Rabbit monoclonal antibody against human PAX8 protein, antibody conjugate and its application

Abstract

The present invention belongs to the technical field of antibody preparation, and in particular, relates to rabbit monoclonal antibodies, antibody conjugates and applications thereof against human PAX8 protein. The amino acid sequences of complementary determining regions CDR1-3 on the light chain variable region of the rabbit monoclonal antibody are shown in SEQ ID NO.3-5, respectively, and the amino acid sequences of complementary determining regions CDR1-3 on the heavy chain variable region are shown in SEQ ID NO.8-10, respectively. The anti-human PAX8 protein antibody of the present invention can specifically recognize the natural PAX8 protein on the surface of cells / tissues, and when used for immunohistochemical staining, the positive staining is obvious, the background is clean, and there is basically no non-specific staining, and the sensitivity and specificity can be as high as 100%, with the advantages of high specificity, good reliability, strong anti-interference ability, high analytical sensitivity and good detection accuracy, which is conducive to avoiding false positive and false negative results.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and in particular to rabbit monoclonal antibodies against human PAX8 protein, antibody conjugates and applications thereof. Background Art

[0002] Paired box (PAX) proteins are a family of transcription factors that play different important roles in animal development. Nine PAX proteins (PAX1-9) have been found in human and other mammalian cells. They have an amino-terminal paired domain, which includes two helix-turn-helix motifs and has DNA binding activity. PAX proteins are divided into four structurally distinct subfamilies (I-IV) based on the presence or absence of a carboxyl-terminal homeodomain and a central octapeptide domain. Subfamily I (PAX1 and 9) contains an octapeptide domain but lacks a homeodomain; subfamily II (PAX2, 5, and 8) contains an octapeptide domain and a truncated homeodomain; subfamily III (PAX3 and 7) contains an octapeptide domain and a complete homeodomain; subfamily IV (PAX4 and 6) contains a complete homeodomain but lacks an octapeptide domain. PAX protein is located in the cell nucleus. It is a type of transcriptional regulatory factor related to early embryonic development and tissue-directed differentiation. It can regulate the transcriptional network responsible for embryonic formation and organ formation. Many members are still expressed after development and maturity, and show certain tissue and cell expression specificity. Therefore, the PAX protein family is a group of valuable molecular markers in molecular pathology. Gene mutations that lead to abnormal expression of PAX genes are related to many cancer subtypes. Among them, members of subfamilies II and III have been found to be potential regulators of tumor progression.

[0003] As a transcription factor required for the expression of thyroid cell-specific genes, PAX8 protein plays a key role in regulating the thyroid-specific gene expression profile and helps maintain the special functions of thyroid cells. Studies have found that Pax-8 gene mutations are associated with thyroid dysplasia, thyroid follicular carcinoma, and atypical thyroid follicular adenomas. In addition, PAX8 plays an important role in the development of kidneys, Müllerian duct-derived tissues (cervix, fallopian tubes, and endometrium), and male reproductive tissues (spermatic vesicles and epididymis). At the same time, PAX8 is also positively expressed in different types of malignant tumors, such as thyroid cancer, renal cancer, epithelial ovarian cancer, and endometrial cancer. Based on the expression characteristics of PAX8, studies have shown that PAX8 is a sensitive and specific marker for primary tumors and metastatic tumors from the thyroid, kidney, and Müllerian duct, and is of great significance in the pathological diagnosis of related diseases.

[0004] At present, immunohistochemistry (IHC) is commonly used in clinical practice to detect the expression of specific proteins in pathological tissue samples. However, the core of IHC is the monoclonal antibody that specifically binds to the protein, and its performance directly determines the sensitivity and specificity of the entire test. Therefore, the development of human PAX8 protein monoclonal antibodies with strong specificity and good anti-interference ability has become an urgent problem to be solved. Summary of the invention

[0005] In view of the problems that the anti-human PAX8 protein antibodies in the prior art have poor specificity and anti-interference ability and are difficult to use for immunological detection, the present invention provides rabbit monoclonal antibodies and antibody conjugates against human PAX8 protein, and further provides the use of the rabbit monoclonal antibodies and antibody conjugates in the preparation of human PAX8 protein immunoassay kits and related immunoassay kits. To achieve the aforementioned objectives, the present invention is specifically implemented through the following technical solutions:

[0006] In a first aspect, the present invention provides a rabbit monoclonal antibody against human PAX8 protein, comprising 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; and 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.

[0007] Furthermore, 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.

[0008] Furthermore, the amino acid sequence of the light chain of the rabbit 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.

[0009] Furthermore, the rabbit 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.

[0010] The second aspect of the present invention provides an antibody conjugate, comprising the rabbit monoclonal antibody against human PAX8 protein as described above, and a detection label connected to the rabbit monoclonal antibody.

[0011] The third aspect of the present invention provides a nucleic acid molecule, a recombinant vector or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule is used to encode the rabbit monoclonal antibody against human PAX8 protein as described above.

[0012] Furthermore, the recombinant vector is a mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell.

[0013] The fourth aspect of the present invention provides the use of the above-mentioned rabbit monoclonal antibody or antibody conjugate against human PAX8 protein in the preparation of a human PAX8 protein immunoassay kit.

[0014] A fifth aspect of the present invention provides a human PAX8 protein immunoassay kit, the kit comprising the rabbit monoclonal antibody or antibody conjugate against human PAX8 protein as described above.

[0015] Furthermore, the kit is an immunohistochemistry kit.

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

[0017] The rabbit monoclonal antibody against human PAX8 protein of the present invention can specifically recognize natural PAX8 protein on the surface of cells / tissues, has good specificity and high affinity, and when used for immunohistochemical staining, the positive staining is obvious, the background is clean, there is basically no non-specific staining, and the sensitivity and specificity can be as high as 100%. It has the advantages of high specificity, good reliability, strong anti-interference ability, high analytical sensitivity and good detection accuracy, etc., which is conducive to avoiding false positive and false negative results, and is suitable for immunodetection of PAX8 protein in various cell / tissue and other pathological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 A map of the mammalian expression vector pRB322 used to construct an anti-human PAX8 protein rabbit monoclonal antibody expression vector in Example 1 of the present invention, from left to right, respectively, vectors carrying the antibody light chain constant region and heavy chain constant region in advance;

[0020] Figure 2 This is a result diagram of the use of anti-human PAX8 protein antibodies to detect the expression of PAX8 in human kidney tissue samples by immunohistochemistry in Example 2 of the present invention;

[0021] Figure 3 This is a result diagram of the use of anti-human PAX8 protein antibodies to detect the expression of PAX8 in human thyroid cancer tissue samples by immunohistochemistry in Example 2 of the present invention;

[0022] Figure 4 This is a graph showing the results of detecting the expression of PAX8 in human thyroid tissue samples by immunohistochemistry using anti-human PAX8 protein antibodies in Example 2 of the present invention. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] The meaning of the terms "include", "comprising", "containing", "having" and the like is non-restrictive, that is, other steps and other ingredients that do not affect the result may be added. The term "and / or" should be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is regarded as including the following situations: (i) A, (ii) B and (iii) A and B.

[0028] The terms "rabbit monoclonal antibody", "monoclonal antibody", "rabbit antibody" and "rabbit monoclonal antibody" and other similar terms have the same meaning and, unless otherwise specified, refer to antibodies that specifically bind to human paired box protein 8 (PAX8). The modifier "rabbit" indicates that the complementarity determining region (CDR) of the antibody is derived from a rabbit immunoglobulin sequence.

[0029] An antibody is an immunoglobulin molecule that can specifically bind to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, as long as they exhibit the desired antigen-binding activity.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] (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.

[0036] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0037] (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.

[0038] (iv) (Fv)2: consists of two Fv fragments covalently linked together.

[0039] (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.

[0040] (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.

[0041] 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 mouse 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 mouse-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 mouse antibody framework (FR) sequence, and this framework sequence is derived from a single or multiple other mouse antibody variable region framework sequences. In the present invention, the CDR region in the chimeric antibody or humanized antibody is derived from a rabbit-derived CDR region.

[0042] 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.

[0043] 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.

[0044] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0045] An embodiment of the present invention provides a rabbit monoclonal antibody against human PAX8 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 both include three complementary determining regions (CDRs), respectively named CDR1, CDR2 and CDR3, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0046] The present invention adopts the 250-280aa amino acid fragment of human PAX8 protein as an immunogen to prepare a rabbit-derived monoclonal antibody against human PAX8 protein. The obtained antibody can specifically recognize the natural PAX8 protein on the surface of cells / tissues, has good specificity and high affinity. The antibody of the present invention is used as a primary antibody, and multiple positive samples expressing PAX8 protein and negative samples not expressing PAX8 protein are detected by immunohistochemical staining. The results show that the positive samples are obviously stained, the background is clean, and there is basically no non-specific staining, and there is no staining in all negative samples. The sensitivity and specificity can reach 100%, indicating that the antibody has high specificity, good reliability, excellent anti-interference ability and analytical sensitivity in immunodetection of PAX8 protein, good detection accuracy, and can effectively avoid false positive and false negative results, providing an antibody raw material with excellent performance for high-quality immunodetection, and is suitable for immunodetection of PAX8 protein in various cell / tissue pathological samples, especially suitable for immunohistochemical detection of tissue sections.

[0047] 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 antibody of the present invention is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.7.

[0048] Optionally, the antibody of the present invention further comprises a light chain constant region and a heavy chain constant region, CL and VL constitute a complete light chain (FL), and CH and VH constitute a complete heavy chain (FH). The constant region of an antibody can usually be obtained by public query, such as: searching for rabbit IgG gamma C reign to obtain CH through the IMGT online database (www.imgt.org), and searching for rabbit IgG Kappa C reign to obtain CL. Specifically, the amino acid sequence of the antibody light chain is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain is shown in SEQ ID NO.6, and the antibody is of IgG type.

[0049] Optionally, the antibody is 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 of the antibody. 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.

[0050] Another embodiment of the present invention provides an antibody conjugate, comprising the rabbit monoclonal antibody against human PAX8 protein as described above, and a detection label connected to the rabbit monoclonal antibody.

[0051] The detection label is used to generate a recognizable signal change to identify the sample to be tested according to the signal change. The detection label includes, but is not limited to: biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (such as allophycocyanin, phycoerythrin, PerCP and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies or combinations thereof.

[0052] Another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule is used to encode the rabbit monoclonal antibody against human PAX8 protein as described above.

[0053] Nucleic acid molecules 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.

[0054] The sequence of the nucleic acid molecule can be derived from the antibody AA sequence by conventional means such as codon coding rules. The full-length sequence of the nucleic acid molecule or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. The obtained nucleic acid molecule is inserted into an expression vector, then introduced into a host cell, and cultured under specific conditions to express the antibody.

[0055] The original vector for constructing the recombinant vector is any conventional vector in the art, as long as it can carry 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 molecules of the present invention can be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule. This is a well-known technique in the art and will not be described in detail herein.

[0056] The nucleic acid molecules encoding the antibodies FL and FH of the present invention can be inserted into two vectors, respectively, which can be introduced into the same or different host cells. When the heavy chain and the light chain are expressed in different host cells, each chain can be separated from the host cell expressing it, and the separated heavy chain and light chain are mixed and incubated under 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.

[0057] 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.

[0058] 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.

[0059] In a preferred embodiment, the recombinant vector is a mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell (293F cell).

[0060] Another embodiment of the present invention provides the use of the rabbit monoclonal antibody or antibody conjugate against human PAX8 protein as described above in the preparation of a human PAX8 protein immunoassay kit.

[0061] The advantages of using the rabbit monoclonal antibody or antibody conjugate against human PAX8 protein in preparing a human PAX8 protein immunoassay kit are the same as the advantages of the rabbit monoclonal antibody against human PAX8 protein over the prior art as described above, and will not be repeated here.

[0062] Based on the same inventive concept, an embodiment of the present invention further provides a human PAX8 protein immunoassay kit, which includes the rabbit monoclonal antibody or antibody conjugate against human PAX8 protein as described above.

[0063] It should be noted that the antibody of the present invention can be used alone or combined or coupled with a detection marker 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 PAX8 protein in the sample to be tested, and then produces a recognizable signal change through the detection marker connected thereto to achieve qualitative or quantitative detection of the PAX8 protein. In other embodiments, the anti-human PAX8 protein antibody (as a primary antibody or capture antibody) is not labeled, and the detection marker 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-human PAX8 protein antibody is a rabbit IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody, thereby generating a recognizable signal change after the secondary antibody coupled to the detection marker specifically binds to the antibody of the present invention, thereby achieving qualitative or quantitative detection.

[0064] The above-mentioned immunoassay methods include but are not limited to: enzyme immunoassay (EIA), enzyme linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), Western blot (WB), immunoprecipitation (IP) and flow cytometry (FC).

[0065] In a preferred embodiment, the immunodetection method is an immunohistochemistry method, and the kit is an immunohistochemistry kit.

[0066] 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.

[0067] Example 1 Preparation of rabbit monoclonal antibody against human PAX8 protein

[0068] The paired box 8 (PAX8) protein 250-280aa amino acid fragment was used as a hapten and coupled to the carrier protein - hemocyanin (KLH) to form a complete antigen. New Zealand white rabbits were immunized, and then single antigen-specific B lymphocytes were selected based on the monoclonal antibody development technology of single B lymphocyte screening and culture. The antibody encoding gene in the cell was amplified by PCR technology, recombinant expression was performed, and the target antibody strain was screened.

[0069] The antibody sequencing was completed by Jinkairui Biotechnology Co., Ltd. The amino acids (AA) of the antibodies are shown in Table 1 , where LCDR1-3 represent the complementary determining regions CDR1-3 on the light chain, and HCDR1-3 represent the complementary determining regions CDR1-3 on the heavy chain.

[0070] Table 1 Sequence information of rabbit monoclonal antibodies in this example

[0071]

[0072] 1.1 Antigen preparation

[0073] The hapten used is the 250-280aa fragment of the PAX8 protein. The amino acid sequence of the full-length PAX8 protein can be found in Uniprot protein number: Q06710, GeneID number: 7849, NCBI protein accession number: NP_003457.1, NCBI gene accession number: NM_003466.3). The sequence of the 250-280aa fragment of the PAX8 protein is as follows:

[0074] ASPSHTKGEQGLYPLPLLNSTLDDGKATLTP (see SEQ ID NO. 11).

[0075] The preparation of the complete antigen by coupling the 250-280aa fragment of PAX8 protein to hemocyanin (KLH) includes the following steps:

[0076] 1) Preparation of SMCC solution: Dissolve 50 mg SMCC in 10 mL 1× PBS and heat in a 55°C water bath for 5-6 min to prepare a 5 mg / mL SMCC solution;

[0077] 2) KLH and thiol coupling: KLH was weighed according to the total mass of the polypeptide to be coupled, polypeptide: KLH = 2:1, dissolved in 1×PBS, the concentration was kept at 3 mg / mL, and shaken well. Then, according to the mass of KLH, an appropriate volume of SMCC solution was taken, KLH: SMCC = 10:1.5, and added to the dissolved KLH. At room temperature, it was placed on a rotating incubator for mixing and shaking for 1 hour to activate; 1×PBS (pH 7.4) was used at a volume ratio of V PBS :V KLH =15:1 dialyzed for 1 h to remove free SMCC;

[0078] 3) Activated KLH coupled hapten: Add 100 μL 1×PBS to the small tube containing the hapten polypeptide for trial dissolution. If it cannot be completely dissolved, use 8M urea to dissolve the protein and prepare a 10 mg / mL polypeptide solution, which is slowly added to the activated KLH (dialysis completed) in multiple times. Place it in a small shaker or rotary incubator at the lowest speed and observe it every half an hour. If precipitation occurs, continue to add urea and incubate at room temperature for 4 hours or at 4°C overnight.

[0079] 1.2 Animal Immunization

[0080] Four New Zealand white rabbits were immunized with a 250-280aa fragment of the PAX8 protein coupled to KLH; each rabbit was immunized with 300 μg, and before the first immunization, the antigen was mixed with an equal amount of complete Freund's adjuvant to form an emulsifier, which was injected subcutaneously at multiple points in the rabbit's abdomen and back, and booster immunization was performed twice; 3 weeks later, 150 μg of the antigen was mixed with an equal amount of incomplete Freund's adjuvant to form an emulsifier, which was injected subcutaneously at multiple points in the rabbit's abdomen and back, and after three immunizations, rabbit serum samples were collected, and the titer against human PAX8 protein was determined by enzyme-linked immunosorbent assay (ELISA), and the serum after the last immunization was purified into antibodies, and endogenous samples (including positive tissue samples expressing PAX8 protein and negative tissue samples not expressing PAX8 protein) were detected by immunohistochemistry, and rabbits with high serum titers and the best endogenous detection were taken, and 150 μg of the immunogen was injected subcutaneously at multiple points for booster immunization once, and the animals were sacrificed three days later and the spleen was taken.

[0081] 1.3. Isolation and sorting of B lymphocytes from spleen

[0082] Conventional methods are used to isolate B lymphocytes from the spleen, and antigen-specific B lymphocytes are sorted. For related methods, please refer to the published patents "Method for efficiently isolating 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] 1.4. Cloning of the gene encoding rabbit monoclonal antibody

[0084] The supernatant of cultured B cells was used to identify positive clones by antigen-coated ELISA. The cells of positive clones were collected and lysed and then analyzed by Quick-RNA TM RNA was extracted using a Micro Prep kit (purchased from ZYMO, catalog number R1051) and reverse transcribed into cDNA. The reverse transcription system included: 1 μL of Oligo(dT)12-18primer(Life), 1 μL of dNTPs(10 mM), and 1 μL of RNA; after PCR at 65°C for 5 min, 4 μL of 5×FS Buffer (from ABconal), 1 μL of DTT(100 mM), 1 μL of RNase OUT(40 U / μL), and 1 μL of ABScriptⅡRT(200 U / μL, from ABconal) were added to the above product, mixed well, and reacted at 42°C for 1 h and 85°C for 5 min to obtain cDNA.

[0085] Using the aforementioned cDNA as a template, the naturally paired rabbit monoclonal antibody light chain variable region (VL) and heavy chain variable region (VH) were amplified from the cDNA of the corresponding positive clones by PCR and sequenced. The PCR reaction system included: 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 PCR amplification program included: 98°C pre-denaturation 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 kept at 72°C for 5 min, and the resulting reaction solution was stored at 4°C. The primer sequences (5'-3') for amplifying VL and VH genes are shown below, where F and R represent forward primers and reverse primers, respectively.

[0086] VL-Primer-F: 5'-tgaattcgagctcggtacccatggacacgagggccccac-3' (SEQ ID NO. 12);

[0087] VL-Primer-R: 5'-cacacacgatggtgactgttccagttgccacctgatcag-3' (SEQ IDNO.13);

[0088] VH-Primer-F: 5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3' (SEQ IDNO.14);

[0089] VH-Primer-R: 5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3' (SEQ ID NO. 15).

[0090] 1.5. Expression and large-scale production of rabbit monoclonal antibodies

[0091] In order to obtain multiple rabbit monoclonal antibodies that recognize human PAX8 protein, the mammalian expression vector pBR322 carrying the light chain constant region (CL) and heavy chain constant region (CH) genes was linearized with XbaI and NheI restriction enzymes respectively. The VL and VH genes containing the signal peptide amplified by the above PCR were purified and then constructed into the above expression vector by homologous recombination to obtain an expression vector containing complete light chain and heavy chain genes. The successful construction of the expression vector was verified by sequencing. The map of the mammalian expression vector pBR322 carrying the rabbit monoclonal antibody light chain constant region (CL) and heavy chain constant region (CH) genes used is shown in Figure 1In the figure, pBR322 origin and f1 origin 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 the light chain constant region (left figure), and Heavy chain constant is the nucleic acid sequence of the heavy chain constant region (right figure). CL and CH genes were obtained by searching the IMGT online database (www.imgt.org) to search for rabbit IgG gamma C reign to obtain CH, and rabbit IgG Kappa C reign to obtain CL.

[0092] The signal peptide of this embodiment can adopt the antibody expression signal peptide sequence commonly used in the art, such as the patent "Anti-human interferon α2 rabbit monoclonal antibody 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)". The light chain variable region upstream has a signal peptide "MDTRAPTQLLGLLLLWLPGATF", and the heavy chain variable region upstream has a signal peptide "METGLRWLLLVAVLKGVQC".

[0093] The constructed expression vectors containing the light chain (FL) gene and the heavy chain (FH) gene were transfected into 293F cells together. After transfection, the cells were cultured for 72-96 hours to obtain the culture supernatant containing the recombinant rabbit monoclonal antibody that recognizes the human PAX8 protein. The target antibody was purified from the culture supernatant using proteinA affinity gel resin (purchased from Tiandi Renhe, catalog number SA015100). The antibody concentration was 0.57 mg / mL and the purity was greater than 95% using 4-12% SDS-PAGE gradient gel electrophoresis. The purified antibody was aliquoted and stored at -20°C for later use.

[0094] Example 2 Immunohistochemical Analysis (IHC) of Rabbit Monoclonal Antibodies Against Human PAX8 Protein

[0095] Human PAX8 protein is mainly expressed in thyroid acinar cells in thyroid tissue and renal tubular cells in kidney tissue, and subcellular localization is in the nucleus. In this example, the positive samples expressing PAX8 protein selected include human kidney and thyroid cancer tissues, a total of 2 positive samples, and the negative samples not expressing PAX8 protein selected include human heart, liver, spleen, lung, brain, testis, skin, tonsil, lung cancer, colon, placenta, cervix and cervical cancer tissues, a total of 13 negative samples.

[0096] Immunohistochemistry (IHC) staining specifically includes the following steps:

[0097] 1) Sample preparation and baking: Place the paraffin sections on the slice rack in the same direction and bake them in a 56°C thermostat for 30 min. At the same time, place a cylinder of dewaxing solution (purchased from Wuxi Jiangyuan Industrial Technology Trade Co., Ltd.) in the 56°C thermostat.

[0098] 2) Dewaxing to water: Put the paraffin sections together with the section rack into the dewaxing liquid 1 cylinder, then take them out of the thermostat and place them at room temperature. After 5 minutes, take out the sections and immerse them in the room temperature dewaxing liquid 2 cylinder, and put the paraffin sections into the cylinders in the order of dewaxing liquid 2, dewaxing liquid 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3. When placed in the dewaxing liquid reagent cylinder, each cylinder is placed for 5 minutes, and when placed in the anhydrous ethanol reagent cylinder, each cylinder is placed for 3 minutes; wash the sections with running water for 3 minutes;

[0099] 3) Antigen retrieval: high pressure heat retrieval with 0.01 M sodium citrate retrieval solution (pH 9.0);

[0100] 4) Inactivation of endogenous peroxidase: Wash with PBS buffer three times, 1 min each time, remove the buffer on the slices; immerse the slices completely in 3% hydrogen peroxide solution and incubate at room temperature for 10 min;

[0101] 5) Blocking: Wash with PBS buffer 3 times, 3 minutes each time, and remove the buffer on the slices; circle the tissue area to be tested on the slide with an immunohistochemical pen, and add PBS blocking solution to the circled area; place the slices horizontally in an incubation wet box with water at the bottom, and incubate at room temperature for 30 minutes, starting from the time when the blocking solution is added;

[0102] 6) Primary antibody incubation: remove the blocking solution, add the anti-PAX8 protein rabbit monoclonal antibody (primary antibody working concentration 0.633 μg / mL) diluted 1:900 with antibody diluent-PBS working solution on the tissue section, place it horizontally in the incubation wet box, and incubate it at room temperature for 60 minutes; remove the antibody working solution, rinse quickly with PBS buffer once, and soak and wash three times with PBS buffer, each time for 3 minutes; during the soaking and washing period, it is necessary to repeatedly pull up and down several times;

[0103] 7) Secondary antibody incubation: Add ready-to-use secondary antibody working solution (purchased from Jackson, catalog number AS074) to the tissue sections and place them horizontally in an incubation humidified box for incubation at room temperature for 25 minutes; remove the secondary antibody working solution from the sections, quickly rinse once with PBS buffer, and soak and wash three times with PBS buffer, each time for 3 minutes; during the soaking and washing period, lift up and down repeatedly;

[0104] 8) Color development: Add color developing solution to the slices, 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 terminate the color development. After the color development is terminated, wash the slices in running water for 10 minutes;

[0105] 9) Counterstaining: Immerse the slightly drained tissue sections in Mayer's hematoxylin for counterstaining for 1 minute, then wash with running water for 3 minutes;

[0106] 10) Blueing: Immerse the slightly drained slices in a saturated aqueous solution of lithium carbonate for 3 seconds, and then rinse with running water for 3 minutes;

[0107] 11) Dehydration: Soak 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; place them in a constant temperature air drying oven at high temperature (54-58°C) to completely dry;

[0108] 12) 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 the coverslip is added, the tissue should be completely covered and no glue should overflow;

[0109] 13) Perform slice scanning.

[0110] Immunohistochemical staining results are divided into: positive and negative. Positive expression must be brown in the specific antigenic sites of cells and tissues, and the background color of cells and tissues that should not be stained in theory is low to be considered positive. Blue staining indicates nuclear localization. For example, in the positive sample renal tissue, the nuclei of renal tubular cells highly express PAX8 protein, and the corresponding parts appear brown after IHC staining (see Figure 2 ); In positive sample thyroid cancer tissue, the nuclei of thyroid acinar cells highly expressed PAX8 protein, and the corresponding parts appeared brown after IHC staining (see Figure 3 ); PAX8 protein was not expressed in the negative sample thyroid tissue, and the whole body was not stained brown (see Figure 4 ).

[0111] The monoclonal antibody against human PAX8 protein prepared in Example 1 was used to detect 2 positive samples, all of which were positively stained, with a sensitivity of 2 / 2=100%, and 13 negative samples were negatively stained, with a specificity of 13 / 13=100%. Specifically, among the positive control samples, 1 / 1 human kidney and 1 / 1 human thyroid cancer were positive; among the negative control samples, 1 / 1 human heart, 1 / 1 human skin, 1 / 1 human liver, 1 / 1 human spleen, 1 / 1 human tonsil, 1 / 1 human lung, 1 / 1 human lung cancer, 1 / 1 human brain, 1 / 1 human testis, 1 / 1 human colon, 1 / 1 human placenta, 1 / 1 human cervix, and 1 / 1 human cervical cancer were negative. The results showed that the anti-human PAX8 rabbit monoclonal antibody staining was accurate in positioning, clear in staining, without nonspecific staining, and with a clean background, which could effectively avoid false positive results and meet the immunohistochemical detection of pathological samples.

[0112] 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 rabbit monoclonal antibody against human PAX8 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 rabbit monoclonal antibody against human PAX8 protein 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 rabbit monoclonal antibody against human PAX8 protein according to claim 2, characterized in that: The amino acid sequence of the light chain of the rabbit 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 rabbit monoclonal antibody against human PAX8 protein according to claim 1, characterized in that: The rabbit 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. An antibody conjugate, characterized in that: The method comprises the rabbit monoclonal antibody against human PAX8 protein according to any one of claims 1 to 4, and a detection label connected to the rabbit monoclonal antibody.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the rabbit monoclonal antibody against human PAX8 protein according to any one of claims 1 to 4.

7. Use of the rabbit monoclonal antibody against human PAX8 protein according to any one of claims 1 to 4, or the antibody conjugate according to claim 5, in the preparation of a human PAX8 protein immunoassay kit.

8. A human PAX8 protein immunoassay kit, characterized in that: Comprising the rabbit monoclonal antibody against human PAX8 protein according to any one of claims 1 to 4, or the antibody conjugate according to claim 5.

9. The human PAX8 protein immunoassay kit according to claim 8, characterized in that: The kit is an immunohistochemistry kit.

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