Monoclonal antibody against human BCOR protein, hybridoma cell line thereof and application

Abstract

This application relates to the field of immunoglobulin technology, specifically to anti-human BCOR protein monoclonal antibodies, hybridoma cell lines, and applications thereof. This application provides an anti-human BCOR protein monoclonal antibody secreted by the hybridoma cell line OTI3D11. The hybridoma cell line OTI3D11 was deposited with the General Microbiology Center of the China General Microbiology Culture Collection (CGMCC) on November 28, 2024, with the deposit number CGMCC No. 46244. The anti-human BCOR protein monoclonal antibody secreted by the hybridoma cell line OTI3D11 described in this application has high specificity and sensitivity. Therefore, the anti-BCOR monoclonal antibody secreted by the hybridoma cell line OTI3D11 can be used in immunohistochemical assays to detect BCOR expression in related tumors, providing a basis for clinicians to diagnose patients.

Description

Technical Field

[0001] The present application relates to the technical field of immunoglobulins, and in particular to anti-human BCOR protein monoclonal antibodies and hybridoma cell lines and applications thereof. Background Art

[0002] The BCOR (BCL6 co-repressor) gene, located on chromosome Xp11.4, is a POZ / zinc finger transcriptional repressor composed of 1755 amino acids. The function of the BCOR protein is related to its two main domains: the BCL6-binding domain, which binds to BCL6 and co-represses gene transcription, exerting an anti-apoptotic effect; and the PUFD domain, which binds to the polycomb repressive complex 1 (PRC1), inhibits the ubiquitination of histone H2A, thereby repressing gene transcription through epigenetic mechanisms. It plays an important role in normal early embryonic development and is expressed in a variety of tumors. The changes in the BCOR gene in tumors mainly include BCOR rearrangement and BCOR internal tandem duplication. It has been found that the partner genes that rearrange with BCOR include CCNB3, MAML3, ZC3H7B and many others. Tumors with BCOR gene abnormalities include BCOR-altered sarcoma, BCOR-CCNB3 sarcoma, BCOR-ITD sarcoma, renal clear cell sarcoma, high-grade endometrial stromal sarcoma, central nervous system tumors (including high-grade CNS-PNET with BCOR abnormalities, medulloblastoma, retinoblastoma, glioma, etc.), blood system-related tumors (such as MDS in myeloid tumors, MDS with multilineage abnormalities, MDS with increased primitive cells, AML with trisomy 11, AML with trisomy 13, etc.), etc.

[0003] BCOR expression has differential diagnostic value in a variety of tumors. For example, BCOR is a specific marker for high-grade endometrial stromal sarcoma (HGESS) with a YWHAE-NUTM2 fusion. BCOR expression also aids in the diagnosis of small round blue cell sarcoma with BCOR gene abnormalities. In renal clear cell sarcoma, BCOR-positive expression is part of its characteristic histomorphology. BCOR is primarily localized to the cell nucleus in immunohistochemical testing. In BCOR-CCNB3 sarcomas, BCOR demonstrates nuclear positivity, often with strong and diffuse nuclear labeling. This nuclear positivity helps distinguish BCOR-associated tumors from other tumor types.

[0004] Therefore, it is crucial to develop highly specific and sensitive monoclonal antibodies that recognize BCOR protein for the differential diagnosis of related tumors. Summary of the Invention

[0005] In view of this, the present application provides an anti-human BCOR protein monoclonal antibody, a hybridoma cell line capable of secreting the anti-human BCOR protein monoclonal antibody, and applications of the anti-human BCOR protein monoclonal antibody and hybridoma cell line.

[0006] On the one hand, the present application provides a monoclonal antibody or an antigen-binding fragment thereof against human BCOR protein, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1-3, the amino acid sequence of LCDR1 comprises QSLFNSRTRKNY (SEQ ID NO. 4), the amino acid sequence of LCDR2 comprises DTS (SEQ ID NO. 5), and the amino acid sequence of LCDR3 comprises KQSY (SEQ ID NO. 6).

[0007] Furthermore, the heavy chain variable region comprises HCDR1-3, the amino acid sequence of the HCDR1 comprises GFSIKDTY (SEQ ID NO.8), the amino acid sequence of the HCDR2 comprises IDPANGDT (SEQ ID NO.9), and the amino acid sequence of the HCDR3 comprises DYRGEWFVY (SEQ ID NO.10).

[0008] Furthermore, the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO.3, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminal modification in the amino acid sequence shown in SEQ ID NO.3, preferably, an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0009] Furthermore, the light chain variable region has a total length of 109 amino acids, the number of amino acids in the four FR domains are 26, 17, 36 and 11 respectively, the number of amino acids in the three domains of LCDR1-3 are 12, 3 and 4 respectively, and the regions of LCDR1, LCDR2 and LCDR3 are 27aa-38aa, 56aa-58aa and 95aa-98aa respectively.

[0010] Furthermore, the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO.7, or an amino acid sequence with more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminal modification in the amino acid sequence shown in SEQ ID NO.7, preferably, an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0011] Furthermore, the full length of the heavy chain variable region is 113 amino acids, the amino acid numbers of the four domains of FR are 24, 17, 36 and 11 respectively, the amino acid numbers of the three domains of HCDR1-3 are 8, 8 and 9 respectively, and the HCDR1, HCDR2 and HCDR3 are 25aa-32aa, 50aa-57aa and 94aa-102aa respectively.

[0012] On the other hand, the present application also provides a hybridoma cell line capable of secreting anti-human BCOR protein monoclonal antibodies or antigen-binding fragments, the hybridoma cell line being the mouse anti-human BCOR monoclonal hybridoma cell line OTI3D11, whose deposit number is CGMCC No. 46244, and which was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on November 28, 2024. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] On the other hand, the present application also provides a monoclonal antibody or antigen-binding fragment thereof against human BCOR protein, wherein the monoclonal antibody or antigen-binding fragment thereof against human BCOR protein is obtained by secretion of the hybridoma cell line OTI3D11.

[0014] On the other hand, the present application also provides an application of the monoclonal antibody or its antigen-binding fragment, characterized in that the application is any of the following: 1) labeling of human BCOR protein in cells or tissues; 2) preparing an immunohistochemical detection kit for human BCOR protein.

[0015] Furthermore, the cells are cancer cells or normal tissue cells, preferably endometrial stromal sarcoma tissue.

[0016] On the other hand, the present application also provides an immunohistochemical detection kit for human BCOR protein, wherein the immunohistochemical detection kit comprises an antibody detection reagent containing the monoclonal antibody or an antigen-binding fragment thereof.

[0017] Furthermore, the kit also includes antigen repair solution, endogenous peroxidase blocker, ultrasensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color development solution, and hematoxylin staining solution.

[0018] Furthermore, the kit also includes antigen repair solution, endogenous peroxidase blocker, ultrasensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color development solution, and hematoxylin staining solution; preferably, the secondary antibody reagent is ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0019] Preservation Information

[0020] Biomaterial: OTI3D11

[0021] Classification and naming: Mouse anti-human BCOR monoclonal hybridoma cell line

[0022] Accession number: CGMCC No.46244

[0023] Deposit date: November 28, 2024

[0024] Depository: China General Microbiology Center (CGMCC)

[0025] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0026] Compared to the prior art, the anti-human BCOR protein monoclonal antibodies or antigen-binding fragments provided in this application are capable of specifically binding to human BCOR protein, significantly improving the specificity, accuracy, and reliability of BCOR protein immunoassays. Furthermore, the hybridoma cell line OTI3D11 provided herein can also stably secrete anti-human BCOR protein monoclonal antibodies. Secondly, using the anti-human BCOR protein monoclonal antibodies and kits of this application to detect tissues such as endometrial stromal sarcomas using IHC, BCOR protein expression in cells can be clearly detected, indicating that the anti-human BCOR protein monoclonal antibodies of this application are well suited for immunohistochemical detection of BCOR protein in tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific implementation of the present application, the following is a brief introduction to the drawings required for describing the specific implementation.

[0028] Figure 1 This is a graph showing the Western blot detection results of the recombinant BCOR protein in Example 2. The expression of the recombinant BCOR protein in E. coli cells was detected using an anti-HIS antibody. Lane L shows the detection result using the lysate of E. coli cells transfected with an empty vector as the antigen, and lane R shows the detection result using the lysate of E. coli cells transfected with the pET23a-rBCOR plasmid as the antigen.

[0029] Figure 2 This is the SDS-PAGE result of BCOR protein in Example 2. The recombinant BCOR protein was purified using a nickel affinity chromatography column, and the purified protein was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining.

[0030] Figure 3 This is a schematic diagram of the results of IHC detection of endometrial stromal sarcoma using the monoclonal antibody secreted by the hybridoma cell line OTI3D11 in Example 6. DETAILED DESCRIPTION

[0031] The present application will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, conditions described in laboratory manuals, or conditions recommended by the manufacturer.

[0032] Example 1 Construction of BCOR protein recombinant expression plasmid

[0033] The BCOR gene NM_001123385 was selected from Genebank. Based on the amino acid sequence characteristics of BCOR, the amino acid fragment of BCOR at positions 1225-1465 was designed as an immunogen (the corresponding nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2). Primers were designed, and restriction endonuclease sites SgfI and MluI were introduced on both sides of the gene, respectively. The gene was inserted into the expression vector pET23a-N-His to construct the recombinant expression plasmid pET23a-rBCOR.

[0034] Example 2 Expression and purification of recombinant BCOR protein

[0035] (1) Transformation of E. coli cells: Thaw 100 μl of competent cells on ice, then add the recombinant plasmid DNA and mix gently. After ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, and then continue to ice-bath for 1-2 minutes. Add 500 μl of fresh antibiotic-free LB medium in a clean bench, incubate on a shaker at 37°C for 45 minutes, and then evenly spread an appropriate amount of bacterial solution on a plate containing antibiotics. Place the culture dish upside down in a 37°C constant temperature incubator and culture overnight.

[0036] (2) Cell lysis: Pick a single clone and place it in fresh culture medium. Cultivate at 37°C and 200 rpm until the OD value reaches 0.4-0.6. Add IPTG (final concentration 1 mM) and induce the culture for 7 hours. Collect the cells by centrifugation, then resuspend the cells in lysis buffer, ultrasonically disrupt them for 20 minutes, and centrifuge them at 12000 rpm at 4°C for 20 minutes. Collect the supernatant. Take a small amount of supernatant protein and use anti-His antibody for Western blotting. Figure 1 .

[0037] Depend on Figure 1 The results showed that the E. coli cell lysate transfected with the pET23a-rBCOR plasmid was located in lane R, with an obvious specific band at 31kD, and the molecular weight was consistent with the theoretical molecular weight of the selected amino acid fragment. However, there was no band of the corresponding size in lane 1 of the control lysate transformed with the empty vector, indicating that the cells specifically expressed the recombinant BCOR protein.

[0038] (3) Nickel affinity chromatography column purification: Equilibrate the nickel column with buffer, filter the supernatant with a 0.45 μm filter membrane, apply the sample, collect the outflow, elute with buffer to remove unbound proteins, and finally elute with eluent containing different concentrations of imidazole. After collection, the eluted proteins that meet the requirements are combined and 10% glycerol is added. The purified recombinant BCOR protein is identified by SDS-PAGE electrophoresis, see Figure 2 .

[0039] Depend on Figure 2 The results showed that the purified protein had an obvious specific band at 31kD in the SDS-PAGE electrophoresis gel image, which was consistent with the theoretical predicted molecular weight of the immunogen amino acid fragment, indicating that a recombinant BCOR protein with good purity had been obtained.

[0040] Example 3 Preparation and Screening of Monoclonal Antibodies Secreted by OTI3D11 Against Human BCOR Protein

[0041] BALB / c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were immunized with purified recombinant BCOR protein (hereinafter referred to as BCOR antigen) according to standard methods. The specific method is as follows:

[0042] (1) Animal immunization: Purified BCOR antigen was emulsified in complete Freund's adjuvant and immunized into 6-8 week old BALB / c mice by subcutaneous or intraperitoneal injection at a dose of 60 μg / mouse. Two weeks later, a second immunization was performed using incomplete Freund's adjuvant emulsified at a dose of 30 μg / mouse. After the second immunization, tail blood was collected and serum titers were determined by serial dilution ELISA. Based on the results, the decision on whether to boost immunization was made, and the mouse with the highest antibody titer was selected for cell fusion.

[0043] (2) Cell fusion: Myeloma cells were sp2 / 0 derived from BALB / c cells, which were in the logarithmic growth phase at the time of fusion. The spleen of immunized mice was taken to prepare a single-cell suspension of lymphocytes. Mouse spleen lymphocytes and myeloma cells were mixed at a ratio of 1:5-1:10, and 1 mL of 50% PEG (pH 8.0) preheated at 37°C was added dropwise. Incomplete culture medium and stop solution were added, and the supernatant was discarded after centrifugation. HAT culture medium was added to suspend and mix thoroughly. The MC volume was adjusted to 50 mL, and the cells were divided into 3.5 cm culture dishes, placed in a humidified box, and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0044] (3) Screening and cloning: Within 7-10 days of fusion, hybridoma cell clones were selected and tested by ELISA using purified recombinant BCOR protein. The cell line number was marked. The cells in the positive wells were subjected to limiting dilution. The ELISA value was measured 5-6 days after each limiting dilution. The monoclonal wells with higher OD280 positive values were selected for limiting dilution until the ELISA results of the entire 96-well plate were positive. The monoclonal wells with higher positive values were selected for identification. The corresponding fusion plate cell line was OTI3D11.

[0045] (4) Preparation and purification of monoclonal antibodies on cells: Hybridoma cell line OTI3D11 was cultured in DMEM containing 15% serum in a 10 cm culture dish and expanded to about 4 × 10 7 When the cells reached 100,0 ... 5 After 1-2 weeks of culture, when the cell death rate reaches 60%-70% (the cell density is about 1-2´10 6 The cell suspension was collected and centrifuged at 6000 rpm for 20 minutes. The supernatant was purified by affinity chromatography using a column material corresponding to the antibody subtype (IgG2b, protein G column material). The purified anti-human BCOR protein monoclonal antibody was concentrated, lyophilized, and aliquoted (100 μg / tube) for storage at -20°C.

[0046] Example 4 Analysis of variable region genes and amino acid sequences of anti-human BCOR protein monoclonal antibodies

[0047] Purchase SMARTer from Takara Bio USA ® The RACE 5' / 3' kit uses 5'RACE (Rapid Amplification of cDNA Ends) technology to amplify the variable region light chain and heavy chain gene sequences of functional antibodies in hybridoma cells. For detailed experimental procedures, please refer to the SMARTer ® RACE 5' / 3'Kit User Manual.

[0048] Since the antibody is of IgG2b subtype, specific gene primers pRace-H-GSP and pRace-K-GSP were designed for the 3' end of its Ig and Kappa constant regions. The primer sequences are as follows:

[0049] pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO: 11)

[0050] pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO: 12)

[0051] mRNA was extracted from the hybridoma cell line OTI3D11 and reverse-transcribed into cDNA. DNA fragments of the heavy and light chains of the anti-human BCOR protein monoclonal antibody were amplified by RACE. The amplified light and heavy chains were ligated into the cloning vector PUC119 via enzyme digestion. Positive clones were picked by blue-white staining, and the positive plasmids were purified and sequenced using an ABI 3730 sequencer with universal primers M13F and M13R.

[0052] M13F: 5'-TGTAAAACGAGCGGCCAGT-3' (SEQ ID NO:13) M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO:14)

[0053] The nucleotide sequence of the light chain and heavy chain of the anti-human BCOR protein monoclonal antibody was analyzed using IMGT / V-QUEST analysis software on the Internet at http: / / www.imgt.org. The amino acid sequence of the light chain variable region of the anti-human BCOR protein monoclonal antibody was shown as SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region was shown as SEQ ID NO. 7.

[0054] The total length of the light chain variable region is 109 amino acids, the number of amino acids in the four FR domains are 26, 17, 36 and 11 respectively, the number of amino acids in the three LCDR domains are 12, 3 and 4 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27aa-38aa, 56aa-58aa and 95aa-98aa respectively, and their amino acid sequences are QSLFNSRTRKNY (SEQ ID NO.4), DTS (SEQ ID NO.5) and KQSY (SEQ ID NO.6), respectively.

[0055] The full length of the heavy chain variable region is 113 amino acids, with the four FR domains containing 24, 17, 36 and 11 amino acids respectively, the three HCDR domains containing 8, 8 and 9 amino acids respectively, HCDR1, HCDR2 and HCDR3 containing 25aa-32aa, 50aa-57aa and 94aa-102aa respectively, and their amino acid sequences are GFSIKDTY (SEQ ID NO.8), IDPANGDT (SEQ ID NO.9), and DYRGEWFVY (SEQ ID NO.10), respectively.

[0056] Example 5 Immunohistochemistry detection kit containing anti-human BCOR protein monoclonal antibody

[0057] An immunohistochemical detection kit containing an anti-human BCOR protein monoclonal antibody comprises an antigen retrieval solution [1 mM EDTA, 10 mM Tris buffer (pH 8.0)], the anti-human BCOR protein monoclonal antibody prepared and purified in Example 3, an endogenous peroxidase blocker (hydrogen peroxide), a supersensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, a DAB substrate buffer, a DAB concentrated color development solution, and a hematoxylin staining solution.

[0058] Example 6 Immunohistochemical Detection Using Anti-Human BCOR Protein Monoclonal Antibody as Primary Antibody

[0059] The immunohistochemical detection kit in Example 5 was used to detect BCOR protein in tissues, and the specific steps are as follows.

[0060] (1) Formalin-fixed endometrial stromal sarcoma tissue blocks were obtained, embedded in paraffin, and sliced using a Leica tissue slicer with a tissue thickness of 4 μm.

[0061] (2) Dewaxing and hydration: Analytical grade xylene for 10 min 3 times, anhydrous ethanol for 1 min 3 times, 95% ethanol for 1 min, 85% ethanol for 1 min, 75% ethanol for 1 min, and deionized water for 2 min 3 times.

[0062] (3) Antigen repair: Add antigen repair solution and perform high-pressure heat repair in a pressure cooker for 3 minutes. When the temperature of the pressure cooker drops to about 90°C, open the pressure cooker, take out the slices, and then cool them naturally to room temperature. Soak in deionized water for 2 minutes three times.

[0063] (4) Inactivation: Use 3% hydrogen peroxide to inactivate endogenous peroxidase in the tissue, let it stand at room temperature for 15 minutes, and soak it in deionized water for 2 minutes 3 times.

[0064] (5) Use an immunohistochemical pen to draw a frame around the tissue and wash once with 0.1% PBST for 2 minutes.

[0065] (6) Incubation with primary antibody: Add 200 μl of diluted anti-human BCOR protein monoclonal antibody secreted by hybridoma cell line OTI3D11 (concentration: 0.39 μg / mL) and incubate in a humidified chamber at 37°C for 60 min. Wash with 0.1% PBST for 2 min or three times.

[0066] (7) Incubation with secondary antibody: Add 100 μl of secondary antibody PV-8000 and incubate at 37°C for 30 min. Wash with 0.1% PBST for 2 min or three times.

[0067] (8) DAB color development: Add 120 μl of DAB color development solution, let it stand at room temperature for 5 min, rinse under tap water to stop color development, and rinse with tap water three times.

[0068] (9) Hematoxylin counterstaining, differentiation, and blueing: Place the sample in the hematoxylin solution for 10-12 seconds, rinse three times with tap water to stop the color development, differentiate in a 1% hydrochloric acid ethanol solution, rinse three times with tap water to stop the color development, place in a freshly boiled Tris-EDTA disodium solution at pH 8.0 to blue the sample, then place in a room temperature Tris-EDTA disodium solution at pH 9.0 for a few seconds, and rinse three times with tap water. Observe the staining under a microscope. If the staining is normal, stop and recover the hematoxylin staining solution. If the staining is excessive, repeat the above steps until the staining is acceptable.

[0069] (10) Dehydration and transparency: 75% ethanol for 1 min, 85% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 1 min 3 times, xylene for 1 min 3 times, and sealing with neutral gum.

[0070] (11) Microscopic examination, e.g. Figure 3 shown.

[0071] Hybridoma cell OTI3D11 secreted an anti-human BCOR protein monoclonal antibody, detected by IHC in endometrial stromal sarcoma tissue. The tumor cells indicated by the asterisk (illustrative example only) are arranged in oval, short spindle-shaped sheets. IHC revealed a brown color, localized to the nucleus, consistent with the theoretical subcellular localization of BCOR, suggesting that the tumor cells in this tissue sample express BCOR protein. The vascular endothelium (arrowheads) indicated by the arrows (illustrative example only), as well as infiltrating lymphocytes and other stromal cells, showed no positive staining, indicating no BCOR protein expression. This result is consistent with the clinical diagnosis of this case. The monoclonal antibody concentration used in this assay was 0.39 μg / mL. Under these conditions, the positive signal intensity on the tumor cells meets clinical criteria, and there is no background staining that could compromise the diagnosis, demonstrating the good specificity and high sensitivity of the antibody secreted by hybridoma cell OTI3D11.

[0072] These results demonstrate that the anti-human BCOR protein monoclonal antibody secreted by the hybridoma cell line OTI3D11 described herein has high specificity and sensitivity. Therefore, the anti-BCOR monoclonal antibody secreted by the hybridoma cell line OTI3D11 can be used in immunohistochemical assays to detect BCOR expression in related tumors, providing a basis for clinicians to diagnose patients.

[0073] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against human BCOR protein, characterized in that: The monoclonal antibody or its antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1-3, the amino acid sequence of LCDR1 is QSLFNSRTRKNY, the amino acid sequence of LCDR2 is DTS, and the amino acid sequence of LCDR3 is KQSY; the heavy chain variable region comprises HCDR1-3, the amino acid sequence of HCDR1 is GFSIKDTY, the amino acid sequence of HCDR2 is IDPANGDT, and the amino acid sequence of HCDR3 is DYRGEWFVY.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The light chain variable region comprises the amino acid sequence shown in SEQ ID NO.

3.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.

7.

4. A hybridoma cell line capable of secreting anti-human BCOR protein monoclonal antibodies, characterized in that: The hybridoma cell line is the mouse anti-human BCOR monoclonal hybridoma cell line OTI3D11, whose preservation number is CGMCC No.46244, and it was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on November 28, 2024.

5. A monoclonal antibody or antigen-binding fragment thereof against human BCOR protein, characterized in that: The anti-human BCOR protein monoclonal antibody or antigen-binding fragment thereof is obtained by secretion from the hybridoma cell line according to claim 4.

6. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or claim 5, characterized in that: The application is to prepare an immunohistochemical detection kit for human BCOR protein.

7. An immunohistochemical detection kit for human BCOR protein, characterized in that: The immunohistochemical detection kit includes a primary antibody detection reagent, which contains the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or claim 5.

8. The kit according to claim 7, characterized in that The kit also includes antigen repair solution, endogenous peroxidase blocker, ultrasensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color development solution, and hematoxylin staining solution.

9. The kit according to claim 8, characterized in that The ultrasensitive secondary antibody reagent is an ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

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