Monoclonal antibodies against human transcription factor pu.1 and uses thereof

Abstract

The application belongs to the technical field of antibody preparation, and particularly relates to a monoclonal antibody against human transcription factor PU.1 and application thereof.The amino acid sequences of CDR1-3 on the light chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of CDR1-3 on the heavy chain variable region are respectively shown as SEQ ID NO.8-10.The antibody provided by the application can specifically and sensitively recognize human transcription factor PU.1 expressed in human cells / tissues, has good affinity to PU.1, is excellent in anti-interference capability, and has no cross reaction with homologous proteins of other species, which is beneficial to improving the accuracy and reliability of immune detection of human PU.1, improving the coincidence degree of detection results and actual conditions, effectively avoiding false positive or false negative results, and providing an effective antibody tool and detection means for high-specificity, high-accuracy and high-sensitivity detection of PU.1 protein.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation technology, and in particular to monoclonal antibodies against human transcription factor PU.1 and their applications. Background Technology

[0002] PU.1, also known as SPI1, Spi.1, SPI-1, SPI-A, OF, or SFPI1, belongs to the ETS (E26 transformation specific) transcription factor family. Its structure includes an amino-terminal transactivation domain (TAD), a PEST region, and a carboxyl-terminal ETS region. PU.1 can recognize and bind to purine-rich PU-box sequences (such as 5'-GAGGAA-3') near the promoter of target genes through its ETS domain, regulating the transcription of target genes. It can also recruit other transcription factors and cofactors, influencing the expression of target genes.

[0003] PU.1 is primarily expressed in hematopoietic cells, including monocytes, macrophages, B lymphocytes, neutrophils, mast cells, and erythroblasts, playing a crucial role in the differentiation and development of myeloid and lymphoid cells. High levels of PU.1 expression favor the differentiation of macrophages, neutrophils, and monocytes, while low levels favor B lymphocyte differentiation. It is not expressed in mature megakaryocytes, erythrocytes, or T lymphocytes. Different levels of PU.1 expression determine the different functional cell types that hematopoietic stem cells ultimately differentiate into. Abnormal PU.1 expression can directly lead to abnormal differentiation or dysfunction of hematopoietic cells, triggering related diseases such as acute myeloid leukemia (AML), Kostmann syndrome, and myelodysplastic syndrome (MDS). Furthermore, the transcription factor PU.1 regulates the expression of cytokines, receptors, and effector enzymes secreted by immune cells during inflammatory responses, helping the body resist pathogen infection and maintain immune homeostasis. Recent research has found that PU.1 is significantly expressed in various lymphomas and is associated with the development of diseases such as mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, diffuse large cell lymphoma, and diffuse large B-cell lymphoma. Therefore, PU.1 / SPI1 has become a potential diagnostic and therapeutic target for various diseases. Clinical detection technologies targeting this protein and targeted therapies may play an important role in the prevention and control of related diseases.

[0004] Immunoassay methods, including immunoblotting, immunofluorescence, immunohistochemistry, and flow cytometry, utilize the specific binding properties of antigens and antibodies to quantitatively or qualitatively detect characteristic antigens. The core material in these methods is the antibody, and its performance directly determines the accuracy and reliability of the test results. Currently, there are few commercially available antibodies against human PU.1 / SPI1 proteins suitable for cell and / or tissue detection applications, and those that exist suffer from relatively weak affinity, low specificity, and low detection sensitivity, making them unsuitable for clinical testing. Given the roles and functions of PU.1 / SPI1 in various related diseases, developing a widely applicable, highly efficient, and specific antibody targeting human PU.1 / SPI1 is of significant practical importance for achieving immunoassay detection of human PU.1 / SPI1 proteins and / or developing more effective pathological diagnostic techniques for diseases related to PU.1 / SPI1 abnormalities. Summary of the Invention

[0005] To address the lack of monoclonal antibodies capable of specifically and sensitively binding to human transcription factor PU.1 in existing technologies, this invention provides a monoclonal antibody against human transcription factor PU.1, and further provides the application of this antibody or its antibody-conjugate in the preparation of a human transcription factor PU.1 immunoassay kit and related immunoassay kits. To achieve the aforementioned objectives, this invention specifically utilizes the following technical solutions:

[0006] The first aspect of the present invention provides a monoclonal antibody against human transcription factor PU.1, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown in SEQ ID NO.3-5, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8-10, 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 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 monoclonal antibody is a full-length antibody or its antigen-binding region; the antigen-binding region is selected from at least one of the Fab fragment, F(ab)2 fragment, Fv fragment, (Fv)2 fragment, scFv fragment, and sc(Fv)2 fragment.

[0010] A second aspect of the present invention provides a nucleic acid molecule that encodes a monoclonal antibody against human transcription factor PU.1 as described above.

[0011] Furthermore, the nucleic acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.12 or a complementary sequence thereto, and the nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO.14 or a complementary sequence thereto.

[0012] Furthermore, the nucleic acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO.11 or is complementary to it, and the nucleic acid sequence of the heavy chain is as shown in SEQ ID NO.13 or is complementary to it.

[0013] A third aspect of the present invention provides the use of the monoclonal antibody against human transcription factor PU.1 or its antibody-drug conjugate as described above in the preparation of a human transcription factor PU.1 immunoassay kit, wherein the antibody-drug conjugate is formed by conjugating the monoclonal antibody with a detection label.

[0014] Furthermore, the immunoassay kit is selected from immunoblotting kits, immunofluorescence kits, immunohistochemistry kits, or flow cytometry kits.

[0015] A fourth aspect of the present invention provides an immunoassay kit for human transcription factor PU.1, the immunoassay kit comprising a monoclonal antibody against human transcription factor PU.1 as described above or an antibody-drug conjugate thereof, wherein the antibody-drug conjugate is formed by conjugating the monoclonal antibody with a detection label.

[0016] Furthermore, the immunoassay kit also includes a fluorescently conjugated anti-rabbit IgG secondary antibody.

[0017] The advantages and positive effects of this invention are as follows:

[0018] The antibody provided by this invention can specifically and sensitively recognize the naturally expressed human transcription factor PU.1 (PU.1 / SPI1) in human cells / tissues. It does not specifically react with complex non-target antigen components in cells / tissues, has good affinity for PU.1 / SPI1 protein, excellent anti-interference ability, and no cross-reactivity with homologous proteins of other species. This is beneficial to improving the accuracy and reliability of immunodetection of human PU.1 / SPI1, increasing the degree of agreement between the detection results and the actual situation, and effectively avoiding false positive or false negative results. It provides an effective antibody tool and detection method for high-specificity, high-accuracy, and high-sensitivity detection of PU.1 / SPI1 protein, and has good applicability in multiple immunodetection systems such as immunoblotting, immunofluorescence, immunohistochemistry, and flow cytometry. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a graph showing the results of detecting the immune serum titer of rabbits immunized with human transcription factor PU.1 in Example 1 of the present invention;

[0021] Figure 2 The image shows the flow cytometry results of human THP-1 cell samples combined with immune serum from rabbits immunized with human transcription factor PU.1 in Example 1 of this invention. From left to right, the serum dilutions are 1:500 and 1:2000, respectively.

[0022] Figure 3 The image shows the flow cytometry results of human Jurkat cell samples combined with immune serum from rabbits immunized with human transcription factor PU.1 in Example 1 of this invention. From left to right, the serum dilutions are 1:500 and 1:2000, respectively.

[0023] Figure 4 This is a map of the monoclonal antibody expression vector used to construct the anti-human transcription factor PU.1 in Example 1 of the present invention. From left to right, it is the pRB322 vector pre-loaded with the antibody light chain constant region and heavy chain constant region.

[0024] Figure 5 This is a graph showing the immunoblotting results of human positive and negative cell / tissue samples using a monoclonal antibody against human transcription factor PU.1 in Example 2 of the present invention.

[0025] Figure 6 This is an image showing the immunofluorescence detection results of human THP-1, HEL, and spleen cells using a monoclonal antibody against human transcription factor PU.1 in Example 2 of the present invention.

[0026] Figure 7 This is an image showing the immunohistochemical detection results of human follicular lymphoma, human tonsil, and human appendix tissue sections obtained by using a monoclonal antibody against human transcription factor PU.1 in Example 2 of the present invention.

[0027] Figure 8 This is a flow cytometry result of human HAP1 and THP-1 cell samples obtained using a monoclonal antibody against human transcription factor PU.1 in Example 2 of this invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0029] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0030] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, or other quantities should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0031] Additionally, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0032] The terms “including,” “contains,” “includes,” “has,” and similar words are non-restrictive and can include other steps and other components that do not affect the result.

[0033] The term “and / or” should be considered 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 considered to include (i) A, (ii) B, and (iii) A and B.

[0034] The terms "rabbit monoclonal antibody," "monoclonal antibody," "rabbit-derived antibody," and "rabbit monoclonal antibody," etc., have the same meaning and, unless otherwise specified, refer to antibodies that specifically bind to the human transcription factor PU.1. The terms "transcription factor PU.1," "PU.1," "SPI1," "PU.1 / SPI1," "SPI1 / PU.1," "Spi.1," and "SPI-1," etc., have the same meaning. The modifier "rabbit" indicates that the antibody's complementarity-determining region (CDR) is derived from a rabbit immunoglobulin sequence.

[0035] An antibody is an immunoglobulin molecule that specifically binds to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In this invention, the term "antibody" is to be interpreted in the broadest sense and includes various antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and their genetic or chemical modifications, provided they exhibit the desired antigen-binding activity. An antibody fragment may be one or more portions or fragments of a full-length antibody, retaining the antibody's ability to specifically bind to a target antigen.

[0036] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be classified into two types: κ chains and λ chains; heavy chains can be classified into five types: μ, δ, γ, α, and ε chains, with antibodies defined as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of both the heavy and light chains vary considerably, while the amino acid sequences of other parts are relatively constant. The regions with significant amino acid sequence variation near the N-terminus in both the light and heavy chains are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The variable regions of the heavy chain (VH) and light chain (VL) are usually the most variable parts of the antibody and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (HVR) and framework regions (FR). The hypervariable region, also known as the complementarity-determining region (CDR), is a ring structure. Heavy chain CDRs and light chain CDRs are tightly joined together by the FR region and cooperate to form a surface that is complementary to the three-dimensional structure of the target antigen or epitope, determining the antibody's specificity and serving as the site for antibody recognition and antigen binding. The FR regions are the more conserved parts of the VH and VL, generally exhibiting a β-sheet configuration, linked by three CDRs forming a connecting loop. Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0037] CDRs and FRs can be identified according to Kabat definitions, Chothia definitions, the sum of Kabat and Chothia definitions, AbM definitions, contact definitions, IMGT unique numbering definitions and / or conformational definitions, or any CDR determination method known in the art. As used in this invention, they are defined by the Kabat numbering system.

[0038] The light chain constant region (CL) and heavy chain constant region (CH) do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The CL lengths of different Ig types (κ or λ) are generally consistent, but the CH lengths differ among Ig classes. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well-known in the art and can be obtained by searching the IMGT database.

[0039] Full-length antibodies are the most complete antibody molecular structures, with a typical Y-type molecular structure. Therefore, in the context of this invention, "full-length antibody," "complete antibody," and "Y-type antibody" have the same meaning and can be used interchangeably.

[0040] An antibody fragment is one or more portions or segments of a full-length antibody that substantially retain the same biological function or activity as the full-length form. Specifically, an antibody fragment includes at least the same CDR region as the full-length antibody, and more preferably the same variable region, thereby retaining complete antigen recognition and binding sites, enabling it to bind to the same antigens, especially the same epitopes, as the full-length antibody. Typical examples of antibody fragments include Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, and sc(Fv)2, which can be obtained using conventional techniques in the art.

[0041] (i) Fab: An antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable and constant regions) and a portion of a heavy chain (variable and first constant region). Fragments such as Fab, F(ab')2, and Fab' can be obtained by protease cleavage of a full-length antibody. 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. Because Fab possesses an antigen-binding region and a portion of a constant region, it not only has antibody-antigen affinity and excellent tissue penetration like scFv, but also has a more stable structure.

[0042] (ii)F(ab)2: Contains a bivalent segment consisting of two Fabs connected by a disulfide bridge in the hinge region.

[0043] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment. It contains only the variable region and consists of a variable region of one light chain and one heavy chain. It is a non-covalently bound dimer of VH and VL (VH-VL dimer). The three CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind antigens, although the affinity is lower than that of the intact antibody.

[0044] (iv)(Fv)2: Consists of two Fv segments covalently linked together.

[0045] (v)scFv: A single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, consisting of a heavy chain variable region (VH) and a light chain variable region (VL) linked by a flexible linker (typically composed of 10-25 amino acids). It retains the original antibody's specificity for binding to the antigen. The linker in this invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked to its two ends. Compared to full-length antibodies, scFv has a smaller molecular weight, thus exhibiting higher penetration and lower immune side effects.

[0046] The (vi)sc(Fv)2 segment is formed by connecting two heavy chain variable regions and two light chain variable regions through a joint, etc.

[0047] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include a CDR region and a FR region derived from a rabbit immunoglobulin sequence. In other embodiments, the antibody may contain amino acid residues encoded by a non-rabbit immunoglobulin sequence, such as humanized antibodies, chimeric antibodies, etc., to reduce the body's rejection response while maintaining the desired specificity and affinity. The term "chimeric antibody" refers to an antibody in which a portion is derived from a specific source or species, while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody containing the CDR region of a non-human antibody, such as a rabbit antibody, and a FR region derived from a human antibody. In some cases, the variable region of the non-human antibody binds to the constant region of a human antibody, such as in human-rabbit chimeric antibodies; in other cases, the CDR region of the non-human antibody binds to both the FR region and the constant region derived from a human antibody sequence, i.e., grafting the CDR region of the non-human antibody onto a human antibody frame (FR) sequence derived from the FR sequence of one or more other human antibody variable regions. In this invention, the CDR region in the chimeric antibody or humanized antibody is derived from the rabbit CDR region.

[0048] The terms "monoclonal antibody" or similar terms are used interchangeably and refer to a homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for a small number of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). A "monoclonal antibody" is highly specific, exhibiting 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 group of antibodies and should not be interpreted as limiting the source or method of preparation of the antibody. This antibody can be prepared by a variety of methods, including but not limited to hybridoma, phage display, yeast display, recombinant DNA, single-cell screening, or single-cell sequencing.

[0049] The term “specific binding” is a well-known term in the art. A molecule exhibits “specific binding” if it reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity for a particular target antigen or epitope than it reacts with other target antigens or epitopes. “Specific binding”, or “preferred binding”, does not necessarily require (although may include) exclusive binding.

[0050] To make the objectives, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0051] This invention provides a monoclonal antibody against human transcription factor PU.1, comprising a light chain variable region and a heavy chain variable region. Both the light chain variable region and the heavy chain variable region include three complementarity-determining regions (CDRs), named CDR1, CDR2, and CDR3, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0052] This invention uses a synthetic polypeptide corresponding to the 1-100 amino acid sequence of human PU.1 / SPI1 as an immunogen to immunize rabbits to prepare monoclonal antibodies. After obtaining single B lymphocytes, the antibody gene is amplified and cloned by PCR, constructed into an expression vector, and finally expressed through a mammalian expression system to obtain the desired antibody, i.e., a monoclonal antibody against human transcription factor PU.1 (PU.1 / SPI1). This antibody exhibits good performance. Rabbits have large spleens, high gene diversity, and unique advantages in small-molecule antigen presentation, making it easier to screen for high-quality antibodies. Furthermore, compared to the traditional method of obtaining antibodies by obtaining hybridoma cells and then preparing ascites fluid, this method uses a recombinant expression vector to prepare antibodies, avoiding the large-scale sacrifice of animals. The recombinant antibody expression vector has higher preservation stability, is easier to scale up production, and the produced antibodies have small batch-to-batch variability and stable and reliable performance.

[0053] The antibody provided by this invention can specifically recognize and bind to human transcription factor PU.1 (PU.1 / SPI1), and has good applicability in multiple immunoassay systems, especially immunoblotting, immunofluorescence, immunohistochemistry and flow cytometry systems. Specifically, Western blotting was used to detect Daudi B lymphoblasts and THP-1 human acute monocytic leukemia cells, which highly express PU.1 / SPI1 protein. A single band of the expected size was detected in the positive cell lysate, while no reaction was observed in negative cells. Immunofluorescence was used to detect THP-1 human acute monocytic leukemia cells, HEL human erythroleukemia cells, and human spleen tissue expressing PU.1 / SPI1 protein. The antibody bound to the PU.1 / SPI1 antigen was accurately localized, and the fluorescence signal was clear and bright. No fluorescence signal was observed in areas without the target antigen. Immunohistochemistry was used to detect human follicular lymphoma, human tonsils, and human appendix tissue samples. The staining sites in positive tissues were accurate and without nonspecific staining, with low background interference. Flow cytometry was used to detect PU.1 / SPI1 protein expressed by cells. A significant fluorescence transition and a single signal intensity were observed in positive cells, while the signal on negative cells that did not express the target antigen was similar to that of the isotype control, which could accurately distinguish between positive and negative cell populations. These results indicate that the antibody of this invention can specifically and sensitively recognize naturally expressed PU.1 / SPI1 proteins in human cells / tissues, without specifically reacting with complex non-target antigen components in cells / tissues. It exhibits good affinity for PU.1 / SPI1 proteins, excellent anti-interference ability, and no cross-reactivity with homologous proteins from other species. This is beneficial for improving the accuracy and reliability of immunodetection of PU.1 / SPI1, increasing the consistency between detection results and actual conditions, and effectively avoiding false positive or false negative results. It provides an effective antibody tool and detection method for high-specificity, high-accuracy, and high-sensitivity detection of PU.1 / SPI1 proteins, and has broad application prospects in clinical diagnosis and scientific research.

[0054] Optionally, both the light chain variable region and the heavy chain variable region include four frame regions (FRs), which are arranged in an alternating sequence with three core parameters (CDRs) to form the variable region. The amino acid sequence of the light chain variable region (VL) of the antibody of the present invention is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.7.

[0055] Optionally, the antibody of the present invention further includes a light chain constant region (CL) and a heavy chain constant region (CH), wherein CL and VL constitute a complete light chain (FL), and CH and VH constitute a complete heavy chain (FH). The constant regions of the antibody can usually be obtained through public searches, such as searching for rabbit-derived IgG gamma C reign to obtain CH and searching for rabbit-derived IgG Kappa C reign to obtain CL through the IMGT online database (www.imgt.org).

[0056] Specifically, the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6. Correspondingly, CL represents the κ type, and CH represents the IgG1 type.

[0057] 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 the antigen-binding region of the full-length antibody; the antigen-binding region refers to a polypeptide that substantially retains the same biological function or activity as the full-length form. Specifically, the antigen-binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining an intact antigen recognition and binding site, capable of binding 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.

[0058] Another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector containing the nucleic acid molecule, or a host cell containing the nucleic acid molecule, wherein the nucleic acid molecule encodes a monoclonal antibody against human transcription factor PU.1 as described above.

[0059] Nucleic acid molecules can be in the form of DNA (such as cDNA, genomic DNA, or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0060] The sequence of a nucleic acid molecule can be derived from the antibody AA sequence using conventional methods such as codon coding rules. The full-length sequence of a nucleic acid molecule or its fragments can usually be obtained using PCR amplification, recombination, or artificial synthesis.

[0061] For example, the nucleic acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.12 or a complementary sequence thereto, and the nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO.14 or a complementary sequence thereto.

[0062] For example, the nucleic acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO.11 or a complementary sequence thereto, and the nucleic acid sequence of the heavy chain is as shown in SEQ ID NO.13 or a complementary sequence thereto.

[0063] Those skilled in the art will understand that, due to the degeneracy of the genetic code, nucleic acid molecules other than those in the above examples can also encode the antibodies of the present invention. Therefore, the nucleic acid molecules in the above examples should not be regarded as limiting the scope of protection of the present invention.

[0064] The original vector used to construct the recombinant vector can be any vector conventional in the art, as long as it can contain 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), viscera, and mini-chromosomes. The vector can be a cloning vector (i.e., used to transfer nucleic acid molecules into a host and multiply them in host cells) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecule inserted into the vector to be expressed in the host cell). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid molecule, which is then introduced into a host cell and cultured under specific conditions to express and obtain an antibody. This is a well-known technique in the art and will not be described in detail here.

[0065] The nucleic acid molecules encoding the antibodies FL and FH of this invention can be inserted into two vectors, which can be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain can be isolated from the host cell expressing it, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to form antibodies. In other embodiments, the nucleic acid molecules encoding antibodies FL and FH can also be cloned into a single vector, with each nucleic acid sequence ligated downstream of a suitable promoter; for example, each nucleic acid sequence encoding the heavy and light chains can be operatively ligated to different promoters, or the nucleic acid sequences encoding the heavy and light chains can be operatively ligated to a single promoter, such that both the heavy and light chains can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibodies.

[0066] Recombinant vector transfection or transformation into host cells is performed using conventional techniques. When the host is a prokaryote such as *E. coli*, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with CaCl2 or MgCl2; alternatively, microinjection, electroporation, or liposome packaging can be used. When the host is a eukaryote, the following DNA transfection methods can be used to achieve gene delivery: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, or gene gun bombardment.

[0067] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in this invention include, but are not limited to, *Escherichia coli* (e.g., DH5α, JM109, BL21, W3110), *Bacillus* spp. (e.g., *Bacillus subtilis*, *Bacillus thuringiensis*), *Enterobacterium* strains (e.g., *Salmonella typhimurium*, *Serratia marcescens*), and *Pseudomonas* spp. Examples of eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining host cells transfected or transformed with the recombinant vector described above, they can be cultured under suitable conditions to express antibodies, which can then be isolated to obtain purified antibodies.

[0068] In a preferred embodiment, the recombinant vector described above is the mammalian expression vector pBR322, and the host cell is human renal epithelial cells (293F cells).

[0069] In a typical implementation, the preparation method of monoclonal antibodies includes: tandemly loading the heavy chain gene, light chain gene, and signal peptide of the antibody onto the expression vector pBR322, co-transfecting 293F cells, culturing and collecting the cell culture supernatant, and purifying to obtain the target antibody. The selection of the signal peptide is designed based on the host cell, and this invention does not have any special limitations in this regard.

[0070] Another embodiment of the present invention provides the application of the monoclonal antibody against human transcription factor PU.1 or its antibody-drug conjugate as described above in the preparation of a human transcription factor PU.1 immunoassay kit, wherein the antibody-drug conjugate comprises the monoclonal antibody against human transcription factor PU.1 as described above and a detection marker linked to the monoclonal antibody.

[0071] The advantages of the monoclonal antibody against human transcription factor PU.1 or its antibody-drug conjugate in the preparation of human transcription factor PU.1 immunoassay kits are the same as the advantages of the monoclonal antibody against human transcription factor PU.1 over the prior art as described above, and will not be repeated here.

[0072] It is important to emphasize that the antibodies of this invention can be used alone or linked (covalently or non-covalently) with detection markers to form antibody-conjugates. In some embodiments, the antibodies of this invention are used as antigen-binding (or capture) antibodies that specifically recognize and bind to PU.1 in the sample to be tested. PU.1 is then qualitatively or quantitatively detected by analyzing the signal of the detection marker linked to it. In other embodiments, the antibody against the transcription factor PU.1 is not labeled (as a primary antibody or capture antibody), but 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-PU.1 antibody is a rabbit-derived IgG antibody, then the secondary antibody can be an anti-rabbit IgG antibody. PU.1 is then qualitatively or quantitatively detected by analyzing the change in the detection marker signal generated after the secondary antibody specifically binds to the antibody of this invention, as exemplified by the multiple detection systems established in Example 2 of this invention below.

[0073] The detection markers are used to generate identifiable signal changes to identify the antibodies or secondary antibodies of the present invention based on these signal changes, thereby identifying the expression of the PU.1 antigen in the sample to be tested through a specific antigen-antibody reaction. Detection markers include, but are not limited to: biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride), fluorescent proteins (such as isophycocyanin, phycoerythrin, PerCP, and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, and avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.

[0074] The aforementioned immunoassay methods include, but are not limited to: enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), Western blot (WB), immunoprecipitation (IP), and flow cytometry (FC).

[0075] Based on the same inventive concept, embodiments of the present invention also provide a human transcription factor PU.1 immunoassay kit, the kit comprising a monoclonal antibody against human transcription factor PU.1 or an antibody-drug conjugate thereof as described above.

[0076] Optionally, the immunoassay kit is an enzyme-linked immunosorbent assay (ELISA) kit, an ELISA kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit, or a flow cytometry kit.

[0077] Preferably, the immunoassay kit is an immunoblotting kit, an immunofluorescence kit, an immunohistochemistry kit, or a flow cytometry kit, and the kit also includes a fluorescently conjugated anti-rabbit IgG secondary antibody.

[0078] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0079] Example 1: Preparation of rabbit monoclonal antibody against human transcription factor PU.1 / SPI1

[0080] 1.1 Antigen Preparation: In this embodiment, rabbits were immunized with the synthesized SPI1 antigen peptide. Based on single-cell labeling and sorting technology, B cells capable of recognizing the target antigen were enriched and sorted from the rabbit spleen. These cells were then cultured in single-cell form to obtain the monoclonal antibody secreted by the B cells. The amino acid sequence of the full-length PU.1 / SPI1 protein is shown in NCBI accession number: NP_003111.2, Uniprot number: P17947. The antigen peptide corresponds to the 1-100 amino acid sequence of human PU.1 / SPI1, as shown below:

[0081] MLQACKMEGFPLVPPPSEDLVPYDTDLYQRQTHEYYPYLSSDGESHSDHYWDFHPHH VHSEFESFAENNFTELQSVQPPQLQQLYRHMELEQMHVLDTPM (see SEQ ID NO. 15).

[0082] 1.2 Animal Immunization: Four New Zealand white rabbits were immunized with 200 μg / rabbit of synthetic human PU.1 / SPI1 protein 1-100AA fragment. Before the first immunization, the antigen was mixed with an equal volume of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsion, which was injected subcutaneously at multiple sites on the abdomen and back of the rabbits. After the first immunization, every 3 weeks, 100 μg of immunogen was mixed with an equal volume of incomplete Freund's adjuvant (purchased from Sigma) to prepare an emulsion, which was injected subcutaneously at multiple sites on the abdomen and back of the rabbits for two booster immunizations. After four immunizations, serum was collected from the rabbits and diluted at a ratio of 1:243000. The titer of targeting PU.1 / SPI1 was determined by enzyme-linked immunosorbent assay (ELISA). Rabbits with OD450nm greater than 0.2 were given a booster immunization with 200 μg of immunogen injected subcutaneously at multiple sites. Three to four days later, the spleen was harvested, and the final immunized serum was diluted and the affinity and specificity of the spleen to the test cell sample were determined by flow cytometry (FC).

[0083] The ELISA method for determining the titer of immune serum includes the following steps: (1) Add 25 μL / well of recombinant Human serum to the microplate to a final concentration of 1 μg / mL. PU.1 / SPI1 protein (1-100AA), coated overnight at 4℃; (2) Add 75μL / well of washing buffer (PBS containing 0.05% (v / v) Tween-20) and wash 5 times, then add 50μL / well of blocking buffer (PBS containing 1% BSA, 0.5% gelatin and 5% sucrose) and incubate at room temperature for 1h; (3) Repeat the washing process of (2) to wash the plate, add 25μL / well of serum diluent and incubate overnight at 4℃; The serum to be tested starts at 1:1000 and is serially diluted three times with dilution buffer (PBS containing 1% BSA) for a total of 8 gradients; (4) Repeat the washing process of (2) to wash the plate, add 25μL / well of 1:5000 horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (purchased from Jackson) diluted 1:5000. ImmunoResearch (catalog number 111-035-045), incubate at room temperature in the dark for 1 hour; (5) repeat the washing process of (2) to clean the plate, add 100 μL / well of TMB for color development, incubate at 37℃ in the dark for 10 min, add 100 μL / well of 0.5 M oxalic acid solution to terminate the reaction, measure the absorbance at 450 nm, use rabbit serum before immunization as negative control, and the detection system without immune serum as blank control (NC), and the ratio of the measured value to the control value ≥2.1 is positive immune serum.

[0084] Serum titer test results are shown in Figure 1 N20584-N20587 are rabbit identification numbers. It is evident that a strong immune response was generated in the rabbits after the third and fourth booster immunizations, resulting in high titers of neutralizing antibodies. The serum titer from the fourth immunization was the highest and can be used for antibody isolation.

[0085] The specificity of FC detection in immune serum includes the following steps: (1) Disinfect the ultra-clean workbench with ultraviolet irradiation for 15-20 min, turn on the fan for 5 min, and prepare for aseptic work; (2) Collect and wash cells, determine the total number of cells, and check that the cell viability is 90%-95%; the cell samples to be tested include human monocytic leukemia cells THP-1 (a monocytic cell line that highly expresses PU.1 / SPI1 protein) and human T lymphocyte leukemia cells Jurkat (a T lymphocyte cell line, with lower PU.1 / SPI1 protein expression levels compared to monocytic cells and B lymphocytes); (3) Resuspend the cells in PBS buffer to approximately 3×10 6 -5×10 6(3) Distribute cells at 100 μL / well into 96-well V-plates and wash once with 1×PBS; (4) Dilute the L / D staining solution of Zombie NIR Fixable Viability Kit (purchased from Biolegend, catalog number 423105) at a ratio of 1:1500 and distribute the diluted staining solution at 100 μL / well into the wells, resuspend the cells in the wells, and react at room temperature for 15 min; (5) Centrifuge at 400g for 5 min, discard the supernatant, and wash twice with FACS buffer (formulation: 1000 mL PBS, 40 mL FBS and 1 mL 0.5 MEDTA); (6) Distribute 1×IntracelluLar Fixation solution at 100 μL / well into 96-well V-plates and wash once with 1×PBS. (7) Dispense the buffer into the well plate, resuspend the cells in each well, and react at room temperature for 30 min; (8) Centrifuge at 400g for 5 min, discard the supernatant, and wash twice with 1×Permeablization buffer; (9) Dispense 100 μL / well of the immune serum (as primary antibody) diluted with 1×Perm buffer into the well plate, resuspend the cells in each well, and react at room temperature for 30 min; (10) Centrifuge at 400g for 5 min, discard the supernatant, and wash twice with 1×Permeablization buffer; (11) Dispense 100 μL / well of the fluorescent secondary antibody (Fluorescein(FITC)AffiniPure F(ab')2FragmentGoatAnti-Rabbit IgG, purchased from Jackson, catalog number 111-096-046) diluted with 1×Permeablization buffer into the well plate, resuspend the cells in the well, and react at room temperature for 30 min; (12) Resuspend the cells in the well plate, and react at room temperature for 30 min; (33) Wash twice with 1×Permeablization buffer. Wash twice with buffer, then resuspend cells in each well with 200 μL FACS buffer and store in the dark; (12) Perform analysis according to Beckman Cytoflex flow cytometer operation and maintenance SOP-105-AND-CA-008. Rabbit IgG isotype control (from ABclonal, catalog number A23821) was used as isotype control.

[0086] The results of FC detection of immune serum bound to THP-1 cells and Jurkat cells are shown in the figures below. Figure 2-3The left graph shows a 1:500 serum dilution, and the right graph shows a 1:2000 serum dilution. The horizontal axis represents relative fluorescence intensity, and the vertical axis represents relative cell count. The red curve represents the negative control, the blue curve represents the isotype control, and the green curve represents N20584 rabbit serum. The graphs show that the negative control showed no specific fluorescence reaction, while the immune serum specifically bound to the positive cell samples, exhibiting good fluorescence transition. This indicates that highly specific and high-affinity antibodies suitable for flow cytometry detection have been generated, and the immunized animal can proceed to the spleen cell isolation stage.

[0087] 1.3 Isolation of B cells and sorting of antigen-specific B cells in the spleen: For relevant methods, please refer to the published patents “Method for efficient isolation of single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)” and “An in vitro culture system for B lymphocytes and its application (Publication No.: CN111518765A, Publication Date: 2020-08-11)”.

[0088] 1.4 Cloning of the gene encoding a rabbit-derived monoclonal antibody: The supernatant from cultured B cells was used to identify antigen-specific B cells using an antigen-coated ELISA. Lysed cells were collected and analyzed using Quick-RNA... TM RNA was extracted using the Micro Prep kit (ZYMO, catalog number R1051) and reverse transcribed into cDNA. Using cDNA as a template, naturally paired rabbit antibody light chain variable region (VL) and heavy chain variable region (VH) genes were amplified by PCR and sequenced. The PCR system consisted of: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria HiFi (ABclonal, catalog number RK20717), and 6.5 μL H2O. The PCR program consisted of: 98℃ for 30 s, followed by 40 cycles of 98℃ for 10 s, 64℃ for 30 s, and 72℃ for 30 s, with a final temperature of 72℃ for 5 min. The reaction solution was stored at 4℃. The primer sequences (5'-3') for amplifying the VL and VH genes are shown below, where F and R represent the forward and reverse primers, respectively.

[0089] VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO. 16);

[0090] VL-R: cacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 17);

[0091] VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 18);

[0092] VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 19).

[0093] 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, resulting in antibody 6E10 with the complete light chain (FL) shown in SEQ ID NO.1 and the complete heavy chain (FH) shown in SEQ ID NO.6.

[0094] Antibody sequencing was performed by Kinkai Biotechnology Co., Ltd. The amino acid (AA) and nucleotide (DNA) sequences of the antibodies are shown in Table 1. In the table, LCDR1-3 represent complementarity-determining regions CDR1-3 on the light chain variable region, and HCDR1-3 represent complementarity-determining regions CDR1-3 on the heavy chain variable region. The variable region numbering system is the Kabat numbering system.

[0095] Table 1. Sequence information of rabbit-derived monoclonal antibody 6E10 in this embodiment.

[0096]

[0097]

[0098] 1.5 Expression and Large-Scale Production of Antibody 6E10: Using recombinant expression technology, the amplified VL and VH genes were inserted in tandem with the light chain constant region (CL) and heavy chain constant region (CH) into an expression vector. Monoclonal antibody 6E10 was then mass-produced through recombinant expression of the antibody genes. In this embodiment, the CL and CH genes were pre-inserted into the mammalian expression vector pBR322, and its expression profile is shown below. Figure 4In the figure, pBR322 origin and f1 origin are replication promoters, Ampcillin is the resistance gene, CMV promoter is the transcription promoter, SV40 PA terminator is the tailing signal, Light chain constant is the nucleotide sequence of CL (left figure), and Heavy chain constant is the nucleotide sequence of CH (right figure). Then, the VL and VH genes were ligated to the expression vector pBR322 carrying the CL and CH genes, linearized with XbaI (955bp) and NheI (949bp) restriction endonucleases respectively, via homologous recombination to obtain complete light chain (FL) and heavy chain (FH) gene expression vectors. Sequencing confirmed the successful construction of the vectors.

[0099] To facilitate antibody purification, a signal peptide is added upstream of the VL and VH genes to achieve secretory expression of the antibody. The signal peptide can be a commonly used antibody expression signal peptide in the field, such as the patent "Rabbit Monoclonal Antibody against Human Interferon α2 and its Application (Publication No.: CN116063487A, Publication Date: 2023-05-05)" and the patent "High Affinity Human..." The IL-5 rabbit monoclonal antibody and its application (Publication No.: CN115819578A, Publication Date: 2023-03-21) has a signal peptide "MDTRAPTQLLGLLLLWLPGATF (encoding gene in this embodiment is atggacacgagggcccccactcagctgctgggactcctgttgctgtggctgccgggcgccactttt)" upstream of VL and "METGLRWLLLVAVLKGVQC (encoding gene in this embodiment is atggagactgggctgcgctggcttctcctggtcgcggtgctcaagggggtccagtgt)" upstream of VH. Of course, those skilled in the art can replace the signal peptide with another after obtaining the antibody sequence of this invention for antibody expression; therefore, the signal peptide sequence is not shown in Table 1 of this embodiment.

[0100] The successfully constructed FL and FH expression vectors were co-transfected into 293F cells. After transfection and culture for 72-96 h, the culture supernatant contained recombinant rabbit monoclonal antibodies recognizing human PU.1 / SPI1 protein. The target antibody was purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, catalog number SA023100). The antibody purity was verified to be ≥95% and the concentration to be 1 mg / mL by 12% polyacrylamide gel electrophoresis (SDS-PAGE). The purified antibody was aliquoted and stored at -20℃ for later use.

[0101] Example 2: Establishment and efficacy evaluation of an immunoassay method based on rabbit-derived antibody 6E10 against human PU.1.

[0102] This embodiment establishes an immunoblotting, immunofluorescence, immunohistochemistry, and flow cytometry analysis system for rabbit monoclonal antibody 6E10 to evaluate the antibody's application performance.

[0103] 2.1 Establishment of an immunoblotting (WB) detection system

[0104] The WB experimental analysis procedure is as follows: (1) Electrophoresis: Lyse the cells or tissues to obtain protein lysis buffer and perform 7% polyacrylamide gel electrophoresis (SDS-PAGE); (2) Transfer membrane: Transfer the gel protein band to the PVDF membrane in the electrotransfer system according to the conventional method; (3) Blocking: Place the PVDF membrane in TBST blocking solution containing 3% skim milk powder and block at room temperature for 1 h; (4) Primary antibody incubation: Add the antibody 6E10 solution prepared in Example 1 (the working concentration of the primary antibody is 1 μg / mL) and incubate overnight at 4°C; (5) Secondary antibody incubation: Wash the membrane 3-4 times with TBST, add goat anti-rabbit IgG (H+L) secondary antibody (from Abclonal, catalog number AS014, secondary antibody dilution ratio 1:5000), and incubate at room temperature for 1 h; (6) Color development: Wash the membrane 3-4 times with TBST, add ECL ultrasensitive color development solution, and develop.

[0105] Positive samples for Western blotting included Daudi B lymphoblasts highly expressing PU.1 / SPI1 protein, THP-1 human acute monocytic leukemia cells, RAW 264.7 mouse monocytic macrophage leukemia cells, and mouse liver and rat lung tissue cell samples. Negative samples included HL-60 human acute promyelocytic leukemia cells and HAP1 human near-haploid cells derived from male chronic myeloid leukemia. Western blotting results are shown below. Figure 5 As shown in the figure, the antibody prepared in this invention detected a single, bright target band in Daudi and THP-1 cell lysates. The actual detected molecular weight matched the theoretical size of PU.1 / SPI1, and no non-specific bands were observed. No band was found in negative cell samples, indicating a negative signal. The Western blot results were consistent with expectations, indicating high antibody specificity, good affinity for PU.1 / SPI1 protein, good recognition sensitivity and resistance to interference from cellular components, and high detection accuracy. Furthermore, the antibody showed negative results in murine positive samples, indicating that it does not recognize murine homologous proteins and has high species specificity for human PU.1 / SPI1.

[0106] 2.2 Establishment of Immunofluorescence (IF) Detection System

[0107] The IF detection method is as follows: (1) Sample processing: Prepare cell sections, add 4% neutral formaldehyde fixative to fix at room temperature for 10-15 min, and wash with PBS 3 times for 5 min each time after fixation; (2) Blocking: Add PBS buffer containing 5% goat serum to the cell sections, incubate at room temperature for 30 min and then remove the blocking solution; (3) Primary antibody incubation: Add antibody 6E10 solution prepared in Example 1 (primary antibody working concentration is 5 μg / mL), incubate at 4℃ overnight; take it out and warm it to room temperature for 15 min, remove the primary antibody working solution, and then wash with PBST; (4) Secondary antibody incubation: Add Cy3 fluorescent secondary antibody working solution (Cy3-conjugated Goat serum) anti-RabbitIgG (H+L), from ABclonal, catalog number AS007, secondary antibody dilution ratio 1:500), incubated at 37℃ in the dark for 1 h; remove the secondary antibody working solution, and then wash with PBST; (5) staining the nucleus: add DAPI nuclear dye working solution, stain for 10 min, remove DAPI working solution, add anti-fluorescence attenuation mounting medium, and observe and collect images under a fluorescence microscope.

[0108] Positive samples for IF included THP-1 cells highly expressing PU.1 / SPI1 protein, human erythroleukemia cells (HEL), and human spleen cells. IF test results are shown below. Figure 6 From left to right, the images show THP-1 cells, HEL cells, and human spleen tissue samples. DAPI counterstaining of cell nuclei results in a blue color, while the antibody of this invention, after binding to PU.1 / SPI1 protein, stains red after secondary antibody development. Therefore, the top image shows confocal imaging of each sample using the anti-PU.1 / SPI1 protein antibody (red), and the bottom image shows nuclear staining of each sample using DAPI (blue) with red-blue merging to demonstrate the localization of PU.1 / SPI1 protein. The images show that the antibody of this invention can specifically bind to PU.1 / SPI1 protein in the cell nucleus, consistent with its theoretical localization (PU.1 protein is mainly located in the cell nucleus). This further demonstrates that the antibody of this invention has advantages such as high binding sensitivity, good specificity, accurate localization, and effective resistance to interference from complex components of cellular tissues.

[0109] 2.3 Construction of Immunohistochemistry (IHC) Detection System

[0110] The IHC detection steps are as follows: (1) Baking: Immerse the paraffin tissue sections baked at 56℃ for 30 min in dewaxing solution 1. After 5 min, take out the sections and immerse them in dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3 in sequence. Place them in the dewaxing solution for 5 min and in the anhydrous ethanol solution for 3 min. Then wash the sections with running water for 3 min. Dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.; (2) Antigen retrieval: 0.01M Tris-EDTA retrieval solution (pH 1-3) 9.0) High-pressure heat repair; (3) Endogenous peroxidase inactivation: Wash with PBS buffer 3 times, 3 min each time, and remove the buffer from the slide; then immerse the slide 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, then remove the buffer, delineate the area to be tested on the slide, add PBS blocking solution to the area to be tested, and incubate at room temperature for 30 min; (5) Primary antibody incubation: Remove the blocking solution, add antibody 6E10 dilution solution (final primary antibody concentration is 0.33 μg / mL), and incubate at room temperature for 60 min; remove the antibody working solution, rinse quickly 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 (Dako REAL EnVision Detection) System, Peroxidase / DAB, Rabbit / Mouse, HRP;), incubate at room temperature for 25 min; remove the secondary antibody working solution, rinse quickly once with PBS buffer, and soak and wash three times, 3 min each time; (7) staining: add staining working solution, closely observe the color change under a microscope, and after obtaining a suitable staining intensity, immerse the section in a large amount of distilled water to stop staining, and then rinse with running water for 10 min; (8) counterstaining: immerse the slightly drained tissue section in Mayer's solution. (9) Blueing: Immerse the slightly drained sections in a saturated lithium carbonate solution for 3 seconds, and rinse with running water for 3 minutes; (10) Dehydration: Immerse the sections in anhydrous ethanol twice, lifting them up and down several times during the immersion process, and remove them after 10 seconds; dry the sections at high temperature (54-58℃); (11) Mounting: Add an appropriate amount of neutral resin to the center of the section and cover it with a coverslip. The amount of resin should be appropriate. After covering with a coverslip, the tissue should be completely covered and there should be no resin overflow. Finally, scan the sections.

[0111] Positive samples for IHC include tissue samples from human follicular lymphoma, human tonsil, and human appendix. IHC staining results are as follows: Figure 7As shown, from left to right, the samples are human follicular lymphoma, human tonsils, and human appendix tissue. Human PU.1 / SPI1 protein is mainly expressed in hematopoietic and immune system-related cells in the human samples, with subcellular localization in the cell nucleus. Figure 6 Specific brown staining was observed in human follicular lymphoma tissue samples, human tonsil tissue samples, and human appendix tissue samples. The stained areas corresponded to the actual expression of PU.1 / SPI1 antigen in the tissue samples. The staining was clear and without non-specific staining, with a clean background, indicating a positive signal. This demonstrates that the antibody of the present invention can recognize naturally expressed PU.1 / SPI1 protein in tissue samples. The antibody exhibits high specificity and sensitivity, and its binding to the antigen is not affected by tissue complexity, ensuring the accuracy and reliability of the detection results.

[0112] 2.4 Establishment of a Flow Cytometry (FC) Detection System

[0113] The FC detection method is the same as in Example 1, except that the primary antibody is antibody 6E10 and the concentration of the primary antibody is 2 μg / mL; the positive cell sample is THP-1 and the negative cell sample is HAP1.

[0114] FC test results are shown below Figure 8 In the figure, the horizontal axis represents relative fluorescence intensity, and the vertical axis represents relative cell number. HAP1 and THP-1 cells were stained intracellularly with a rabbit monoclonal antibody against human PU.1 / SPI1 protein (yellow curve) or an isotype control antibody (used to eliminate background staining caused by non-specific antibody binding to cells, blue curve), and then... 594-conjugated goat anti-rabbit IgG (H+L) staining (from ABclonal, catalog number AS039), with non-fluorescent cells serving as a blank control (red line curve). The figure shows a significant difference in fluorescence signal transition between positive and negative samples with antibody 6E10, specifically binding to THP-1 positive cells and not to HAP1 negative cells. This confirms its specific recognition of native PU.1 / SPI1 protein. The antibody exhibits high affinity and specificity for native human PU.1 / SPI1 protein, with no non-specific recognition.

[0115] 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 within the protection scope of the present invention.

Claims

1. A monoclonal antibody against human transcription factor PU.1, characterized in that, It includes a light chain variable region and a heavy chain variable region. The amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are 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.

2. The monoclonal antibody against human transcription factor PU.1 according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

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

6.

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

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a monoclonal antibody against human transcription factor PU.1 as described in any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleic acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.12 or is complementary to it, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO.14 or is complementary to it.

7. The nucleic acid molecule according to claim 6, characterized in that, The nucleic acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.11 or is complementary to it, and the nucleic acid sequence of the heavy chain is shown in SEQ ID NO.13 or is complementary to it.

8. The use of the monoclonal antibody against human transcription factor PU.1 as described in any one of claims 1-4 in the preparation of a human transcription factor PU.1 immunoassay kit.

9. A human transcription factor PU.1 immunoassay kit, characterized in that, Including monoclonal antibodies against human transcription factor PU.1 as described in any one of claims 1-4.

10. The human transcription factor PU.1 immunoassay kit according to claim 9, characterized in that, The immunoassay kit is selected from immunoblotting kits, immunofluorescence kits, immunohistochemistry kits, or flow cytometry kits.

Images