Monoclonal antibodies binding to human chromosome remodeling molecules and their applications

Abstract

The present application relates to the technical field of human chromosome remodeling molecules, and specifically to monoclonal antibodies and applications that bind to human chromosome remodeling molecules. A rabbit monoclonal antibody with high affinity against human chromosome remodeling molecules has been successfully developed through single B cell screening and culture technology. It has high affinity and specificity for human chromosome remodeling molecules, and the monoclonal antibody can be used to prepare immunoblotting kits and immunohistochemistry kits for detecting human chromosome remodeling molecules. The monoclonal antibody also has important clinical value for the diagnosis and treatment of human chromosome remodeling molecules and related diseases.

Description

Technical Field

[0001] The present application relates to the technical field of human chromosome remodeling molecules, and specifically to monoclonal antibodies binding to human chromosome remodeling molecules and their applications. Background Art

[0002] The yeast switch in mating type / sucrose nonfermentation (SWI / SNF) complex is a highly conserved, ATP-dependent, multi-subunit chromatin structure regulatory complex in the evolutionary process. The main biological function of the SWI / SNF complex is to change and reshape the interaction between histones and DNA in nucleosomes by using the energy obtained by hydrolyzing ATP by its ATPase subunits (BRM and BRG1), thereby affecting the degree of chromatin openness in specific regions of the genome and regulating gene expression.

[0003] Chromosome remodeling molecule (SMARCB1) is one of the core subunits of SWI / SNF complex and is required for BRG1 to effectively remodel nucleosomes in vitro. Loss, mutation or epigenetic modification of SMARCB1 gene leads to loss of function, loss of histone methyltransferase (enhancer ofzeste homolog 2, EZH2) inhibition, leading to cell proliferation and tumorigenesis. EZH2 is the catalytic subunit of histone lysine methyltransferase inhibitory complex PRC2 (polycomb repressor complex).

[0004] The human chromosome remodeling molecule (SMARCB1) is prone to mutation or deletion in malignant rhabdoid tumors (MRT). The overall survival of MRT is much lower than that of medulloblastoma and primitive neuroectodermal tumors. Therefore, it is necessary to distinguish MRT from medulloblastoma and primitive neuroectodermal tumors. SMARCB1 is usually missing in MRT, while most medulloblastomas and primitive neuroectodermal tumors express it. Epithelioid sarcomas in soft tissues often show rhabdoid features histologically, and neither expresses SMARCB1. Sometimes it is difficult to distinguish between the two. However, epithelioid sarcomas are positive for CD34 and β-catenin, while MRTs are negative for both. Therefore, it is of great significance to develop a highly sensitive human chromosome remodeling molecule (SMARCB1) detection methodology. Summary of the invention

[0005] The present application embodiment provides a class of rabbit monoclonal antibodies with high affinity for human chromosome remodeling molecules to solve one of the above technical problems to a certain extent. The embodiment successfully developed a rabbit monoclonal antibody with high affinity against human chromosome remodeling molecules through single B cell screening and culture technology, which has high affinity and specificity for human chromosome remodeling molecules. Therefore, the monoclonal antibody can be used to prepare a kit for detecting the level of human chromosome remodeling molecules. The monoclonal antibodies provided in this application have important clinical value for the diagnosis and treatment of human chromosome remodeling molecules and related diseases.

[0006] To this end, this application discloses at least the following technical solutions:

[0007] In a first aspect, the embodiment discloses a monoclonal antibody, which specifically binds to a human chromosome remodeling molecule, and the monoclonal antibody comprises:

[0008] three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VL CDR1 as set forth in SEQ ID NO:5, VL CDR2 as set forth in SEQ ID NO:6, and VL CDR3 as set forth in SEQ ID NO:7;

[0009] and / or

[0010] Three complementarity determining regions (CDRs) of the heavy chain variable region (VH) are defined according to the Kabat numbering system: VH CDR1 as shown in SEQ ID NO:8, VH CDR2 as shown in SEQ ID NO:9, and VH CDR3 as shown in SEQ ID NO:10.

[0011] In a second aspect, the embodiments disclose a monoclonal antibody that specifically binds to a human chromosome remodeling molecule, wherein the monoclonal antibody comprises a light chain as shown in SEQ ID NO:1 and a heavy chain as shown in SEQ ID NO:2.

[0012] In a third aspect, the present application discloses a monoclonal antibody that specifically binds to a human chromosome remodeling molecule, wherein the monoclonal antibody comprises a light chain variable region (VL) as shown in SEQ ID NO:3 and a heavy chain variable region (VH) as shown in SEQ ID NO:4.

[0013] In a fourth aspect, the present application discloses an immunoblotting detection kit for detecting human chromosome remodeling molecules, wherein the kit comprises the monoclonal antibody described in any one of the first to third aspects.

[0014] In a fifth aspect, the present application discloses an immunohistochemical detection kit for detecting human chromosome remodeling molecules, wherein the kit comprises the monoclonal antibody described in any one of the first to third aspects.

[0015] In a sixth aspect, the embodiments of the present application disclose the use of the monoclonal antibody described in any one of the first to third aspects in the preparation of a molecular reagent or kit for detecting human chromosome remodeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 In this example, the immunoblotting method was used to detect the recognition specificity of rabbit monoclonal antibodies against SMARCB1 in cell samples.

[0017] Figure 2 For the purpose of this example, an immunoprecipitation method was used to detect the recognition specificity of a rabbit monoclonal antibody against SMARCB1 in a cell sample 293F.

[0018] Figure 3 The staining localization diagram of SMARCB1 in human colon cancer tissue samples detected by immunohistochemistry is shown in the example.

[0019] Figure 4 The present invention uses immunohistochemistry to detect the staining localization of SMARCB1 in human undifferentiated esophageal cancer tissue samples.

[0020] Figure 5 The staining localization of SMARCB1 in human epithelioid sarcoma tissue (INI-1 deletion) samples detected by immunohistochemistry is shown in the example.

[0021] Figure 6 The staining localization diagram of SMARCB1 in mouse heart tissue samples detected by immunohistochemistry is shown in the example.

[0022] Figure 7 The staining localization diagram of SMARCB1 in rat lung tissue samples detected by immunohistochemistry is shown in the example.

[0023] Figure 8 Schematic diagram of the structure of the expression vector containing the heavy chain constant region of the rabbit monoclonal antibody constructed in the embodiment. In the figure, pBR322 origin and f1origin are replication promoters in Escherichia coli (E.Coli), Ampcillin is a plasmid resistance gene, CMV immearly promoter is a promoter in eukaryotes, SV40 PAterminator is a tailing signal, and Heavy chain constant is the nucleotide sequence of the heavy chain constant region of the rabbit monoclonal antibody.

[0024] Fig. 9Schematic diagram of the structure of the expression vector containing the light chain constant region of a rabbit monoclonal antibody constructed in the embodiment of the present application. In the figure, pBR322 origin and f1 origin are replication promoters in Escherichia coli (E.Coli), Ampcillin is a plasmid resistance gene, CMV immearly promoter is a promoter in eukaryotes, SV40 PAterminator is a tailing signal, and Light chain constant is the nucleotide sequence of the light chain constant region of a rabbit monoclonal antibody. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0026] Explanation of terms

[0027] In this application, the term "antibody" should be interpreted in the broadest sense, with various antibody structures, including but not limited to Y-shaped antibodies, so-called full-length antibodies, antigen-binding portions of Y-shaped antibodies, and genetic or chemical modifications thereof. Among them, "antigen-binding portion" refers to one or more parts or fragments of a Y-shaped antibody that can retain the ability of the antibody to specifically bind to human chromosome remodeling molecules.

[0028] In the present application, the term "monoclonal antibody" (mAb) includes a highly homogeneous antibody population with substantially identical antigenic determinants. That is, in this antibody population, individual antibodies are substantially identical, except for a small amount of mutations that may occur naturally. Monoclonal antibodies can show a single binding specificity and affinity for a specific epitope on an antigen. Compared with polyclonal antibodies that usually contain antibodies for different epitopes, each monoclonal antibody can be directed to the same or substantially the same epitope on the antigen. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous antibody population and cannot be interpreted as an antibody that needs to be made by any specific method. The antibody can be prepared by a variety of methods, including but limited to hybridoma method, recombinant DNA method, phage antibody library, B cell screening and cultivation and similar methods.

[0029] In the present application, the modifier "rabbit" in the terms "rabbit antibody", "anti-human chromosome remodeling molecule rabbit monoclonal antibody", "rabbit monoclonal antibody to human chromosome remodeling molecule" or similar terms indicates that the complementarity determining region (CDR) of the antibody is derived from a rabbit immunoglobulin sequence. In one embodiment, the rabbit monoclonal antibody to human chromosome remodeling molecule may comprise the CDR and framework region (FR) of an antibody from a rabbit immunoglobulin sequence. In one embodiment, the rabbit antibody or rabbit monoclonal antibody to human chromosome remodeling molecule may comprise the CDR of an antibody from a rabbit immunoglobulin sequence. In one embodiment, the rabbit monoclonal antibody to human chromosome remodeling molecule may be an antibody whose CDR region is derived from a rabbit immunoglobulin sequence and whose FR is derived from the germline immunoglobulin sequence of other mammals (such as mouse or human). The term "rabbit monoclonal antibody to human chromosome remodeling molecule" may also include antibodies having amino acid residues encoded by non-rabbit immunoglobulin sequences, for example, mutations introduced by random mutation or site-specific mutation in vitro, or mutations introduced by somatic mutation in vivo.

[0030] In the present application, the term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains, of which the two chains with larger molecular weight are called heavy chains (H), and the two chains with smaller molecular weight are called light chains (L). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. The sequences of about 110 amino acids near the N-terminus of the heavy and light chains vary greatly, while the sequences of the other parts are relatively constant. Therefore, the regions of the light and heavy chains where the amino acid sequences near the N-terminus vary greatly are called variable regions (V), accounting for 1 / 4 and 1 / 2 of the heavy and light chains, respectively; the regions where the amino acid sequences near the C-terminus are relatively stable are called constant regions (C), accounting for 3 / 4 and 1 / 2 of the heavy and light chains, respectively.

[0031] The V region of the heavy chain and the V region of the light chain are called VL and VH, respectively. VL and VH each contain three regions with highly variable amino acid composition and arrangement order, called hypervariable regions (HVR) or complementarity determining regions (CDR), including HVR1 (CDR1), HVR2 (CDR2) and HVR3 (CDR3), among which HVR3 (CDR3) has a higher degree of variation. Generally, VH includes three complementary determining regions VH CDR1, VH CDR2 and VH CDR3, and VL includes three complementary determining regions VL CDR1, VL CDR2 and VLCDR3. The three complementary determining regions of VL and VH constitute the antigen-binding site of the antibody alone or together, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. In the V region, the amino acid composition and arrangement order of the region outside the CDR are relatively conservative, called the framework region (FR). VH or VL each has four framework regions, represented by FR1, FR2, FR3 and FR4. By replacing or changing the framework regions, multiple antibodies with the same specificity can be produced while ensuring that the site of antibody recognition and antigen binding remains unchanged.

[0032] The C region of the heavy chain and light chain is called CH and CL respectively. The length of CL of different types (κ or λ) of Ig is basically the same, but the length of CH of different classes of Ig is different. For example, IgG, IgA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4.

[0033] In the present application, the term "framework region" or "framework region" residues refers to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0034] In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. The intensity or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0035] Antibody

[0036] The embodiments of the present application disclose an antibody that specifically binds to a human chromosome remodeling molecule.

[0037] In some embodiments, the antibody comprises three complementary determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VL CDR1, VL CDR2 and VL CDR3. VL CDR1 is shown in SEQ ID NO:5, VL CDR2 is shown in SEQ ID NO:6, and VL CDR3 is shown in SEQ ID NO:7.

[0038] In some embodiments, the antibody comprises three complementary determining regions (CDRs) of the heavy chain variable region (HL) defined according to the Kabat numbering system: VH CDR1, VH CDR2 and VH CDR3. VL CDR1 is shown in SEQ ID NO:8, VL CDR2 is shown in SEQ ID NO:9, and VL CDR3 is shown in SEQ ID NO:10.

[0039] In some embodiments, the antibody comprises three complementary determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VL CDR1, VL CDR2 and VL CDR3, and three complementary determining regions (CDRs) of the heavy chain variable region (HL) defined according to the Kabat numbering system: VH CDR1, VH CDR2 and VH CDR3. VL CDR1 is shown in SEQ ID NO: 5, VL CDR2 is shown in SEQ ID NO: 6, and VL CDR3 is shown in SEQ ID NO: 7. VL CDR1 is shown in SEQ ID NO: 8, VLCDR2 is shown in SEQ ID NO: 9, and VL CDR3 is shown in SEQ ID NO: 10.

[0040] In certain embodiments, the antibody against human chromosome remodeling molecules may have a Y-shaped molecular structure. In one embodiment, the antibody against human chromosome remodeling molecules may include a pair of heavy chains and a pair of light chains. The heavy chain may include a heavy chain variable region and one or more heavy chain constant regions. Mammalian antibodies generally include five types of heavy chains: γ, δ, α, μ and ε, and the corresponding antibodies are called IgG, IgD, IgA, IgM and IgE. The light chain may be a polypeptide subunit that is smaller than the heavy chain. The light chain may include a light chain variable region and a light chain constant region. VL is usually the N-terminal part of the light chain, which shows higher variability in the amino acid sequence. VL between different antibodies has a specific amino acid sequence. In one embodiment, the heavy chain variable region VH and the light chain variable region VL can both be used to recognize and bind to human chromosome remodeling molecules.

[0041] In some embodiments, the CDRs in the VL and VH regions may be separated from each other by FRs. FRs are conserved regions in the sequence structure. FRs can generally serve as a scaffold to allow CDRs to form a three-dimensional structure that can specifically bind to an antigen (e.g., a human chromosome remodeling molecule). The three-dimensional structure of FRs can be conserved in different antibodies.

[0042] In some embodiments, the light chain amino acid sequence of the rabbit monoclonal antibody is shown as SEQ ID NO.1, and the heavy chain amino acid sequence is shown as SEQ ID NO.2.

[0043] In some embodiments, the amino acid sequence of the light chain variable region of the rabbit monoclonal antibody is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region thereof is shown as SEQ ID NO.4.

[0044] Antibody preparation

[0045] The monoclonal antibodies provided in the embodiments of the present application can be prepared by a variety of methods, including but not limited to the hybridoma method, recombinant DNA method, phage antibody library, single B cell screening and culture, and similar methods.

[0046] In some embodiments, the preparation process of the monoclonal antibody includes: immunizing New Zealand white rabbits with recombinantly expressed human chromosome remodeling molecules as immunogens; sorting antigen-specific B lymphocytes from spleen cells of white rabbits; screening positive B lymphocytes that can secrete specific antibodies after antibody function verification; amplifying to obtain antibody heavy chain and light chain genes of positive B lymphocytes, and then recombinantly expressing them to obtain rabbit monoclonal antibodies against human SMARCB1.

[0047] In some embodiments, the sequence of the amino acid fragment from position 110 to position 135 of the human SMARCB1 protein synthesized by Sangon Biotechnology Co., Ltd. (SMARCB1 polypeptide) is as follows: VSISTEPPTYLREQKAKRNSQWVPTL (SEQ ID NO: 11).

[0048] In some embodiments, a thiol-coupled SMARCB1 polypeptide is also provided. The coupling process includes:

[0049] Use PBS to accurately prepare 3 mg / mL KLH (purchased from Merck) solution and 5 mg / mL SMCC (a bifunctional coupling agent containing N-hydroxysuccinimide (NHS) active ester and maleimide, Thermo Company) solution;

[0050] Take 180 μL of SMCC solution and add it to 2 mL of KLH solution. Place it on a rotating incubator and mix and shake it for 1 hour at room temperature. White flocs will appear.

[0051] The free SMCC was removed by dialysis for 1 h with 1× PBS at pH 7.4, with a volume ratio of V(PBS):V(KLH)=15:1;

[0052] Use PBS to accurately prepare a 10 mg / mL SMARCB1 peptide solution, slowly add activated KLH (dialysis completed) to the solution in multiple times, and place it in a small rotating incubator at the lowest speed at 4°C overnight.

[0053] In some embodiments, the method for preparing monoclonal antibodies to human chromosome remodeling molecules comprises:

[0054] (1) Animal immunization

[0055] The KLH-coupled SMARCB1 polypeptide obtained in the above example was used as an immunogen, and each New Zealand white rabbit was immunized with 700 μg of the immunogen. Before the first immunization, the immunogen was mixed with an equal amount of complete Freund's adjuvant (purchased from Sigma) to prepare an emulsifier, which was injected subcutaneously at multiple points in the rabbit's abdomen and back. After the first immunization, 350 μg of the immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) to prepare an emulsifier every 3 weeks, and injected subcutaneously at multiple points in the rabbit's abdomen and back for four booster immunizations. After five immunizations, rabbit serum samples were collected, and the titer against human SMARCB1 protein was determined by ELISA. The serum was diluted 1:243000 and the titer was measured by ELISA. Rabbits with OD450nm exceeding 0.2 were taken and boosted once with 200 μg of the immunogen by multiple subcutaneous injections, and the spleen was taken 3 days later.

[0056] (2) Isolation of splenocytes

[0057] Under a sterile environment, the rabbit spleen obtained above was washed with basal culture medium, excess connective tissue and fat were cut off, and the cells were ground in a cell screen. The grinding liquid (i.e., the intramembrane cells) was collected, centrifuged at 400g for 5 minutes at room temperature, the supernatant was removed, the cells were retained, 13mL of normal temperature RBC lysing solution (purchased from BioGems) was added, and the erythrocytes were lysed for 1 minute. 37mL of basal culture medium was added, and the erythrocytes were lysed. Centrifuged at 400g for 5 minutes at room temperature, the supernatant was removed, 40mL of normal temperature basal culture medium was added, and the cell clusters were gently blown away with a pipette to resuspend the cells. Centrifuged at 400g for 5 minutes at room temperature again, the supernatant was removed, the cells were retained, 20mL of normal temperature basal culture medium was added, and the cell clusters were gently blown away with a pipette to resuspend the cells; the resuspended cells were filtered again through a cell screen to remove the clumping cells, and then the cells were counted.

[0058] (3) B lymphocyte sorting and culture

[0059] For the B lymphocyte screening method, please refer to the method in paragraphs 0030 to 0044 of the CN110016462B specification.

[0060] (4) Cloning of the gene encoding rabbit monoclonal antibody

[0061] The supernatant of cultured B cells was used to identify positive clones by antigen-coated ELISA. After the cells of the positive clones were collected and lysed, RNA was extracted using the Quick-RNATM MicroPrep kit (purchased from ZYMO) and reverse transcribed into cDNA. Using cDNA as a template, the naturally paired rabbit monoclonal antibody chain variable region (VL) and heavy chain variable region (VH) genes were amplified from the cDNA of the corresponding positive clones by PCR, and several clones were selected for sequencing.

[0062] The PCR reaction system was as follows: 4 μL cDNA, 1 μL, 10 mM forward primer, 1 μL, 10 mM reverse primer, 12.5 μL 2×Gloria HiFi (provided by Wuhan Abotek Biotechnology Co., Ltd.), 6.5 μL N.F H2O. PCR amplification procedure: 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. The resulting reaction solution was stored at 4°C.

[0063] Among them, the light chain variable region primer pair is: VL-F: SEQ ID NO: 12; VL-R: SEQ ID NO: 13.

[0064] The heavy chain variable region primer pair is: VH-F: SEQ ID NO: 14; VH-R: SEQ ID NO: 15.

[0065] (5) Construction of heavy chain and light chain expression vectors

[0066] The heavy chain genes and light chain genes of several rabbit monoclonal antibodies selected in the above steps were loaded onto expression vectors respectively. The mammalian expression vector pBR322 used was shown in Figure 8 and Fig. 9 Among them, the selected light chain coding gene is shown in SEQ ID NO.16, and the selected light chain variable region coding gene is shown in SEQ ID NO.17. The selected heavy chain coding gene is shown in SEQ ID NO.18, and the selected heavy chain variable region coding gene is shown in SEQ ID NO.19.

[0067] (6) Monoclonal antibody preparation and purification

[0068] The constant region of the heavy chain of rabbit monoclonal antibody ( Figure 8 ) and the light chain constant region ( Fig. 9 ) mammalian cell (such as CHO) expression vectors were conventionally linearized using NheI and XbaI restriction endonucleases, respectively.

[0069] After purifying the PCR products amplified in the above steps, the heavy chain variable region genes and the light chain variable region genes are constructed into corresponding mammalian expression vectors by homologous recombination. After sequencing verification, the expression vectors containing the corresponding rabbit monoclonal antibody light chain genes and heavy chain genes are transfected into 293F cells together. The cells are transfected for 72 to 96 hours, and the obtained transformants are continuously cultured. The culture fluid is collected and centrifuged using protein A affinity gel resin. The rabbit monoclonal antibody that recognizes human SMARCB1 is purified and harvested from the supernatant of the centrifugation. The purity of the antibody is verified by 12% SDS-PAGE gel electrophoresis. The purified rabbit monoclonal antibody to human SMARCB1 protein is subjected to amino acid sequencing and stored at -20°C for future use.

[0070] As a result, it can be seen that the light chain amino acid sequence of the obtained rabbit monoclonal antibody is shown in SEQ ID NO.1, the heavy chain amino acid sequence is shown in SEQ ID NO.2, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4, the light chain complementary determining region VL CDR1 amino acid sequence is shown in SEQ ID NO.5, the light chain complementary determining region VL CDR2 amino acid sequence is shown in SEQ ID NO.6, the heavy chain complementary determining region VL CDR3 amino acid sequence is shown in SEQ ID NO.7, the heavy chain complementary determining region VH CDR1 amino acid sequence is shown in SEQ ID NO.8, the heavy chain complementary determining region VH CDR2 amino acid sequence is shown in SEQ ID NO.9, and the heavy chain complementary determining region VH CDR3 amino acid sequence is shown in SEQ ID NO.10.

[0071] In some detection examples, the specificity of the rabbit monoclonal antibody against human SMARCB1 protein prepared in the above examples was tested using 293F (Shanghai Cell Bank, Chinese Academy of Sciences), K562 (Shanghai Cell Bank, Chinese Academy of Sciences), HeLa (Shanghai Cell Bank, Chinese Academy of Sciences) that highly expressed SMARCB1, the cell sample HeLa-SMARCB1-C3 (homozygous) (ABclonal, RM02409) that did not express SMARCB1, NIH / 3T3 (Shanghai Cell Bank, Chinese Academy of Sciences) that highly expressed SMARCB1, Neuro2a (Shanghai Cell Bank, Chinese Academy of Sciences), and rattestis (Wuhan Saimeiyin Biotechnology Co., Ltd.).

[0072] The testing process includes:

[0073] Take cells that highly express SMARCB1, use the immunoblotting method to detect the recognition specificity of the monoclonal antibody, and perform 12% polyacrylamide gel electrophoresis. Transfer the gel protein band to the PVDF membrane in the electrotransfer system according to the conventional method. Place the membrane in a TBST blocking solution containing 3% skimmed milk powder and incubate at room temperature for 1 hour, add the SMARCB1 rabbit monoclonal antibody provided in the example (1:12000 dilution), and incubate at 4°C overnight. After washing the membrane with TBST, add a 1:10000 diluted goat anti-rabbit secondary antibody (Jackson ImmunoResearch) and incubate at room temperature for 1 hour. Wash the membrane with TBST again, add ECL color developing solution, develop, and expose for 90 seconds. See the results. Figure 1 Only the cell sample HeLa-SMARCB1-C3 that does not express SMARCB1 has no band, while the other lanes can detect specific target bands. This shows that the rabbit monoclonal antibody provided in the example has a high binding specificity to human SMARCB1.

[0074] In some detection examples, the monoclonal antibody provided in the examples was used to detect the cell sample 293F (Shanghai Cell Bank of the Chinese Academy of Sciences) that highly expressed SMARCB1, and the recognition specificity of the monoclonal antibody was detected by immunoprecipitation.

[0075] The testing process includes:

[0076] Collect 293F cells, centrifuge, discard the supernatant, collect the precipitate, wash once with PBS, add the cell lysis buffer for IP experiment, there should be no obvious precipitate after sufficient lysis, centrifuge and take the supernatant for later use; take 30μL rProteinA / G Plus MaqPolyBeads (Biyuntian Biotechnology) to wash and remove the magnetic bead protection solution, add 1mL Cell lysis buffer for IP (without inhibitors) (Biyuntian Biotechnology), mix well, place on a magnetic separator, collect the magnetic beads, use a pipette to discard the washing solution, repeat twice, add 1mL of 3% BSA, place on a flip mixer at 4℃ to seal for 1h for later use. Add 3 μg of SMARCB1 rabbit monoclonal antibody (prepared in Experimental Example 2) to the 293F cell lysate, place it in a flip mixer at 4°C overnight, mix the above antibody-antigen binding complex with the spare magnetic beads (Biyuntian Biotechnology), react at 4°C for 2 hours, separate the above magnetic beads-antibody-antigen complex on a magnetic separator, collect the supernatant, and perform subsequent WB detection; in this experiment, the same type of Rabbit IgG is used as a blank control. There are three groups of samples in the WB experiment, Input group: cell lysate without IP, to determine the location of the target protein and the relative degree of enrichment; Isotype group: use the product of Isotye Control IP as the background signal of protein binding to IgG; IP group: IP experimental group, the results are shown in Figure 2 .Depend on Figure 2 It can be seen that the rabbit monoclonal antibody provided in the example can specifically enrich SMARCB1 protein from the 293F sample.

[0077] In some detection examples, a rabbit monoclonal antibody prepared against human SMARCB1 protein was used as the primary antibody, and immunohistochemical staining was used to qualitatively detect SMARCB1 in human colon carcinoma tissue, human undifferentiated carcinoma of esophagus tissue, human epithelioid sarcoma (INI-1deletion), mouse heart tissue, and rat lung tissue.

[0078] The testing process includes:

[0079] (1) Chip selection:

[0080] Immunohistochemistry chip, catalog number TMA000-42, Bios Corporation.

[0081] Human colon cancer tissue samples, human undifferentiated esophageal cancer tissue samples, and human epithelioid sarcoma tissue (INI-1 deletion) samples were obtained from Wuhan Saiweier Biotechnology Co., Ltd.

[0082] Mouse heart tissue and rat lung tissue samples were obtained from Wuhan Saimeiyin Biotechnology Co., Ltd.

[0083] (2) IHC staining and analysis

[0084] Baking: Paraffin sections of these tissue samples were prepared by baking in a constant temperature oven at 56°C for 30 min.

[0085] Dewaxing to water: Put the paraffin sections together with the section rack into the dewaxing liquid 1 cylinder, then take them out of the constant temperature box 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. Each cylinder of dewaxing liquid reagent cylinder is 5 minutes, and each cylinder of anhydrous ethanol reagent cylinder is 3 minutes; wash the sections with running water for 3 minutes.

[0086] Antigen retrieval: 0.01 M sodium citrate retrieval solution (pH 6.0) and high pressure heat retrieval.

[0087] Inactivation of endogenous peroxidase: Wash with PBS buffer three times, 1 min each time, to remove the buffer on the slices; immerse the slices completely in 3% hydrogen peroxide solution, incubate at room temperature for 10 min.

[0088] Blocking: Wash with PBS buffer 3 times, 3 minutes each time, and remove the buffer on the slice; circle the tissue area to be tested on the slide with an immunohistochemistry pen, and add blocking solution - PBS blocking solution in the circled area; place the slice horizontally in an incubation wet box with water at the bottom, incubate at room temperature for 30 minutes, and start timing from the addition of the blocking solution.

[0089] Primary antibody incubation: remove the blocking solution, add the primary antibody diluted with PBS buffer (antibody dilution ratio is 1:1000) on the tissue section, place it horizontally in an incubation wet box, and incubate it at room temperature for 60 minutes; remove the antibody working solution, rinse quickly once with PBS buffer, 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.

[0090] Secondary antibody incubation: Add ready-to-use secondary antibody working solution (Dako REAL EnVision Detection System, Peroxidase / DAB, Rabbit / Mouse, HRP; Dako) to the tissue sections and place them horizontally in an incubation humidified box for incubation at room temperature for 25 minutes; remove the reagents on the sections, quickly rinse once with buffer PBS, and soak and wash three times with buffer PBS, each time for 3 minutes; during the soaking and washing period, lift up and down repeatedly

[0091] Color development: Add color developing solution to the tissue section, observe the color change closely under the microscope, and after obtaining the appropriate staining intensity, immerse the section in a large amount of distilled water to stop the color development. After the color development is stopped, wash the section in running water for 10 minutes.

[0092] Counterstaining: Immerse the slightly drained sections in Mayer's hematoxylin for counterstaining for 1 minute. After counterstaining, wash with running water for 3 minutes.

[0093] Bluing: 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;

[0094] 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;

[0095] 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 adding the coverslip, the tissue must be completely covered and no glue should overflow from the slice for scanning.

[0096] It should be noted that immunohistochemical staining results are divided into: positive and negative. Positive expression must be in specific antigenic sites of cells and tissues to be considered positive.

[0097] Figures 3 to 5 The staining performance in human colon cancer, human undifferentiated esophageal cancer and human epithelioid sarcoma (INI-1 deletion) is shown. This shows that the anti-human SMARCB1 rabbit monoclonal antibody provided in the embodiment has accurate staining positioning, clear staining without nonspecific staining, and a clean background. Combined with the results of immunoblotting and immunoprecipitation, it is shown that the anti-human SMARCB1 rabbit monoclonal antibody specifically recognizes human SMARCB1 protein without nonspecific staining, effectively avoiding false positive results.

[0098] Figures 6-7The staining performance of rabbit monoclonal antibodies in mouse heart and rat lung tissue samples is shown. This shows that the SMARCB1 rabbit monoclonal antibody provided in the embodiment has accurate staining positioning, clear staining without nonspecific staining, and a clean background. Combined with the results of immunoblotting and immunoprecipitation, it is shown that the SMARCB1 rabbit monoclonal antibody specifically recognizes the SMARCB1 protein of rats and mice, without nonspecific staining, and effectively avoids false positive results.

[0099] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A monoclonal antibody that specifically binds to a human chromosome remodeling molecule, the monoclonal antibody comprising: three complementarity determining regions (CDRs) of the light chain variable region (VL) defined according to the Kabat numbering system: VL CDR1 as set forth in SEQ ID NO:5, VL CDR2 as set forth in SEQ ID NO:6, and VL CDR3 as set forth in SEQ ID NO:7; Three complementarity determining regions (CDRs) of the heavy chain variable region (VH) are defined according to the Kabat numbering system: VH CDR1 as shown in SEQ ID NO:8, VH CDR2 as shown in SEQ ID NO:9, and VH CDR3 as shown in SEQ ID NO:

10.

2. A monoclonal antibody that specifically binds to a human chromosome remodeling molecule, the monoclonal antibody comprising: The light chain shown in SEQ ID NO: 1; and The heavy chain is shown in SEQ ID NO:

2.

3. A monoclonal antibody that specifically binds to a human chromosome remodeling molecule, the monoclonal antibody comprising: The light chain variable region (VL) as shown in SEQ ID NO:3; and The heavy chain variable region (VH) is shown in SEQ ID NO:

4.

4. A nucleic acid molecule carrying a gene encoding the monoclonal antibody according to any one of claims 1 to 3.

5. A host cell carrying the nucleic acid molecule according to claim 4 and capable of autonomously expressing the monoclonal antibody according to any one of claims 1 to 3.

6. A kit for detecting human chromosome remodeling molecules, the kit comprising: The monoclonal antibody according to any one of claims 1 to 3.

7. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a molecular reagent or kit for detecting human chromosome remodeling.

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