen-specific B lymphocytes from spleen cells (publication number: CN110016462A, publication date: 2019-07-16)" and "A B lymphocyte in vitro culture system and application (publication number: CN111518765A, publication date: 2020-08-11)".

[0083] 3. Cloning of the gene encoding rabbit monoclonal antibody: The supernatant of cultured B lymphocytes was used to identify positive clones by immunogen-coated ELISA. The positive clones of single B lymphocyte culture and proliferation were collected, lysed and then analyzed by Quick-RNA TMRNA was extracted using the MicroPrep kit (purchased from ZYMO, catalog number R1051) and reverse transcribed into cDNA. Using cDNA as a template, the naturally paired rabbit monoclonal antibody VL and VH genes were amplified by PCR. PCR reaction system: 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; amplification program: 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s, and finally 72°C for 5 min. The resulting reaction solution was stored at 4°C. The primer sequences (5'-3') for amplifying the VL and VH genes are as follows, where F and R represent 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 product was sequenced to obtain the VL sequence shown in SEQ ID NO.2 and the VH sequence shown in SEQ ID NO.7; then the IMGT online database (www.imgt.org) was queried to obtain the sequence of the constant region, and 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 on 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, and the expression profile is shown in 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, Light chain constant is the nucleic acid sequence of CL (left figure), and Heavy chain constant is the nucleic acid sequence of CH (right figure). Then, the amplified VL and VH genes were connected to the expression vector pBR322 carrying CL and CH genes linearized with restriction endonucleases XbaI (955bp) and NheI (949bp) respectively by homologous recombination to obtain complete light chain (FL) and heavy chain (FH) gene expression vectors, and the vector construction was successfully verified by sequencing.

[0090] In order to achieve secretory expression of antibodies, signal peptides can be added to the front end of VL and VH genes. The signal peptides can be antibody expression signal peptides commonly used in the art, such as the patent "rabbit monoclonal antibody against human interferon α2 and its application (publication number: CN116063487A, publication date: 2023-05-05)" and the patent "high affinity human IL-5 rabbit monoclonal antibody and its application (publication number: CN115819578A, publication date: 2023-03-21)" VL upstream has a signal peptide "MDTRAPTQLLGLLLLWLPGARC", and VH upstream has a signal peptide "METGLRWLLLVAVLKGVQC". 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 reflected in the antibody sequence of Table 1 of this embodiment.

[0091] The constructed FL and FH expression vectors were co-transfected into 293F cells, and cultured for 72-96 hours after transfection to obtain rabbit monoclonal antibodies that recognize human p21 protein in the culture supernatant. Protein A affinity gel resin (purchased from Tiandi Renhe, catalog number SA023100) was used to purify the target antibody from the culture supernatant. 12% polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify that the antibody purity was ≥95% and the concentration was 1mg / mL. The purified antibody was divided and stored at -20℃ for later use.

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

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

[0094] Positive cells / tissue samples expressing p21 and negative cells / tissue samples not expressing p21 were lysed to obtain protein lysates, and 10% SDS-PAGE electrophoresis was performed. The gel protein band was transferred to the NC membrane in an electrotransfer system according to a conventional method; the membrane was placed in a TBST blocking solution containing 3% skim milk powder and incubated at room temperature for 1 hour, and the anti-p21 protein antibody prepared in Example 1 (primary antibody dilution ratio 1:1000, final concentration 1μg / mL) was added and incubated at room temperature for 1 hour; then the membrane was washed 3-4 times with TBST, and HRP-conjugated Goat anti-Rabbit IgG (H+L) (purchased from Jackson, item number AS014, secondary antibody dilution ratio 1:5000) was added and incubated at room temperature for 1 hour; the membrane was washed again with TBST for 3-4 times; finally, ECL ultrasensitive colorimetric solution was added for development. β-actin protein was used as an internal reference.

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

[0096] From the results of protein immunoblotting analysis, it can be seen that the antibody of the present invention can specifically bind to human p21 protein. Only one clear band is detected in HeLa, MCF7 and 293T cells, and the band size is consistent with the theoretical positioning. At the same time, no band is detected in CDKN1A gene knockout homozygotes, indicating that the antibody has good recognition specificity, can effectively avoid false positive results, and ensure the high credibility of the test results. In addition, the antibody of the present invention can simultaneously recognize p21 proteins in some mouse and rat samples, and has certain applicability to p21 proteins of different species.

[0097] 2. Immunohistochemistry (IHC) of monoclonal antibodies

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

[0099] IHC staining was performed as follows: 1) Sample preparation and baking: paraffin sections were placed in a 56°C constant temperature baking chamber for 30 min; at the same time, dewaxing solution 1 was placed in a 56°C constant temperature box, and paraffin sections and section racks were placed in dewaxing solution 1, and then taken out of the constant temperature box and placed at room temperature. After 5 min, the sections were taken out and immersed in room temperature dewaxing solution 2, and the paraffin sections were immersed in the order of dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3. The sections were placed in the dewaxing solution for 5 min and in the anhydrous ethanol for 3 min; then the sections were washed with running water for 3 min; dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology Trade Co., Ltd.; 2) Antigen repair: 0.01 M Tris-EDTA repair solution (pH 9.0) High-pressure heat repair; 3) Inactivation of endogenous peroxidase: Wash with PBS buffer 3 times, 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 3 times, 3 min each time, remove the buffer on the slice; circle the tissue area to be detected on the slide with an immunohistochemical pen, and add PBS blocking solution in the circled area; place the slice horizontally in an incubation humid box with water at the bottom, start timing 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) on the tissue slice, and incubate at room temperature for 60 min; remove the antibody working solution, quickly rinse once with PBS buffer, and soak and wash 3 times, 3 min each time; 6) Secondary antibody incubation: add ready-to-use secondary antibody working solution (purchased from DAKO, item number K5007) on the tissue slice ), incubate at room temperature for 25 minutes; remove the secondary antibody working solution on the slice, quickly rinse once with PBS buffer, and soak and wash three times, each time for 3 minutes; 7) Color development: add color development working solution to the slice, observe the color change closely under a microscope, and after obtaining the appropriate staining intensity, immerse the slice in a large amount of distilled water to stop the color development, and then wash it in running water for 10 minutes; 8) Counterstaining: immerse the slightly drained tissue slice in Mayer's hematoxylin counterstaining for 1 minute, and wash it with running water for 3 minutes. min; 9) Blueing: Immerse the slightly drained slices in a saturated aqueous solution of lithium carbonate for 3 seconds, and wash with running water for 3 minutes; 10) Dehydration: Immerse the washed slices in anhydrous ethanol once, lift them up and down several times during the soaking period, and take them out after 10 seconds; Dry the slices at high temperature (54-58℃); 11) Sealing: Add an appropriate amount of neutral gum to the center of the slice, and cover it with a coverslip. The amount of glue added should be appropriate. After sealing the coverslip, the tissue should be completely covered, and no glue should overflow. Finally, scan the slices.

[0100] Immunohistochemical staining results are divided into: positive and negative, wherein positive expression must be brown-colored and low or no background in the specific cell antigen position of the corresponding tissue to be considered positive, and negative refers to no brown-colored staining in the cells of the specific tissue. In the present embodiment, human esophagus, human colon, human hepatocellular carcinoma, human lung, mouse placenta and rat colon are positive tissue samples, and positive cells are colon and tonsil cells, and p21 protein subcellular localization is in the nucleus, so the positive result is shown as the aforementioned cells appearing moderate to strong nuclear staining reaction. Normal human liver tissue is a negative sample, without coloring. Figure 3 From left to right and from top to bottom, the IHC staining results of human esophagus, human lung, human colon, human hepatocarcinoma, and human liver are representatively 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 dilution 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 location of p21 in the corresponding samples, the positive staining is located in the cell nucleus, the staining is clear, the background is clean, and 1 negative sample has no nonspecific staining; indicating 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, and 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 protection scope of the present invention.

Claims

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

2. The anti-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 anti-p21 protein monoclonal antibody 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 p21 protein according to any one of claims 1 to 4.

6. An antibody conjugate, characterized in that: The invention comprises the monoclonal antibody against p21 protein as claimed in 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 assay 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 monoclonal antibody against p21 protein according to any one of claims 1 to 4 or the antibody conjugate according to claim 6.

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