Anti-ezh2 protein monoclonal antibody and related products and uses thereof

Abstract

The present application relates to a kind of anti-EZH2 protein monoclonal antibody and its related product and purposes, belong to biological medicine technical field, anti-EZH2 protein monoclonal antibody includes heavy chain and light chain, heavy chain includes heavy chain variable region, the amino acid sequence of heavy chain variable region is respectively as shown in SEQ ID NO:1;Light chain includes light chain variable region, the amino acid sequence of light chain variable region is respectively as shown in SEQ ID NO:3;The present antibody has higher sensitivity and staining intensity, the comprehensive detection performance is more excellent, not only can effectively prevent the problem of false negative, but also can significantly improve the staining intensity and positive detection rate of low expression tumor sample, effectively avoid the problem of missed detection caused by staining intensity degradation, to further improve the accuracy and reliability of EZH2 related tumor pathology detection and diagnosis, more satisfy market demand, can be widely used in scientific research and the detection of non-disease diagnosis purposes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a monoclonal antibody against EZH2 protein and its uses. Background Technology

[0002] Zeste enhancer homolog 2 (EZH2), also known as lysine methyltransferase 6, is the core catalytic subunit of the polycomb repressive complex 2 (PRC2). Primarily located in the cell nucleus, EZH2 catalyzes the methylation of histone H3 at position 27 (H3K27), particularly the formation of H3K27 trimethylation (H3K27me3), thereby regulating chromatin state and gene transcription. EZH2 participates in various biological processes, including embryonic development, stem cell maintenance, cell proliferation, differentiation, and immune regulation.

[0003] In tumor tissues, EZH2 can exhibit protein overexpression, gene amplification, gain-of-function mutations, or loss-of-function alterations. Representative studies show that approximately 46% of diffuse large B-cell lymphomas express EZH2 protein, with an overexpression rate of approximately 50% in primary gastrointestinal diffuse large B-cell lymphomas; the high expression rate of EZH2 in extranodal NK / T-cell lymphomas is approximately 55.2%, and it is associated with a higher proliferation index, advanced disease, and poorer survival. In solid tumors, approximately 49.8% of invasive breast cancers show high EZH2 expression, with overexpression rates exceeding 90% in some triple-negative breast cancer cohorts; the positivity rates of EZH2 in prostate cancer and colorectal cancer can reach approximately 87.5% and 69.7%, respectively. Overall, high EZH2 expression is often associated with tumor cell proliferation, high histological grade, increased invasiveness, and poor prognosis. In addition to elevated protein expression, EZH2 can also undergo gain-of-function mutations in some germinal center-derived B-cell lymphomas. Studies show that approximately 21.7% of germinal center B-cell diffuse large B-cell lymphomas and approximately 7.2% of follicular lymphomas exhibit EZH2 Tyr641 mutations, while the EZH2 mutation rate in other follicular lymphoma cohorts is approximately 17%–22%. Related mutations can increase H3K27 trimethylation levels, promote tumor cell proliferation, and inhibit terminal differentiation. However, EZH2 protein expression levels are not entirely consistent with gene mutation status, and total EZH2 protein detection generally cannot replace gene mutation detection or H3K27me3 detection alone.

[0004] With the clear understanding of the role of EZH2 in tumor development and progression, EZH2 and its related epigenetic pathways have become important targets for anti-tumor drugs. Currently available rabbit or mouse monoclonal antibodies against EZH2 protein (such as CST's D2C9 antibody) can be used for immunohistochemistry, Western blotting, immunofluorescence, flow cytometry, immunoprecipitation, and chromatin-related detection. However, existing commercially available anti-EZH2 protein monoclonal antibodies (such as CST's D2C9 antibody) have limited detection sensitivity and are insufficient in capturing lymphoma tumor samples with low and heterogeneous EZH2 expression, which are prevalent in clinical settings. They generally suffer from weak staining intensity and low detection rate of tumor-positive cells, and are prone to staining intensity degradation and signal attenuation, leading to missed detections and distorted interpretation results. In addition, commercially available EZH2 antibodies are prone to false negatives in some tumor samples that truly express the target antigen, which can easily lead to missed tumor diagnoses and misclassifications, seriously affecting diagnostic accuracy. Therefore, the development of a novel anti-EZH2 protein monoclonal antibody with good specificity, staining stability, high sensitivity, effective prevention of false negatives, and suitability for detecting low-expression tumor samples is of great clinical value and application significance for improving the accuracy and reliability of EZH2-related tumor pathological diagnosis, and is an urgent issue to be addressed. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, the first aspect of the present invention provides a monoclonal antibody against EZH2 protein, which not only has good specificity and can specifically recognize cells expressing EZH2 protein, but also has the advantages of high affinity, higher sensitivity and staining intensity. It is beneficial to prevent false negatives and can significantly improve the staining intensity and positive detection rate of low-expression tumor samples, effectively avoiding the problem of missed detection caused by the downgrading of staining intensity.

[0006] A monoclonal antibody against EZH2 protein comprises a heavy chain and a light chain, wherein the heavy chain includes a variable region, the amino acid sequence of which is shown in SEQ ID NO:1; and the light chain includes a variable region, the amino acid sequence of which is shown in SEQ ID NO:3. Compared to existing EZH2 antibodies, the monoclonal antibody against EZH2 protein provided in this protocol not only exhibits high specificity, specifically recognizing cells expressing EZH2 protein, but also possesses high affinity and stable antigen binding. Compared to commercially available antibodies, this antibody demonstrates higher staining intensity and sensitivity, significantly improving the staining intensity and positive detection rate of low-expression tumor samples, effectively avoiding false negatives caused by staining intensity degradation, and preventing false negatives. This antibody possesses excellent comprehensive detection performance, which is beneficial for further improving the accuracy and reliability of EZH2-related tumor pathological detection and diagnosis, and is more suitable for application in scientific research and non-disease diagnostic purposes.

[0007] Furthermore, the complementarity-determining region (CDR) of the antibody is defined according to the IMGT numbering rule; the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is: GYTFTNPY; the amino acid sequence of HCDR2 is: INPYNDEY; and the amino acid sequence of HCDR3 is: QWGPIAY.

[0008] Furthermore, the amino acid sequence of the heavy chain is shown in SEQ ID NO:2.

[0009] Furthermore, the complementarity-determining region (CDR) of the antibody is defined according to the IMGT numbering rule; the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is: QDINSW; the amino acid sequence of LCDR2 is: DVS; and the amino acid sequence of LCDR3 is: LAGYDT.

[0010] Furthermore, the amino acid sequence of the light chain is shown in SEQ ID NO:4.

[0011] A second aspect of this invention also provides a reagent for detecting EZH2 protein, comprising the aforementioned anti-EZH2 protein monoclonal antibody. Because this anti-EZH2 protein monoclonal antibody possesses high affinity, sensitivity, and staining intensity, it can specifically recognize cells expressing EZH2 protein and effectively prevent false negatives and missed detections. Its application in reagents for detecting EZH2 protein facilitates obtaining more accurate detection results.

[0012] A third aspect of this invention also provides a detection tool for detecting EZH2 protein, comprising the anti-EZH2 protein monoclonal antibody or the reagent. Because the anti-EZH2 protein monoclonal antibody has high affinity, sensitivity, and staining intensity, it can specifically recognize cells expressing EZH2 protein and effectively prevent false negatives and missed detections. Applying it to reagents for detecting EZH2 protein facilitates obtaining more accurate detection results.

[0013] Preferably, the detection tool is a reagent kit, a test strip, an antibody chip, or a detection plate. In this embodiment, when the detection tool is a reagent kit, the kit contains the anti-EZH2 protein monoclonal antibody or the reagent for detecting EZH2 protein; when the detection tool is a test strip, the test strip contains the anti-EZH2 protein monoclonal antibody or the reagent for detecting EZH2 protein; when the detection tool is an antibody chip, the antibody chip contains the anti-EZH2 protein monoclonal antibody or the reagent for detecting EZH2 protein; and when the detection tool is a detection plate, the detection plate contains the anti-EZH2 protein monoclonal antibody or the reagent for use in scientific research and non-disease diagnostic purposes for EZH2 protein detection.

[0014] Furthermore, the kit also includes a container in which the anti-EZH2 protein monoclonal antibody or reagent is disposed.

[0015] The fourth aspect of this invention provides the use of the aforementioned anti-EZH2 protein monoclonal antibody in the preparation of tools for detecting EZH2 protein. Because this anti-EZH2 protein monoclonal antibody possesses high affinity, sensitivity, and staining intensity, it can specifically recognize cells expressing EZH2 protein and effectively prevent false negatives and missed detections. Its application in tools for detecting EZH2 protein facilitates obtaining more accurate detection results.

[0016] Furthermore, the tool for detecting EZH2 protein includes reagents, kits, test strips, antibody chips, or detection plates. In this embodiment, when the tool includes reagents, the reagents include the anti-EZH2 protein monoclonal antibody; when the tool includes kits, the anti-EZH2 protein monoclonal antibody can be contained within the kit; when the tool includes test strips, the anti-EZH2 protein monoclonal antibody can be contained within the test strip; when the tool includes antibody chips, the anti-EZH2 protein monoclonal antibody can be contained within the antibody chip; when the tool includes detection plates, the anti-EZH2 protein monoclonal antibody can be contained within the detection plate; all of these methods can achieve the function of detecting EZH2 protein.

[0017] Compared with existing technologies, the anti-EZH2 protein monoclonal antibody and its related products and uses provided by this invention are not only specific, capable of specifically recognizing cells expressing EZH2 protein, but also have higher sensitivity and staining intensity, resulting in superior overall detection performance. This not only effectively prevents false negatives but also significantly improves the staining intensity and positive detection rate of low-expression tumor samples, effectively avoiding missed detections caused by staining intensity degradation. Therefore, it is beneficial to further improve the accuracy and reliability of EZH2-related tumor pathological detection and diagnosis, better meeting market demands and can be widely used in scientific research and non-disease diagnostic testing. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The image shows a comparison of immunohistochemical staining results for a case of diffuse large B-cell lymphoma, where (A) uses the H1381-EZH2 antibody of the present invention; and (B) uses the commercially available EZH2(D2C9) antibody.

[0020] Figure 2 The image shows a comparison of immunohistochemical staining results for a case of follicular lymphoma, where (A) uses the H1381-EZH2 antibody of the present invention; and (B) uses the commercially available EZH2(D2C9) antibody.

[0021] Figure 3 The kinetic curves of the interaction between the H1381-EZH2 antibody and the antigen are shown.

[0022] Figure 4 The images show a comparison of the results of immunoblotting, where (A) uses a commercially available EZH2 antibody (Cell Signaling Technology (CST), D2C9), and (B) uses the H1381-EZH2 monoclonal antibody prepared in this invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] This embodiment provides a method for preparing a monoclonal antibody against EZH2 protein, comprising the following steps:

[0026] Step 1: Antigen preparation.

[0027] In this embodiment, a recombinant human EZH2 protein fragment was selected as the immunogenic antigen, corresponding to the UniProt number Q15910. Detailed information such as its full-length amino acid sequence, domains, and functional annotations can be found in the UniProt database using this number. This method selects amino acids 182-431 (AA182-431) of the EZH2 protein as the immunogenic antigen region, which is the antibody-specific binding region.

[0028] This step involves expressing and purifying recombinant human EZH2 (AA182-431) protein using a prokaryotic expression system. The amino acid sequence of the antigen is as follows:

[0029] NDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTY KRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVE.

[0030] The purified recombinant protein was analyzed for purity and specificity by SDS-PAGE and Western blot, and its concentration was determined. After aliquoting, the protein was stored at low temperature and used as a subsequent animal immunization antigen. For ease of description, this antigen is labeled as H1381 antigen in this invention.

[0031] Step 2: Animal immunization.

[0032] Healthy female Balb / C mice aged 6–8 weeks were selected for immunization.

[0033] First immunization: The purified recombinant EZH2 antigen was thoroughly emulsified with an equal volume of complete Freund's adjuvant and then immunized in multiple mice via subcutaneous injection at multiple points on the back.

[0034] Second and third booster immunizations: Subsequent immunizations were all emulsified with incomplete Freund's adjuvant (to prevent repeated injections of complete Freund's adjuvant from causing severe inflammation and ulcers in mice, which would be detrimental to immunization) and the antigen, with an interval of 14 days between immunizations, for a total of three immunizations.

[0035] Blood was collected from the tail vein of mice on day 10 after three immunizations, and serum was separated. The immunotiter of mouse serum against EZH2 antigen was detected by indirect ELISA and immunohistochemistry. Mice with the highest antibody titer against EZH2 antigen were selected as target animals for further hybridoma cell screening / for subsequent spleen cell extraction and cell fusion.

[0036] Step 3: Monoclonal cell screening.

[0037] This embodiment uses PEG-mediated hybridoma cell fusion technology for monoclonal cell screening. The specific steps are as follows:

[0038] Log-phase Sp2 / 0 myeloma cells were collected, and high-titer mouse spleen lymphocytes were aseptically isolated. The spleen cells and myeloma cells were mixed in a standard ratio, washed with sterile PBS, and centrifuged to precipitate the cells. Preheated PEG4000 fusion buffer was applied to the cell pellet, and after a short period of fusion, complete culture medium was added to terminate the reaction. The fused cells were then collected by centrifugation.

[0039] The fused cells were resuspended in HAT selective medium and evenly seeded into 96-well culture plates, then incubated at 37°C in a 5% CO2 incubator. Unfused myeloma cells, unfused spleen cells, and autofusion cells were discarded according to the HAT selection rules, retaining only hybridoma cells. The medium was changed regularly during culture, and selection was continued for 7–10 days. After that, the medium was replaced with HT medium for continued culture to eliminate selection stress and stabilize cell status.

[0040] After clear monoclonal cell colonies appeared in the wells and grew to a suitable density, the cell culture supernatant was collected from each well. Specificity was detected using ELISA, with recombinant human EZH2 antigen as the coating antigen, to screen for positive cell wells that specifically bind to EZH2 protein. Cells from positive wells were subcloned three times using a limiting dilution method. Specificity was retested by ELISA after each subcloning round. Finally, hybridoma cell lines that stably secrete specific anti-human EZH2 monoclonal antibodies were obtained.

[0041] Step 4: Antibody preparation and purification.

[0042] The hybridoma cell lines screened in the previous step were subjected to large-scale suspension culture in serum-free DMEM medium for approximately 10 days. When the cell death rate decreased to 60%, the cell suspension was collected. The collected cell suspension was centrifuged at 6000 rpm for 20 min to remove cells and cell debris, and the clear supernatant was collected. The supernatant was purified using a protein G affinity chromatography column, and the high-purity antibody was eluted and collected. The purified antibody was then analyzed for concentration, aliquoted, and stored at 2–8°C to obtain a high-purity anti-EZH2 protein monoclonal antibody (i.e., anti-EZH2 recombinant mouse monoclonal antibody). For ease of description, the prepared anti-EZH2 protein monoclonal antibody is labeled as H1381-EZH2.

[0043] Step 5: Antibody gene sequencing.

[0044] Hybridoma cell lines in the logarithmic growth phase were collected, total mRNA was extracted, cDNA was synthesized by reverse transcription, and the antibody heavy chain and light chain variable region gene fragments were amplified using 5'RACE technology.

[0045] The amplified product was cloned into a sequencing vector, and universal primers M13F and M13R were used as sequencing primers. Gene sequencing was performed using an ABI 3730 sequencer. The sequencing results were analyzed by bioinformatics software (such as IMGT / V-QUEST) for sequence alignment, germline gene alignment and feature analysis. Finally, the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the anti-EZH2 protein monoclonal antibody were obtained, and the antibody gene sequence was identified.

[0046] In this embodiment, the prepared anti-EZH2 protein monoclonal antibody comprises a heavy chain and a light chain. The heavy chain includes a variable region of the heavy chain (VH) and a constant region of the heavy chain; the light chain includes a variable region of the light chain (VL) and a constant region of the light chain. The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:1, and is detailed in Table 1. More specifically, the variable region of the heavy chain sequentially includes a first heavy chain framework region (HFR1), a first heavy chain complementarity-determining region (HCDR1), a second heavy chain framework region (HFR2), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain framework region (HFR3), a third heavy chain complementarity-determining region (HCDR3), and a fourth heavy chain framework region (HFR4). The complementarity-determining regions (CDRs) of the antibody are defined according to the IMGT numbering rules.

[0047] The amino acid sequence of HCDR1 is: GYTFTNPY; HCDR1 corresponds to the amino acid sequence shown in positions 26 to 33 of SEQ ID NO:1.

[0048] The amino acid sequence of HCDR2 is: INPYNDEY; HCDR2 corresponds to the amino acid sequence shown in positions 51 to 58 of SEQ ID NO:1.

[0049] The amino acid sequence of HCDR3 is: QWGPIAY; HCDR3 corresponds to the amino acid sequence shown in positions 95 to 101 of SEQ ID NO:1.

[0050] The amino acid sequence of HFR1 is: EVQLQQSGPELVKPGASVKMSCKAS.

[0051] The amino acid sequence of HFR2 is: IHWVKRKPGQGLEWIGY.

[0052] The amino acid sequence of HFR3 is: NEKFKDKATLTSDKSSSTAYMELSSLPSEDSAVYYC.

[0053] The amino acid sequence of HFR4 is: WGQGTLVTVSA.

[0054] Table 1. Sequence of the heavy chain variable region (VH) in anti-EZH2 protein monoclonal antibodies.

[0055] The amino acid sequence of the complete heavy chain (including the heavy chain variable region and the heavy chain constant region) is shown in SEQ ID NO:2, as detailed in Table 2. The heavy chain constant region corresponds to the amino acid sequence shown from position 113 to position 436 in SEQ ID NO:2.

[0056] Table 2. Sequences of the heavy chain in anti-EZH2 protein monoclonal antibodies

[0057] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:3, and is detailed in Table 3. More specifically, the light chain variable region includes, in sequence, the first light chain framework region (LFR1), the first light chain complementarity-determining region (LCDR1), the second light chain framework region (LFR2), the second light chain complementarity-determining region (LCDR2), the third light chain framework region (LFR3), the third light chain complementarity-determining region (LCDR3), and the fourth light chain framework region (LFR4). The complementarity-determining regions (CDRs) of the antibody are defined according to the IMGT numbering rules.

[0058] The amino acid sequence of LCDR1 is: QDINSW; LCDR1 corresponds to the amino acid sequence shown in positions 27 to 32 of SEQ ID NO:3.

[0059] The amino acid sequence of LCDR2 is: DVS; LCDR2 corresponds to the amino acid sequence shown in positions 50 to 52 of SEQ ID NO:3.

[0060] The amino acid sequence of LCDR3 is: LAGYDT; LCDR3 corresponds to the amino acid sequence shown in positions 89 to 94 of SEQ ID NO:3.

[0061] The amino acid sequence of LFR1 is: DIKMTQSPSSMYASLGERVTLTCKAS.

[0062] The amino acid sequence of LFR2 is: LSWFQQKPGKSPKTLIY.

[0063] The amino acid sequence of LFR3 is: RLVDGVPSRFSGSGSGQDFSLTISSLEYEDMGIYYC.

[0064] The amino acid sequence of LFR4 is: FGGGTKLEIK.

[0065] Table 3. Sequence of the light chain variable region (VL) in anti-EZH2 protein monoclonal antibodies.

[0066] The amino acid sequence of the complete light chain (including the light chain variable region and the light chain constant region) is shown in SEQ ID NO:4, as detailed in Table 4. The light chain constant region corresponds to the amino acid sequence shown from position 105 to position 211 in SEQ ID NO:4.

[0067] Table 4. Light chain sequences in anti-EZH2 protein monoclonal antibodies

[0068] Example 2

[0069] This embodiment uses the anti-EZH2 protein monoclonal antibody (anti-EZH2 recombinant mouse monoclonal antibody) prepared in Example 1 as the primary antibody for immunohistochemical detection. The specific method is as follows:

[0070] (1) Sample preparation: The sections of diffuse large B-cell lymphoma, Hodgkin lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma, etc., which were fixed in formalin and embedded in paraffin, were baked in a constant temperature oven at 60℃ for 1-2 hours and stored for later use.

[0071] (2) Use an automatic repair instrument to dewax, hydrate and repair the sections; during this process, the temperature is preferably controlled at 95℃ and the time is preferably controlled at 30min.

[0072] (3) Immerse in hydrogen peroxide for 10 minutes to block the reaction, then rinse with pure water 3 times. Then circle the tissue to be tested with an immunohistochemical pen, rinse with pure water 2 times, and immerse in phosphate buffer (PBST) for 2-3 minutes.

[0073] (4) Primary antibody incubation: Add 100 μL of anti-EZH2 recombinant mouse monoclonal antibody to completely cover the tissue, incubate at room temperature for 1 h, and then wash with PBST 3 times for 3 min each time.

[0074] (5) Secondary antibody incubation: Incubate with secondary antibody for 30 min according to the instructions of the DAB staining solution kit of the secondary antibody staining system used, and rinse with PBST 3 times, 3 min each time.

[0075] (6) DAB color development: Prepare DAB color development solution according to the kit instructions. Drop an appropriate amount of the prepared DAB color development solution to completely cover the tissue, incubate for 10 min, and rinse with pure water 3 times, 3 min each time.

[0076] (7) Hematoxylin counterstaining: Follow the instructions and suggestions of the hematoxylin manufacturer to counterstain the sections for 2 minutes, and then perform PBST blueing for 5 minutes.

[0077] (8) Dehydrate and become transparent: Soak in alcohol for 2-3 minutes and air dry.

[0078] (9) Mounting: Mount the sample with neutral resin.

[0079] (10) Perform slice scanning.

[0080] (11) Results analysis and statistics.

[0081] Immunohistochemical staining results are generally classified as positive or negative. Positive expression must occur at a specific antigenic site on the cell or tissue to be considered positive. When tissue staining distribution is clear and cell localization is accurate, staining results can be further subdivided based on differences in staining intensity and positive rate, as shown in Table 5 below.

[0082] Table 5. Classification of Immunohistochemical Staining Intensities

[0083] The EZH2 monoclonal antibody prepared in Example 1 and the commercially available EZH2 antibody (Cell Signaling Technology (CST), D2C9) were used to simultaneously detect 25 cases of different subtypes of lymphoma, and the results were compared. The entire experiment was conducted using a double-blind design, and the immunohistochemical results of EZH2 were statistically analyzed. The statistical results are shown in Table 6.

[0084] Table 6. Statistical analysis of EZH2 immunohistochemical results

[0085] First, the results in Table 6 show that H1381-EZH2 has a positive rate and staining intensity that are not inferior to those of commercially available antibodies in immunohistochemical detection. Moreover, in cases with low EZH2 expression, the staining intensity and positive rate are significantly better than those of commercially available antibodies, indicating that the sensitivity of H1381-EZH2 monoclonal antibody is higher than that of commercially available antibodies.

[0086] second, Figure 1 This is a comparison of immunohistochemical staining results for a case of diffuse large B-cell lymphoma. Figure 1 (A) in this invention uses H1381-EZH2; Figure 1 (B) uses commercially available EZH2 (D2C9), from Figure 1 As can be seen, for the same diffuse large B-cell lymphoma tissue, the staining intensity and positive rate of the H1381-EZH2 monoclonal antibody of the present invention are stronger than those of the commercially available EZH2 (D2C9). This antibody is less likely to cause false negatives due to the degradation of staining intensity, which is beneficial to improving the accuracy of diagnosis.

[0087] Third, in addition, such as Figure 2 The image shown is a comparison of immunohistochemical staining results for a case of follicular lymphoma. Figure 2 (A) in this invention uses H1381-EZH2; Figure 2 (B) in this study used commercially available EZH2 (D2C9). The neoplastic follicular cells of this follicular lymphoma sample expressed the EZH2 antigen. Figure 2 As can be seen from the results, the H1381-EZH2 monoclonal antibody prepared in this invention can effectively detect the expression of EZH2, while the commercially available EZH2 (D2C9) antibody failed to detect the signal, showing a false negative result. This further proves that the antibody prepared in this invention has higher sensitivity than the commercially available antibody, which is more conducive to improving the accuracy of detection and diagnosis.

[0088] Example 3

[0089] This embodiment describes the affinity assay of the anti-EZH2 protein monoclonal antibody (anti-EZH2 recombinant mouse monoclonal antibody) prepared in Example 1. In this embodiment, the ability of the antibody to bind to the antigen was characterized using Biacore SPR. Specifically, a CM5 chip (channel 1 is the reference channel, channel 2 is the ligand channel) was used. The antigen, as a ligand, was immobilized at a concentration of 1 μg / ml into channel 2 of the CM5 chip, with a binding time of 30 s and a flow rate of 10 μL / min.

[0090] In this embodiment, human EZH2 antigen was used, and the antibody was used as the analyte. The antigen sample ID was H1381. The monoclonal antibody prepared in this invention was used as the antibody, and the antibody was diluted with running buffer to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.563 nM, 0.781 nM, 0.391 nM and 0 nM respectively.

[0091] In this embodiment, the analyte stream passed through channels 1 and 2, with a binding time of 90 s, a dissociation time of 120 s, and a flow rate of 30 μL / min. After analyte loading, the chip surface was regenerated using glycine buffer at pH 2.0. The ligands needed to be immobilized before each analyte loading, and the chip surface was regenerated with regeneration solution after analyte loading.

[0092] The affinity of EZH2 antibody for human EZH2 antigen at different concentrations was determined using Biacore SPR. Binding and separation were analyzed using a 1:1 kinetic model with corresponding analytical software. The results are shown in Table 7. Figure 3 As shown.

[0093] Table 7. Results of Affinity Measurement

[0094] In Table 7, KD represents the dissociation constant, a comprehensive indicator of affinity. A lower KD value indicates higher intermolecular affinity, meaning a lower concentration is needed to achieve a certain degree of binding, and the two molecules are more likely to combine to form a more stable complex. Ka represents the binding rate constant, indicating the speed of intermolecular binding; a higher value indicates faster binding. Kd represents the dissociation rate constant, indicating the speed of intermolecular dissociation; a higher value indicates faster dissociation, and a lower value indicates a more stable complex and higher affinity. Therefore, from the data in Table 7, we can see that ( Figure 3 (The original chromatograms obtained from the data in Table 7) show that the H1381-EZH2 monoclonal antibody prepared in this invention has a high affinity for human EZH2 antigen, reaching the high affinity level of monoclonal antibodies. The stronger the antibody affinity, the better it is for improving the sensitivity of detection, lowering the detection limit, and reducing false negatives. Combined with the good specificity of this antibody, it can jointly improve the accuracy of detection results and better meet market demands.

[0095] Example 4

[0096] This embodiment uses the anti-EZH2 protein monoclonal antibody (anti-EZH2 recombinant mouse monoclonal antibody) prepared in Example 1 as the primary antibody for Western blotting detection. The specific procedure is as follows:

[0097] (1) Activate the polyvinylidene fluoride (PVDF) membrane with SNK6 / NKYS cell lysis buffer by methanol activation treatment for 1 min, washing 3 times with TBST; block with 5% skim milk for 1 h; TBST is a commonly used washing buffer suitable for experiments such as immunoblotting. It contains three basic components: Tris buffer, salt (usually sodium chloride), and surfactant Tween20.

[0098] (2) Primary antibody incubation: Place the blocked PVDF membrane in a solution of diluted H1381-EZH2 monoclonal antibody and incubate at 4°C overnight. After incubation, remove the membrane and wash it with TBST 5 times, 5 min each time.

[0099] (3) Secondary antibody incubation: Place the cleaned PVDF membrane in a diluted HRP-anti-rabbit IgG (1:10000) solution and shake at room temperature for 1 hour. After incubation, remove the membrane and wash it with TBST 5 times, 5 minutes each time.

[0100] (4) Prepare the developing solution according to the Immobilon Western Developing Kit instructions, and evenly drop it onto the above membrane. Develop the membrane using a GelView+ 6000ProⅡ multi-functional image workstation according to the instructions. The results are as follows: Figure 4 As shown, where, Figure 4 (B) uses the H1381-EZH2 monoclonal antibody prepared according to this invention. Figure 4 (A) uses a commercially available EZH2 antibody (Cell Signaling Technology (CST), D2C9). The theoretical molecular weight of EZH2 protein is around 85.4 kDa. However, due to phosphorylation modifications and other factors, the actual measured molecular weight is greater than the theoretical value. Figure 4 Sample 1 is an SNK cell line that is positive for EZH2 expression, and sample 2 is an NKYS cell line that is negative for EZH2 expression. In the immunoblotting results, the H1381-EZH2 monoclonal antibody prepared in this invention and the commercially available EZH2 antibody (Cell Signaling Technology (CST), D2C9) showed no bands in NKYS cells, while the bands in SNK6 cells were of the same size. This indicates that the anti-EZH2 recombinant mouse monoclonal antibody prepared in the embodiments of this invention can recognize the EZH2 protein with high specificity.

[0101] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against EZH2 protein, characterized in that, It includes heavy chains and light chains, among which, The heavy chain includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1; The light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:

3.

2. The anti-EZH2 protein monoclonal antibody according to claim 1, characterized in that, The complementarity-determining region (CDR) of the antibody is defined according to the IMGT numbering rule; the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, wherein... The amino acid sequence of HCDR1 is: GYTFTNPY; The amino acid sequence of HCDR2 is: INPYNDEY; The amino acid sequence of HCDR3 is: QWGPIAY.

3. The anti-EZH2 protein monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:

2.

4. The anti-EZH2 protein monoclonal antibody according to claim 1, characterized in that, The complementarity-determining region (CDR) of the antibody is defined according to the IMGT numbering rule; the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein, The amino acid sequence of LCDR1 is: QDINSW; The amino acid sequence of LCDR2 is: DVS; The amino acid sequence of LCDR3 is: LAGYDT.

5. The anti-EZH2 protein monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain is shown in SEQ ID NO:

4.

6. A reagent for detecting EZH2 protein, characterized in that, It includes the anti-EZH2 protein monoclonal antibody as described in any one of claims 1-5.

7. A detection tool for detecting EZH2 protein, characterized in that, It comprises the anti-EZH2 protein monoclonal antibody as described in any one of claims 1-5, or the reagent as described in claim 6.

8. The detection tool according to claim 7, characterized in that, The detection tool is a reagent kit, test strip, antibody chip, or detection plate.

9. Use of an anti-EZH2 protein monoclonal antibody as described in any one of claims 1-5 in the preparation of a tool for detecting EZH2 protein.

10. The use according to claim 9, characterized in that, The tools used for detecting EZH2 protein include reagents, kits, test strips, antibody chips, or detection plates.

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