Monoclonal Antibody Against Human MNDA Protein, Hybridoma Cell Line Thereof, and Application

Abstract

This application relates to the technical field of immunoglobulins, specifically to anti-human myeloid nuclear differentiation antigen (MNDA) monoclonal antibodies, their hybridoma cell lines, and applications. This application provides an anti-human myeloid nuclear differentiation antigen (MNDA) monoclonal antibody. At the same time, this application also provides the hybridoma cell line OTI4H6, which was deposited on November 28, 2024, at the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is No. 46245. The hybridoma cell line OTI4H6 provided by this application can stably secrete an anti-human myeloid nuclear differentiation antigen (MNDA) monoclonal antibody, and can specifically bind to the MNDA protein, significantly improving the specificity, accuracy, and reliability of its immunoassay, and is applicable to the labeling of MNDA protein in tissue cells.

Description

Technical Field

[0001] This application relates to the technical field of immunoglobulins, and specifically relates to a monoclonal antibody against human MNDA protein, its hybridoma cell line, 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 crucial 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 highly expressed in the bone marrow and monocyte lineages and is also expressed in B cells, especially in the marginal zone of the spleen and the marginal zone and mantle zone 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 between MZL and follicular lymphoma (FL), and can also be used for the differential diagnosis between myeloid tumors and plasmacytoid dendritic cell tumors. 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 relatively difficult to distinguish from reactive lymphoid hyperplasia.

[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) detection in the pathological diagnosis of extranodal and nodal marginal zone lymphomas. 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 subtypes of marginal zone lymphoma, MNDA has the highest positive rate in extranodal marginal zone lymphoma. Both MNDA and immunoglobulin receptor translocation-associated protein 1 (IRTA1) can be used as markers for the marginal zone and mucosa-associated lymphoid tissue, and their combined application is helpful for the clinical diagnosis of gastric extranodal marginal zone lymphoma. The combined application of CD43 and MNDA can increase the diagnostic sensitivity of MZL from 78% to 88%.

[0005] Therefore, developing a monoclonal antibody that specifically recognizes 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 nuclear differentiation antigen (MNDA), a hybridoma cell line capable of secreting the monoclonal antibody against human MNDA protein, and the applications of the monoclonal antibody against human MNDA protein and the hybridoma cell line.

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

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

[0009] Furthermore, the VL domain comprises the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence having a homology of more than 95% obtained by substituting, deleting and / or adding one or more amino acids and / or terminal modification to any one or more amino acids in the amino acid sequence shown in SEQ ID NO.3, preferably, an amino acid sequence having a homology of 95%, 96%, 97%, 98% or 99%.

[0010] Furthermore, the full length of the VL domain is 100 amino acids, the number of amino acids in the 4 domains of the FR is 26, 17, 28 and 11 respectively, and the number of amino acids in the LCDR1-3 domains is 10, 3 and 5 respectively, which are 27-36aa, 54-56aa and 85-89aa respectively.

[0011] Furthermore, the VH domain comprises the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having a homology of more than 95% obtained by substituting, deleting and / or adding one or more amino acids and / or terminal modification to any one or more amino acids in the amino acid sequence shown in SEQ ID NO.7, preferably, an amino acid sequence having a homology of 95%, 96%, 97%, 98% or 99%.

[0012] Furthermore, the full length of the VH domain is 110 amino acids, and the number of amino acids in the 4 domains of FR is 24, 17, 37, and 11 respectively. The number of amino acids in the HCDR1-3 domains is 8, 8, and 5 respectively, which are 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 a monoclonal antibody or antigen-binding fragment against human MNDA protein. The hybridoma cell line is a murine anti-human MNDA monoclonal hybridoma cell line OTI4H6, and its deposit number is CGMCC No. 46245. It was deposited on November 28, 2024 at the China General Microbiological Culture Collection Center (CGMCC), and the address of the deposit 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, which is secreted and obtained by the above-mentioned hybridoma cell line OTI4H6.

[0015] On the other hand, the present application also provides the application of the above-mentioned monoclonal antibody or antigen-binding fragment, which is characterized in that the application is any one of the following: 1) labeling of human MNDA protein in tissues; 2) preparation of 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. The immunohistochemical detection kit includes a primary antibody detection reagent, and the primary antibody detection reagent contains the above-mentioned monoclonal antibody or antigen fragment.

[0018] Furthermore, the kit also includes an antigen retrieval solution, an endogenous peroxidase blocker, a hypersensitive secondary antibody reagent, a DAB substrate buffer, a DAB concentrated chromogenic solution, and a hematoxylin staining solution.

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

[0020] Deposited Information

[0021] Biological Material: OTI4H6

[0022] Taxonomic Nomenclature: Murine Anti-Human MNDA Monoclonal Hybridoma Cell Line

[0023] Accession Number: CGMCC No.46245

[0024] Date of Deposit: November 28, 2024

[0025] Depositary Institution: China General Microbiological Culture Collection Center (CGMCC)

[0026] Address of Depositary Institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0027] Compared with the prior art, the present application provides monoclonal antibodies that can specifically bind to human MNDA protein, including monoclonal antibodies against human MNDA protein obtained by screening through hybridoma technology. At the same time, the present application also establishes an immunohistochemical detection method and a detection kit based on the monoclonal antibody against human MNDA protein. Using the immunohistochemical detection kit of the present application, specific labeling of human MNDA protein can be achieved in tissues including tonsil, appendix, and follicular lymphoma, etc., and it can also be used for immunohistochemical detection of tissues and for differentiating lymphomas related to the expression of human MNDA protein. Brief Description of the Drawings

[0028] In order to more clearly illustrate the specific embodiments of the present application, the drawings required for use in the description of the specific embodiments will be briefly introduced below.

[0029] Figure 1 It is a Western blot detection result diagram of the recombinant MNDA protein described in Example 2 of the present invention. The expression of the recombinant MNDA protein in E. coli cells was detected with anti-HIS antibody. Among them, the detection result of the lane L is the detection result with the lysate of E. coli cells transfected with the empty vector as the antigen, and the detection result of the lane R is the detection result with the lysate of E. coli cells transfected with the pET23a-His-rMNDA plasmid as the antigen.

[0030] Figure 2 It is an SDS-PAGE result diagram of the MNDA protein described in Example 2 of the present invention. The recombinant MNDA protein was purified with a nickel affinity chromatography column, and the purified protein was subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

[0031] Figure 3 It is a schematic diagram of the IHC detection result of tonsil by the monoclonal antibody secreted by OTI4H6 described in Example 6 of the present invention.

[0032] Figure 4 It is a schematic diagram of the IHC detection result of appendix by the monoclonal antibody secreted by OTI4H6 described in Example 7 of the present invention.

[0033] Figure 5 Schematic diagram of the results of IHC detection of follicular lymphoma with the monoclonal antibody IHC secreted by OTI4H6 described in Example 8 of the present invention. Detailed implementation mode

[0034] The following further elaborates on this application in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate this application and not to limit the scope of this application. The experimental methods without specific conditions noted in the following embodiments are usually in accordance with conventional conditions, the conditions described in laboratory manuals, or the conditions recommended by the manufacturer.

[0035] Example 1 Construction of the recombinant expression plasmid of MNDA protein

[0036] 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 and hydrophobicity, amino acid categories and distributions, homology between human sequences and immunized animals, etc. Finally, 300 amino acids at positions 1-300 of the MNDA protein were determined as the amino acid sequence of the synthetic protein. The corresponding amino acid sequence was designed and the DNA sequence was synthesized. The nucleotide sequence of the MNDA gene was synthesized, and the nucleotide sequence is shown in SEQ ID NO.1. The corresponding amino acid sequence of the MNDA protein is shown in SEQ ID NO.2. Restriction endonuclease sites SgfI and MluI were introduced on both sides of the gene and cloned into the expression vector pET23a-N-His to establish the MNDA recombinant expression plasmid pET23a-His-rMNDA.

[0037] Example 2 Expression and purification of recombinant MNDA protein

[0038] (1) Transformation of E. coli cells: 100 μl of competent cells were placed on ice to melt, and then recombinant plasmid DNA was added and gently mixed. After ice-bathing for 30 min, heat shock was performed at 42 °C for 90 sec, and then it was continued to be ice-bathed for 1.5 min. 500 μl of fresh antibiotic-free LB medium was added in the ultra-clean bench, and after incubation on a shaker at 37 °C for 45 min, an appropriate amount of the bacterial solution was evenly spread on a plate containing antibiotics. The culture dish was inverted and cultured overnight in a 37 °C constant temperature incubator.

[0039] (2) Cell lysis: The next day, monoclonal colonies were picked into fresh medium and cultured at 37 °C and 200 rpm until the OD value reached 0.4-0.6, then IPTG (final concentration 1 mM) was added for induction culture for 7 h. The bacterial cells were collected by centrifugation, and then the bacterial cells were resuspended with lysis buffer. After ultrasonic disruption for 20 min, centrifugation was performed at 12,000 rpm at 4 °C for 20 min, and the supernatant was collected. A small amount of the supernatant protein was identified by WB with anti-His antibody, as shown in Figure 1 .

[0040] from Figure 1 The results showed that there was an obvious specific band (R) in the WB detection of the lysate of E. coli cells transfected with the pET23a-His-rMNDA plasmid, and the molecular weight was consistent with the expected molecular weight. However, there was no corresponding band of the same size in the control lysate (L) transformed with the empty vector. This indicated the specific expression of recombinant MNDA protein in the cells.

[0041] (3) Purification by nickel affinity chromatography column: The supernatant of the centrifuged lysate was filtered through a 0.45 μM, 33 mm PVDF membrane filter and transferred to a 15 mL tube. 1 mL of the mixed Beads was added, and after sealing, it was placed in a 360-degree mixer and bound at 4°C for 2 hours. The 15 mL tube was taken out, and the lysate was poured into a BIO-RAD chromatography column, and the breakthrough solution was collected. After dripping dry, the breakthrough solution was sampled for WB detection. The column material was washed 1 - 2 times with the lysis buffer. After dripping dry, the Beads were washed 3 times with TBST. After dripping dry, it was eluted with 0.1 M Glycine buffer (pH 3.5). The first elution was 200 μL, which was not collected after dripping dry. The second and third elutions were each 500 μL, and the fourth elution was 250 μL, which was collected into a 1.5 mL centrifuge tube. Immediately, NaH2PO4 buffer (pH 11.0) was added to neutralize to about 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, as shown in Figure 2 .

[0042] from Figure 2 The results showed that there was an obvious specific band on the SDS-PAGE gel of the purified protein. The band was clear and complete, and the molecular weight was consistent with the expected molecular weight, indicating that recombinant MNDA protein with good purity had been obtained.

[0043] Example 3 Preparation and screening of monoclonal antibodies secreted by OTI4H6 against human MNDA protein

[0044] According to the standard method, BALB / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized with the purified recombinant MNDA protein (hereinafter referred to as MNDA antigen). The specific method was as follows:

[0045] (1) Animal immunization: The purified MNDA antigen was emulsified with complete Freund's adjuvant and used to immunize 6 - 8-week-old BALB / c mice by subcutaneous or intraperitoneal injection. The immunization dose was 60 μg / mouse. After two weeks, the second immunization was carried out, emulsified with incomplete Freund's adjuvant, and the immunization dose was 30 μg / mouse. After two immunizations, tail blood was taken to measure the serum titer by ELISA in gradient dilution; According to the results, it was determined whether to boost the immunization, and the mouse with the highest antibody titer was selected for cell fusion.

[0046] (2) Cell fusion: The myeloma cells used were sp2 / 0 cells derived from BALB / c, and the cells were in the logarithmic growth phase during fusion. The spleens of immunized mice were taken and made into single-cell suspensions of lymphocytes. Mouse spleen lymphocytes and myeloma cells were mixed at a ratio of 1:7, 1 mL of 50% PEG (pH 8.0) preheated at 37°C was added dropwise, incomplete medium and termination solution were added, and after centrifugation to discard the supernatant, the cells were suspended and mixed with HAT medium, made up to 50 mL with MC, dispensed into 3.5 cm culture dishes, placed in a wet box, and cultured in a constant temperature incubator at 37°C and 5% CO2.

[0047] (3) Screening and cloning: Hybridoma cell clones were selected 8 days after fusion, and ELISA tests were performed using purified recombinant MNDA protein. The cell line numbers were marked, and the cells in the positive wells were subjected to limited dilution. The ELISA values were measured 6 days after each limited dilution, and the monoclonal wells with higher OD280 positive values were selected for limited dilution until the results of the entire 96-well plate in the ELISA assay were positive. Monoclonal strains with high positive values were selected, and the corresponding cell line in the fusion plate was OTI4H6.

[0048] (4) Preparation and purification of monoclonal antibodies on cells: The hybridoma cell line OTI4H6 was cultured in a 10 cm culture dish with DMEM medium containing 15% serum until it expanded to approximately 4×10 7 cells. Then, it was centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were transferred to a 2 L spinner flask. Serum-free medium was added to make the cell density approximately 3×10 5 cells / mL. After continued culture for 1.5 weeks, when the cell mortality rate reached 60%, the cell suspension was collected and centrifuged at 6000 rpm for 20 min. The supernatant was taken, and the supernatant was purified by affinity chromatography. The corresponding column material was selected for purification according to the antibody subtype (the subtype was IgG2a, and protein G column material was used for purification). The concentration of the purified monoclonal antibody was measured, freeze-dried, and dispensed (100 μg / tube), and finally stored at -20°C.

[0049] Example 4 Analysis of the Variable Region Genes and Amino Acid Sequences of Monoclonal Antibodies Against Human MNDA Protein

[0050] Purchase the SMARTer ® RACE 5’ / 3’ Kit from Takara Bio USA, and use 5’ RACE (Rapid Amplification of cDNA Ends) technology to amplify the variable region light and heavy chain gene sequences of the functional antibodies of hybridoma cells. For the specific experimental procedure, refer to the SMARTer ®RACE 5’ / 3’ Kit User Manual.

[0051] According to the antibody being of IgG2a subtype, specific gene primers pRace-H-GSP and pRace-K-GSP targeting the 3'-ends of its Ig and Kappa constant regions were designed, and the primer sequences are as follows:

[0052] pRace-H-GSP: 5'-CATCDGTCTATCCACTGGCCCCTG-3' (SEQ ID NO:11)

[0053] pRace-K-GSP: 5'-CTTCCCACCATCCAGTGAGCAGTT-3' (SEQ ID NO:12).

[0054] Extract mRNA from the hybridoma cell OTI4H6, reverse transcribe it into cDNA, and perform RACE

[0055] Amplify the DNA fragments of the antibody heavy chain and light chain. Respectively ligate the amplified light chain and heavy chain to the cloning vector PUC119 by enzymatic digestion, pick positive clones through blue-white screening, purify the positive plasmids for sequencing, use the sequencer ABI 3730, and the sequencing primers are the universal primers M13F and M13R.

[0056] M13F: 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO:13) M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO:14).

[0057] Using the Internet, on http: / / www.imgt.org, use the IMGT / V-QUEST analysis software 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. The amino acid sequence of the VL domain of the anti-human MNDA protein monoclonal antibody is as shown in SEQ ID NO.3, and the amino acid sequence of the VH domain is as shown in SEQ ID NO.7.

[0058] The full length of the VL domain is 100 amino acids. The number of amino acids in the 4 domains of its FR are 26, 17, 28, and 11 respectively. The number of amino acids in the 3 domains of the LCDR 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 ID NO.5), WQGTH (SEQ ID NO.6).

[0059] The full length of the VH domain is 110 amino acids. The numbers of amino acids in the 4 domains of FR are 24, 17, 37, and 11 respectively. The numbers of amino acids in the 3 domains of HCDR are 25 - 32 aa, 50 - 57 aa, and 95 - 99 aa respectively. The lengths of 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).

[0060] Example 5 Immunohistochemical Detection Kit Containing Monoclonal Antibody Against Human MNDA Protein

[0061] An immunohistochemical detection kit containing a monoclonal antibody against human MNDA protein, comprising an antigen retrieval solution [1 mM EDTA, 10 mM Tris buffer (pH 8.0)], the monoclonal antibody against human MNDA protein prepared and purified in Example 3, an endogenous peroxidase blocker (hydrogen peroxide), a hypersensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer, a DAB chromogenic solution, and a hematoxylin staining solution.

[0062] Example 6 Immunohistochemical Detection of Tonsil Using Monoclonal Antibody Against Human MNDA Protein as the Primary Antibody

[0063] The tonsil tissue was detected using the immunohistochemical detection kit in Example 5. The specific steps are as follows:

[0064] (1) Take the formalin-fixed tonsil tissue block for paraffin embedding, and use a Leica tissue slicer to cut sections with a tissue thickness of 4 μm.

[0065] (2) Deparaffinization and hydration: Immerse in 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.

[0066] (3) Antigen repair: Add the antigen retrieval 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 sections, and then cool them naturally to room temperature. Immerse in deionized water for 2 min, 3 times.

[0067] (4) Inactivation: Use 3% hydrogen peroxide to inactivate the endogenous peroxidase in the tissue, let it stand at room temperature for 15 min, and immerse in deionized water for 2 min, 3 times.

[0068] (5) Use an immunohistochemical pen to draw a frame around the tissue and wash it with 0.1% PBST for 2 min, once.

[0069] (6) Incubate with primary antibody: Add 200 μl of the monoclonal antibody against human MNDA protein secreted by the hybridoma cell line OTI4H6 after dilution into a wet box, and incubate at 37 °C for 60 min. Wash with 0.1% PBST for 2 min, three times.

[0070] (7) Incubate with secondary antibody: Add 100 μl of the 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, three times.

[0071] (8) DAB color development: Add 120 μl of DAB color development solution, let it stand at room temperature for 5 min, rinse with tap water under running water to terminate color development, and rinse with tap water three times.

[0072] (9) Counterstain with hematoxylin, differentiation and bluing: Let it stand in hematoxylin solution for 12 s, rinse with tap water three times to terminate color development, differentiate in 1% hydrochloric acid ethanol solution, rinse with tap water three times to terminate, put it into the freshly boiled Tris-EDTA disodium solution with pH 8.0 for bluing, then put it into the Tris-EDTA disodium solution with pH 8.0 at room temperature for several seconds, and rinse with tap water three times. Observe the staining under a microscope. If it is normal, end and recycle the hematoxylin stain; if over-differentiated, repeat the above steps until the staining is qualified.

[0073] (10) Dehydration and clearing: 75% ethanol for 1 min, 85% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 1 min, three times, xylene for 1 min, three times, seal with neutral gum.

[0074] (11) Microscopic examination, the results are as Figure 3 shown.

[0075] The results showed that MNDA protein was only expressed in the nuclei of B lymphocytes and monocytes in the tonsillar mantle zone. Figure 3 The solid arrows in

[0076] Example 7 Immunohistochemical detection of appendix with monoclonal antibody against human MNDA protein as the primary antibody

[0077] The appendix tissue was detected using the immunohistochemical detection kit in Example 5. The specific steps were the same as in Example 6. The results are as Figure 4 shown, Figure 4 The solid arrows in

[0078] Example 8 Immunohistochemical Detection of Follicular Lymphoma Using Monoclonal Antibody Against Human MNDA Protein as Primary Antibody

[0079] The immunohistochemical detection kit in Example 5 was used to detect follicular lymphoma tissue. The specific steps were the same as those in Example 6, and the results were as Figure 5 shown Figure 5 The solid arrows in indicate monocytes with positive expression of MNDA protein, and the dashed arrows indicate follicular lymphoma cells with negative expression of MNDA protein, and there is no non-specific background staining

[0080] The above results indicate that the monoclonal antibody against human MNDA secreted by the hybridoma cell OTI4H6 described in this application has high specificity and sensitivity, is only expressed in monocytes and scattered B lymphocytes in the mantle zone, and at a concentration of 0.25 μg / ml, the staining intensity is strong and the nuclear staining is clear and definite. Therefore, the MNDA monoclonal antibody secreted by OTI4H6 can be used for immunohistochemical detection and for the study of MNDA-related lymphoma diseases

[0081] This specific embodiment is only an interpretation of this application and does not limit this application. Those skilled in the art can make modifications to this embodiment without creative contributions according to their needs after reading this specification, but as long as they are within the scope of the claims of this application, they are protected by the patent law

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 is QSLLDSDETY, the amino acid sequence of LCDR2 is LTS, and the amino acid sequence of LCDR3 is WQGTH; 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 HCDR1 is GFTFSRYA, the amino acid sequence of HCDR2 is INSGGSVP, and the amino acid sequence of HCDR3 is TTSTT.

2. 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.

3. 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.

4. 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.

5. 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 4.

6. 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 3 or claim 5.

7. The kit according to claim 6, 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.

8. The kit according to claim 7, characterized in that The ultrasensitive secondary antibody reagent is an ultrasensitive enzyme-labeled goat anti-mouse / rabbit IgG polymer.

Images