Anti-human MNDA protein monoclonal antibody and hybridoma cell strain and application thereof

Abstract

The invention relates to the technical field of immunoglobulins, in particular to an anti-human myeloid cell nucleus differentiation antigen (MNDA) monoclonal antibody and a hybridoma cell strain and application thereof. The invention provides an anti-human myeloid cell nucleus differentiation antigen (MNDA) monoclonal antibody, and also provides a hybridoma cell strain OTI4H6, the hybridoma cell strain OTI4H6 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 28, 2024, and the preservation number of the hybridoma cell strain OTI4H6 is No.46245. The invention further provides an anti-human myeloid cell nucleus differentiation antigen (MNDA) monoclonal antibody. The hybridoma cell strain OTI4H6 provided by the invention can stably secrete an anti-human myeloid cell nucleus differentiation antigen (MNDA) monoclonal antibody, and can be specifically combined with MNDA protein, so that the specificity, accuracy and reliability of immunodetection are remarkably improved, and the hybridoma cell strain OTI4H6 can be suitable for marking the MNDA protein in tissue cells.

Description

Technical Field

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

[0002] Myeloid Nuclear Differentiation Antigen (MNDA) is a regulatory protein involved in cell differentiation and apoptosis. The MNDA gene is located on human chromosome 1q22 and is a member of the HIN-200 family, which is essential for the production of interferon. The C-terminus of MNDA contains two adjacent oligonucleotide / oligosaccharide binding (OB) domains, which are involved in nucleic acid recognition, DNA replication, recombination, repair, and telomere maintenance.

[0003] MNDA is usually expressed at high levels in the bone marrow and monocytic lineages, and is also expressed in B cells, especially in the marginal zone of the spleen and the marginal and mantle zones of some lymph nodes. In mature B-cell lymphomas, MNDA is mainly expressed in marginal zone lymphoma (MZL), and can be used for the differential diagnosis of MZL from follicular lymphoma (FL), and can also be used for the differential diagnosis of myeloid neoplasms and plasmacytoid dendritic cell neoplasms. In B-cell non-Hodgkin lymphoma (B-NHL), including mantle cell lymphoma (MCL), FL, small lymphocytic lymphoma / chronic lymphocytic leukemia (CLL / SLL), etc., all have typical immunophenotypes, while MZL lacks specific antibodies, and its pathological diagnosis is usually based on exclusive diagnosis, and it is difficult to differentiate from reactive lymphoproliferation.

[0004] MNDA is considered a potential diagnostic marker for MZL. The Chinese Lymphoma Treatment Guidelines (2023 Edition) recommend MNDA as a secondary target for immunohistochemistry (IHC) testing in the pathological diagnosis of extranodal and intranodal marginal zone lymphoma. Studies have shown that MNDA is positive in 77.9% of MZL, 21.9% of MCL, 28.9% of CLL / SLL, 2.6% of FL, and 25% of lymphoplasmacytic lymphoma (LPL). Among the three marginal zone lymphoma subtypes, MNDA has the highest positive rate in extranodal marginal zone lymphoma. Both MNDA and immune receptor translocation-associated protein 1 (IRTA1) can be used as markers for marginal zone and mucosal lymphoid tissue, and their combined use is helpful for the clinical diagnosis of gastric extranodal marginal zone lymphoma. The combined use of CD43 and MNDA can increase the diagnostic sensitivity of MZL from 78% to 88%.

[0005] Therefore, the development of monoclonal antibodies that specifically recognize MNDA protein is of great significance for the diagnosis and treatment of MZL. Summary of the invention

[0006] In view of this, the present application provides a monoclonal antibody against human myeloid cell nuclear differentiation antigen (MNDA), a hybridoma cell line capable of secreting the anti-human MNDA protein monoclonal antibody, and the application of the anti-human MNDA protein monoclonal antibody and hybridoma cell line.

[0007] On the one hand, the present application provides a monoclonal antibody or antigen-binding fragment against human myeloid cell nuclear differentiation antigen (MNDA), wherein the monoclonal antibody or antigen-binding fragment comprises a VL domain, wherein the VL domain comprises LCDR1-3, wherein the amino acid sequence of LCDR1 comprises QSLLDSDETY (SEQ ID NO.4), the amino acid sequence of LCDR2 comprises LTS (SEQID NO.5), and the amino acid sequence of LCDR3 comprises WQGTH (SEQ ID NO.6).

[0008] Furthermore, the monoclonal antibody or antigen-binding fragment also comprises a VH domain, the VH domain comprises HCDR1-3, the amino acid sequence of the HCDR1 comprises GFTFSRYA (SEQ ID NO.8), the amino acid sequence of the HCDR2 comprises INSGGSVP (SEQ ID NO.9), and the amino acid sequence of the HCDR3 comprises TTSTT (SEQ ID NO.10).

[0009] Further, the VL domain comprises an amino acid sequence as shown in SEQ ID NO.3, or an amino acid sequence with more than 95% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence shown in SEQ ID NO.3, preferably, an amino acid sequence with 95%, 96%, 97%, 98% or 99% homology.

[0010] Furthermore, the VL domain has a total length of 100 amino acids, the amino acid numbers of the four FR domains are 26, 17, 28 and 11 respectively, and the amino acid numbers of the LCDR1-3 domains are 10, 3 and 5 respectively, which are 27-36aa, 54-56aa and 85-89aa respectively.

[0011] Further, the VH domain comprises an amino acid sequence as shown in SEQ ID NO.7, or an amino acid sequence with more than 95% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence as shown in SEQ ID NO.7, preferably, an amino acid sequence with 95%, 96%, 97%, 98% or 99% homology.

[0012] Furthermore, the VH domain has a total length of 110 amino acids, the amino acid numbers of the four FR domains are 24, 17, 37 and 11 respectively, and the amino acid numbers of the HCDR1-3 domains are 8, 8, 5, 25-32aa, 50-57aa and 95-99aa respectively.

[0013] On the other hand, the present application also provides a hybridoma cell line capable of secreting anti-human MNDA protein monoclonal antibodies or antigen-binding fragments, the hybridoma cell line being a mouse anti-human MNDA monoclonal hybridoma cell line OTI4H6, whose deposit number is CGMCC No.46245, and which was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on November 28, 2024. The address of the depository unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] On the other hand, the present application also provides a monoclonal antibody or antigen-binding fragment against human MNDA protein, wherein the monoclonal antibody or antigen-binding fragment against human MNDA protein is obtained by secretion of the hybridoma cell line OTI4H6.

[0015] On the other hand, the present application also provides an application of the monoclonal antibody or antigen-binding fragment, characterized in that the application is any of the following: 1) labeling of human MNDA protein in tissue; 2) preparing an immunohistochemical detection kit for human MNDA protein.

[0016] Furthermore, the tissues include tonsils, appendix, follicular lymphoma and other tissues.

[0017] On the other hand, the present application also provides an immunohistochemical detection kit for human MNDA protein, wherein the immunohistochemical detection kit comprises a primary antibody detection reagent, wherein the primary antibody detection reagent contains the monoclonal antibody or antigen fragment.

[0018] Furthermore, the kit also includes antigen repair solution, endogenous peroxidase blocker, ultrasensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color developing solution, and hematoxylin staining solution.

[0019] Furthermore, the kit also includes antigen repair solution, endogenous peroxidase blocker, ultra-sensitive secondary antibody reagent, DAB substrate buffer, DAB concentrated color development solution, and hematoxylin staining solution; preferably, the secondary antibody reagent is an ultra-sensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0020] Preservation Information Biomaterial: OTI4H6 Classification and naming: Mouse anti-human MNDA monoclonal hybridoma cell line Deposit number: CGMCC No.46245 Deposit date: November 28, 2024 Depository: China General Microbiology Center (CGMCC) Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] Compared with the prior art, the present application provides a monoclonal antibody that can specifically bind to human MNDA protein, including a monoclonal antibody against human MNDA protein obtained by hybridoma technology screening. At the same time, the present application also establishes an immunohistochemical detection method and a detection kit based on the anti-human MNDA protein monoclonal antibody. The immunohistochemical detection kit of the present application can achieve specific labeling of human MNDA protein in tissues including tonsils, appendix and follicular lymphoma, and can also be used for chemical detection of immunohistochemistry and for identifying lymphomas associated with human MNDA protein expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation of the present application, the drawings required for describing the specific implementation are briefly introduced below.

[0023] Figure 1 This is a graph showing the results of Western blot detection of the recombinant MNDA protein described in Example 2 of the present invention, in which the expression of the recombinant MNDA protein in E. coli cells was detected using anti-HIS antibody, wherein lane L is the detection result using the E. coli cell lysate transfected with an empty vector as the antigen, and lane R is the detection result using the E. coli cell lysate transfected with a pET23a-His-rMNDA plasmid as the antigen.

[0024] Figure 2 This is a diagram of the SDS-PAGE result of the MNDA protein described in Example 2 of the present invention. The recombinant MNDA protein was purified using a nickel affinity chromatography column, and the purified protein was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining.

[0025] Figure 3 This is a schematic diagram of the results of IHC detection of tonsils using the monoclonal antibody secreted by OTI4H6 described in Example 6 of the present invention.

[0026] Figure 4 This is a schematic diagram of the results of IHC detection of the appendix using the monoclonal antibody secreted by OTI4H6 described in Example 7 of the present invention.

[0027] Figure 5 This is a schematic diagram of the results of IHC detection of follicular lymphoma using the monoclonal antibody secreted by OTI4H6 described in Example 8 of the present invention. DETAILED DESCRIPTION

[0028] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples that do not specify specific conditions are usually carried out according to conventional conditions, conditions described in laboratory manuals, or conditions recommended by manufacturers.

[0029] Example 1 Construction of MNDA protein recombinant expression plasmid The MNDA gene NM_002432.2 was selected from Genebank, and the amino acid sequence of the MNDA protein was analyzed for antigenic epitopes, mainly evaluating biological characteristics, antigenicity, hydrophilicity, amino acid category and distribution, homology between human sequences and immune animals, etc., and finally determining that the 300 amino acids at positions 1-300 of the MNDA protein were used as the amino acid sequence of the synthetic protein. The corresponding amino acid sequence was designed and the DNA sequence was synthesized, and the MNDA gene was synthesized. The nucleotide sequence is shown in SEQ ID NO.1, and the corresponding MNDA protein amino acid sequence is shown in SEQ ID NO.2. Restriction endonuclease sites SgfI and MluI were introduced on both sides of the gene, respectively, and cloned into the expression vector pET23a-N-His to establish the MNDA recombinant expression plasmid pET23a-His-rMNDA.

[0030] Example 2 Expression and purification of recombinant MNDA protein (1) Transformation of E. coli cells: Thaw 100 μl competent cells on ice, add recombinant plasmid DNA and mix gently. Heat shock at 42°C for 90 seconds after ice bath for 30 minutes, and then continue to ice bath for 1.5 minutes. Add 500 μl fresh antibiotic-free LB medium in a clean bench, incubate at 37°C on a shaker for 45 minutes, take an appropriate amount of bacterial solution and evenly spread it on the plate containing antibiotics, and place the culture dish upside down in a 37°C constant temperature incubator for overnight incubation.

[0031] (2) Cell lysis: On the second day, 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 h. Collect the cells by centrifugation, then resuspend the cells in lysis buffer, ultrasonically disrupt for 20 min, and centrifuge at 12000 rpm at 4°C for 20 min. Collect the supernatant. Take a small amount of supernatant protein and use anti-His antibody for WB identification, see Figure 1 .

[0032] Depend on Figure 1The results showed that after WB detection, there was an obvious specific band (R) in the E. coli cell lysate transfected with pET23a-His-rMNDA plasmid, and the molecular weight was consistent with the expected molecular weight, while there was no band of the corresponding size in the control lysate (L) transformed with an empty vector, indicating that the recombinant MNDA protein was specifically expressed in the cells.

[0033] (3) Nickel affinity chromatography column purification: Filter the supernatant of the centrifuged lysate with a 0.45 μM, 33 mm PVDF membrane filter and transfer it into a 15 mL tube. Add 1 mL of the mixed beads, seal the tube and place it in a 360-degree mixer to bind at 4°C for 2 hours. Take out the 15 mL tube, pour the lysate into the BIO-RAD chromatography column, and collect the flow-through. After dripping, take a sample of the flow-through for WB detection. Rinse the column material with lysis buffer 1-2 times, rinse the beads 3 times with TBST after dripping, and elute with 0.1 M Glycine buffer (pH 3.5) after dripping. The first time is 200 μL, which is not collected until dripped. The second and third times are 500 μL each, and the fourth time is 250 μL. Collect them in a 1.5 mL centrifuge tube and quickly add NaH2PO4 buffer (pH 11.0) was neutralized to pH 7.0, glycerol was added to each tube to a final concentration of 10%, and Tween-80 was added to a final concentration of 0.1%. The purified recombinant MNDA protein was identified by SDS-PAGE, see Figure 2 .

[0034] Depend on Figure 2 The results showed that the purified protein had obvious specific bands on the SDS-PAGE electrophoresis gel. The bands were clear and complete, and the molecular weight was consistent with the expected molecular weight, indicating that the recombinant MNDA protein with good purity had been obtained.

[0035] Example 3 Preparation and screening of monoclonal antibodies secreted by OTI4H6 against human MNDA protein BALB / c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were immunized with purified recombinant MNDA protein (hereinafter referred to as MNDA antigen) according to standard methods. The specific method is as follows: (1) Animal immunization: The purified MNDA antigen was emulsified with 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, the second immunization was performed with incomplete Freund's adjuvant emulsified at a dose of 30 μg / mouse. After the second immunization, tail blood was collected and serum titer was determined by gradient dilution using ELISA. The results determined whether to boost immunization and selected mice with the highest antibody titer for cell fusion.

[0036] (2) Cell fusion: Myeloma cells were sp2 / 0 cells derived from BALB / c cells. The cells were in the logarithmic growth phase during 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 1:7, and 1 mL of 50% PEG (pH 8.0) preheated at 37°C was added dropwise. Incomplete culture medium and stop solution were added. After centrifugation and discarding the supernatant, HAT culture medium was added to suspend and mix. The MC volume was adjusted to 50 mL and dispensed into 3.5 cm culture dishes. The dishes were placed in a humidified box and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0037] (3) Screening and cloning: After 8 days of fusion, hybridoma cell clones were selected and ELISA tests were performed using purified recombinant MNDA protein. The cell lines were marked and limiting dilutions were performed on the cells in the positive wells. The ELISA values ​​were measured 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 finalization, and the corresponding fusion plate cell line was OTI4H6.

[0038] (4) Preparation and purification of monoclonal antibodies on cells: The hybridoma cell line OTI4H6 was cultured in a 10 cm culture dish using DMEM medium containing 15% serum and expanded to approximately 410 μg / mL. 7 When the cell density is about 310 cells, centrifuge at 800 rpm for 5 min, discard the supernatant and transfer the cells to a 2L spinner bottle, add serum-free medium, and make the cell density about 310 5 After 1.5 weeks of continuous culture, when the cell death rate reached 60%, the cell suspension was collected and centrifuged at 6000 rpm for 20 minutes, the supernatant was taken, and the supernatant was purified by affinity chromatography. The corresponding column material was selected according to the antibody subtype for purification (the subtype was IgG2a, and protein G column material was used for purification). The concentration of the purified monoclonal antibody was measured, lyophilized and packaged (100 μg / tube), and finally stored at -20°C.

[0039] Example 4 Analysis of variable region genes and amino acid sequences of anti-human MNDA protein monoclonal antibodies Purchase SMARTer from Takara Bio USA ® 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 the specific experimental process, please refer to Takara Bio USA's SMARTer ® RACE 5' / 3'Kit User Manual.

[0040] Since the antibody is of IgG2a subtype, specific gene primers pRace-H-GSP and pRace-K-GSP targeting the 3' end of its Ig and Kappa constant regions were designed. The primer sequences are as follows: pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO: 11) pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO: 12).

[0041] mRNA was extracted from hybridoma cell OTI4H6, reverse transcribed into cDNA, and RACE Amplify the DNA fragments of antibody heavy and light chains. Connect the amplified light and heavy chains to the cloning vector PUC119 by enzyme digestion, pick positive clones by blue-white staining, purify positive plasmids for sequencing, use sequencer ABI 3730, and use universal primers M13F and M13R for sequencing primers.

[0042] M13F: 5'-TGTAAAACGAGCGGCCAGT-3' (SEQ ID NO: 13) M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 14).

[0043] Using the Internet, IMGT / V-QUEST analysis software was used at http: / / www.imgt.org to perform data analysis on the sequencing results of the nucleotide sequences of the light chain and heavy chain of the anti-human MNDA protein monoclonal antibody, respectively, to obtain the VL domain amino acid sequence of the anti-human MNDA protein monoclonal antibody as shown in SEQ ID NO.3, and the VH domain amino acid sequence as shown in SEQ ID NO.7.

[0044] The total length of the VL domain is 100 amino acids, the number of amino acids in the four FR domains are 26, 17, 28 and 11 respectively, the number of amino acids in the three LCDR domains are 10, 3 and 5 respectively, the regions of LCDR1, LCDR2 and LCDR3 are 27-36aa, 54-56aa and 85-89aa respectively, and their amino acid sequences are: QSLLDSDETY (SEQ ID NO.4), LTS (SEQ IDNO.5), WQGTH (SEQ ID NO.6).

[0045] The total length of the VH domain is 110 amino acids, the amino acid numbers of the four FR domains are 24, 17, 37 and 11 respectively, the amino acid numbers of the three HCDR domains are 25-32aa, 50-57aa and 95-99aa respectively, HCDR1, HCDR2 and HCDR3 are 8, 8 and 5 respectively, and their amino acid sequences are: GFTFSRYA (SEQ ID NO.8), INSGGSVP (SEQ ID NO.9), TTSTT (SEQ ID NO.10).

[0046] Example 5 Immunohistochemical detection kit containing anti-human MNDA protein monoclonal antibody An immunohistochemical detection kit containing an anti-human MNDA protein monoclonal antibody comprises an antigen repair solution [1 mM EDTA, 10 mM Tris buffer (pH 8.0)], the anti-human MNDA 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 colorimetric solution, and a hematoxylin staining solution.

[0047] Example 6 Immunohistochemical Detection of Tonsilla Using Anti-Human MNDA Protein Monoclonal Antibody as Primary Antibody The tonsil tissue was tested using the immunohistochemical detection kit in Example 5, and the specific steps are as follows: (1) Formalin-fixed tonsil tissue blocks were obtained and embedded in paraffin. The sections were sliced ​​using a Leica tissue slicer with a tissue thickness of 4 μm.

[0048] (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.

[0049] (3) Antigen repair: Add antigen repair solution and perform high-pressure heat repair in a pressure cooker for 3 min. 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 them in deionized water for 2 min 3 times.

[0050] (4) Inactivation: Use 3% hydrogen peroxide to inactivate endogenous peroxidase in tissues, let stand at room temperature for 15 min, and soak in deionized water for 2 min 3 times.

[0051] (5) Use an immunohistochemistry pen to draw a frame around the tissue and wash once with 0.1% PBST for 2 min.

[0052] (6) Incubation with primary antibody: Add 200 μl of diluted anti-human MNDA protein monoclonal antibody secreted by hybridoma cell line OTI4H6 and place in a wet box, incubate at 37°C for 60 min. Wash with 0.1% PBST for 2 min 3 times.

[0053] (7) Incubation with secondary antibody: Add 100 μl of secondary antibody ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer and incubate at 37°C for 30 min. Wash with 0.1% PBST for 2 min 3 times.

[0054] (8) DAB color development: Add 120 μl of DAB color development solution, let stand at room temperature for 5 min, rinse under tap water to stop color development, and rinse three times with tap water.

[0055] (9) Hematoxylin re-staining, differentiation and blueing: Place in hematoxylin solution for 12 seconds, rinse with tap water three times to stop color development, differentiate in 1% hydrochloric acid ethanol solution, rinse with tap water three times to stop, place in freshly boiled Tris-EDTA disodium solution at pH 8.0 to blue, then place in room temperature Tris-EDTA disodium solution at pH 8.0 for a few seconds, rinse with tap water three times. Observe the staining under a microscope. If it is normal, stop and recover the hematoxylin staining solution; if it is too differentiated, repeat the above steps until the staining is qualified.

[0056] (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.

[0057] (11) Microscopic examination, the results are as follows Figure 3 shown.

[0058] The results showed that MNDA protein was only expressed in the nucleus of B lymphocytes and monocytes in the tonsil mantle region. Figure 3 The solid arrows in the figure indicate that MNDA protein is positively expressed in B lymphocytes and monocytes in the tonsil mantle region, but B lymphocytes, plasma cells and T cells located in the germinal center do not express MNDA, which is indicated by dotted arrows.

[0059] Example 7: Immunohistochemical detection of appendix using anti-human MNDA protein monoclonal antibody as primary antibody The immunohistochemical detection kit in Example 5 was used to detect the appendix tissue, and the specific steps were the same as in Example 6. The results are as follows: Figure 4 As shown, Figure 4 The solid arrows indicate monocytes and B lymphocytes that are positive for MNDA protein expression, and the dotted arrows indicate glandular epithelial cells and B lymphocytes located in the germinal center that are negative for MNDA protein expression.

[0060] Example 8 Immunohistochemical Detection of Follicular Lymphoma Using Anti-Human MNDA Protein Monoclonal Antibody as Primary Antibody The immunohistochemical detection kit in Example 5 was used to detect follicular lymphoma tissue, and the specific steps were the same as in Example 6. The results are as follows: Figure 5 As shown, Figure 5 The solid arrows in the figure indicate monocytes that are positive for MNDA protein expression, the dotted arrows indicate follicular lymphoma cells that are negative for MNDA protein expression, and there is no nonspecific background staining.

[0061] The above results indicate that the anti-human MNDA monoclonal antibody secreted by the hybridoma cell OTI4H6 described in the present application has high specificity and sensitivity, is only expressed in monocytes and B lymphocytes dispersed in the mantle zone, and at a concentration of 0.25 μg / ml, the staining intensity is strong and the cell nucleus staining is clear and distinct. Therefore, the MNDA monoclonal antibody secreted by OTI4H6 can be used for immunohistochemical detection and for the study of MNDA-related lymphoma diseases.

[0062] 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 modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against human myeloid cell nuclear differentiation antigen (MNDA), characterized in that: The monoclonal antibody or antigen-binding fragment comprises a VL domain, wherein the VL domain comprises LCDR1-3, wherein the amino acid sequence of LCDR1 comprises QSLLDSDETY, the amino acid sequence of LCDR2 comprises LTS, and the amino acid sequence of LCDR3 comprises WQGTH.

2. The monoclonal antibody or antigen-binding fragment according to claim 1, characterized in that The monoclonal antibody or antigen-binding fragment further comprises a VH domain, wherein the VH domain comprises HCDR1-3, wherein the amino acid sequence of the HCDR1 comprises GFTFSRYA, the amino acid sequence of the HCDR2 comprises INSGGSVP, and the amino acid sequence of the HCDR3 comprises TTSTT.

3. The monoclonal antibody or antigen-binding fragment according to claim 1, characterized in that The VL domain comprises the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence having more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence shown in SEQ ID NO.

3.

4. The monoclonal antibody or antigen-binding fragment according to claim 2, characterized in that: The VH domain comprises the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having more than 90% homology obtained by replacing, deleting and / or adding one or more amino acids and / or terminally modifying any one or more amino acids in the amino acid sequence shown in SEQ ID NO.

7.

5. A hybridoma cell line capable of secreting anti-human myeloid cell nuclear differentiation antigen (MNDA) monoclonal antibody, characterized in that: The hybridoma cell line is the mouse anti-human MNDA monoclonal hybridoma cell line OTI4H6, whose preservation number is CGMCC No.46245, and it was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on November 28, 2024.

6. A monoclonal antibody or antigen-binding fragment against human myeloid cell nuclear differentiation antigen (MNDA), characterized in that: The anti-human myeloid cell nuclear differentiation antigen (MNDA) monoclonal antibody or antigen-binding fragment is obtained by secretion from the hybridoma cell line according to claim 5.

7. Use of the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 4 or claim 6, characterized in that: The application is any of the following, 1) Labeling of human MNDA protein in cells or tissues; 2) Prepare an immunohistochemical detection kit for human MNDA protein.

8. An immunohistochemical detection kit for human MNDA protein, characterized in that: The immunohistochemical detection kit comprises a primary antibody detection reagent, and the primary antibody detection reagent contains the monoclonal antibody or antigen fragment according to any one of claims 1 to 4 or claim 6.

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

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

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