IDH2 K155 acetylation modified antibody as well as preparation method and application thereof

Abstract

The invention discloses an IDH2 K155 acetylation modified antibody and a preparation method and application thereof, and the preparation method comprises the following steps: coupling IDH2 K155 acetylation modified polypeptide with KLH immune rabbit to obtain immunized rabbit serum, then determining the antibody titer of the antibody in the immunized rabbit serum, and carrying out purification treatment on the antibody to obtain the IDH2 K155 acetylation modified antibody. The antibody prepared by the invention can help to evaluate GBM diagnosis and prognosis, and when the antibody is adopted for detection, compared with a conventional mass spectrometry method, the detection time is greatly shortened, and the detection difficulty is greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an IDH2 K155 acetylated modified antibody and a preparation method and application thereof. Background Art

[0002] Glioblastoma Multiforme (GBM) is the most invasive and malignant glioma in the brain. The specific mechanism of GBM proliferation and invasion is still unclear. Isocitrate dehydrogenase (IDH) is an important rate-limiting enzyme in the tricarboxylic acid cycle (TCA) of cells. It is a key enzyme involved in cell metabolism, epigenetic regulation, redox and DNA repair. It can catalyze the oxidative decarboxylation of isocitrate to produce α-ketoglutaric acid (α-KG) and convert NADP to + (nicotinamide adenine dinucleoside phosphate) is converted to NADPH (reduced coenzyme II). In recent years, molecular pathological indicators have played an important role in the diagnosis and prognosis of gliomas. For example, the Chinese invention patent with application number 201210234736.6 records that IDH mutation is a very important molecular indicator in the diagnosis and prognosis of gliomas.

[0003] Many studies have shown that IDH mutations are involved in the tumorigenesis of gliomas. Mutated IDH can convert α-KG (α-ketoglutarate) into D-enantiomer of 2-Hydroxyglutarate (D-2HG), which is a tumor metabolite. D-2HG can competitively inhibit α-KG-dependent dioxygenases, such as KDM (histone lysine demethylase) and TETs (thymic epithelial tumors), causing DNA and histone hypermethylation, and further affecting the HIF1-α pathway and promoting the generation of reactive oxygen species (ROS), thereby promoting glioma occurrence and participating in the metabolic reprogramming of gliomas. IDH mutant gliomas can be classified according to genetic characteristics and have different prognoses. However, prognostic heterogeneity can also be detected in IDH wild-type gliomas, but the indicators for predicting the prognosis of IDH wild-type GBM patients are still unclear. Therefore, it is extremely urgent to explore the potential diagnostic and prognostic indicators of IDH wild-type GBM. Summary of the invention

[0004] The technical problem to be solved by the present invention is: an IDH2 K155 acetylated modified antibody capable of detecting the survival rate of IDH wild-type GBM and a preparation method and application thereof.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing an IDH2 K155 acetylated modified antibody, comprising the following steps: using an IDH2 K155 acetylated modified polypeptide coupled to KLH to immunize a rabbit to obtain immunized rabbit serum, then determining the antibody titer of the antibody in the immunized rabbit serum, and purifying the antibody to obtain an IDH2 K155 acetylated modified antibody.

[0006] Another technical solution adopted by the present invention is: the IDH2 K155 acetylated modified antibody is prepared by the above-mentioned method for preparing the IDH2 K155 acetylated modified antibody.

[0007] Another technical solution adopted by the present invention is: use of the above-mentioned IDH2 K155 acetylated modified antibody in the preparation of a kit for diagnosis and prognosis evaluation of glioblastoma.

[0008] The beneficial effect of the present invention is that the antibody prepared by the present invention can help evaluate the diagnosis and prognosis of GBM, and the use of the antibody for detection greatly shortens the detection time and difficulty compared with conventional mass spectrometry analysis methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a mass spectrometry detection diagram of IDH2 K155ac polypeptide A in Example 1 of the present invention; Figure 2 This is a mass spectrometry detection diagram of IDH2 K155ac polypeptide B in Example 1 of the present invention; Figure 3 This is a mass spectrometry image of the non-modified polypeptide in Example 1 of the present invention; Figure 4 The results of serum ELISA (enzyme-linked immunosorbent assay) of R1 to R3 in Example 1 of the present invention; Figure 5 The serum ELISA test results of R4 to R6 in Example 1 of the present invention; Figure 6 The results of the initial screening of serum WB (protein immunoblotting) of R1 to R6 in Example 1 of the present invention; Figure 7 The antibody ELISA test results in Example 1 of the present invention; Figure 8 The antibody Dot blot test result in Example 1 of the present invention; Fig. 9 The antibody IHC (immunohistochemistry) test results in Example 1 of the present invention; Fig.10 Survival curves for IDH2 K155 acetylation in IDH wild-type GBM. DETAILED DESCRIPTION

[0010] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in combination with the implementation modes and the accompanying drawings.

[0011] A method for preparing an IDH2 K155 acetylated modified antibody comprises the following steps: using an IDH2 K155 acetylated modified polypeptide coupled with KLH to immunize a rabbit to obtain immunized rabbit serum, then determining the antibody titer of the antibody in the immunized rabbit serum, and purifying the antibody to obtain the IDH2 K155 acetylated modified antibody.

[0012] From the above description, it can be seen that the beneficial effect of the present invention is that the expression level of IDH2 K155 acetylation modification is negatively correlated with the survival rate of IDH wild-type GBM, so the IDH2 K155 acetylation modification antibody of the present invention can help evaluate GBM diagnosis and prognosis.

[0013] At present, most of the methods for detecting acetylation modification of IDH2 in glioma use mass spectrometry analysis, which requires fresh frozen samples of glioma or glioma cell lines to be lysed and then digested with trypsin into peptides. The peptides are then subjected to mass spectrometry analysis and acetylation modification enrichment before mass spectrometry analysis to detect the level of IDH2 K155 acetylation. This technical method currently has the following disadvantages: 1. The sample needs to be fresh, and the fixed samples currently collected in the clinic cannot be tested, which makes clinical operation difficult. 2. The equipment requirements are high, requiring an ultra-high performance liquid phase system, etc. 3. The amount of data analysis is large, and database searches and bioinformatics analysis are required. 4. The cost is high. The present invention prepares antibodies for IDH2 K155 acetylation and then detects it, which greatly shortens the detection time and difficulty, and previous specimens can also be collected for cohort studies.

[0014] Furthermore, the antibody titer of the antibodies in the sera of the immunized rabbits was determined by enzyme-linked immunosorbent assay.

[0015] Furthermore, when immunizing rabbits, antigens were injected at two points under the skin of both shoulders and at two points in the muscles of both hind legs, with 1 / 4 volume of the immunogen in each area.

[0016] From the above description, it can be seen that when immunization is performed using the above method, the immunogen can persist for a long time, thereby enhancing the immune response.

[0017] Furthermore, the IDH2 K155 acetylation-modified polypeptide includes IDH2 K155ac polypeptide A or IDH2 K155ac polypeptide B.

[0018] Preferably, the IDH2 K155 acetylation-modified polypeptide is IDH2 K155ac polypeptide A.

[0019] Furthermore, the polypeptide sequence of IDH2 K155ac polypeptide A is CREPIIS-(acetyl)K-NIPR, and the polypeptide sequence of IDH2K155ac polypeptide B is REPIIS-(acetyl)K-NIPRLVC.

[0020] From the above description, it can be seen that the antibody of the present invention has a recognition ability for at least one modified polypeptide at a dilution of no less than 1:50K, and does not recognize non-modified polypeptides.

[0021] Further, the purification process is specifically as follows: S1: Filter the immunized rabbit serum, load it onto a protein A (recombinant purified protein A) chromatography column, and then elute it; collect the eluate, add a neutralizing buffer solution and adjust the pH to 7 to obtain crude IgG (immunoglobulin G); S2: Load the crude IgG onto an antigen polypeptide affinity chromatography column to obtain the target antibody; S3: Load the target antibody onto a non-modified affinity chromatography column and collect the flow-through.

[0022] It can be seen from the above description that the purification process of the present invention can effectively remove non-specific antibody components.

[0023] Furthermore, after washing the column with PBS buffer solution in S1, elution was performed with glycine buffer.

[0024] Another technical solution adopted by the present invention is: the IDH2 K155 acetylated modified antibody is prepared by the above-mentioned method for preparing the IDH2 K155 acetylated modified antibody.

[0025] Another technical solution adopted by the present invention is: use of the above-mentioned IDH2 K155 acetylated modified antibody in the preparation of a kit for diagnosis and prognosis evaluation of glioblastoma.

[0026] Furthermore, the kit was used to detect the expression level of IDH2 K155 acetylation modification by immunohistochemistry.

[0027] Furthermore, the kit was used to detect the survival rate of IDH wild-type GBM.

[0028] Furthermore, the kit detected the expression level of IDH2 K155 acetylation modification, and the negative correlation between the expression level of IDH2 K155 acetylation modification and the survival rate of IDH wild-type GBM was used for glioblastoma diagnosis and prognosis evaluation.

[0029] From the above description, it can be seen that the immunohistochemical detection of the present invention found that the expression level of IDH2 K155 acetylation modification was negatively correlated with the survival rate of IDH wild-type GBM. Therefore, detecting the acetylation modification level of IDH2 K155 can be a method to help evaluate the diagnosis and prognosis of GBM.

[0030] Embodiment 1 of the present invention is: a method for preparing an IDH2 K155 acetylated modified antibody, comprising the following steps: 1. Preliminary preparation (1) Antigen design: Based on the protein sequence and modification type, two antigen peptides were designed and synthesized for animal immunization, purification and detection; at the same time, a non-modified control peptide was designed and synthesized for purification and detection. Peptide information is shown in Table 1.

[0031] Table 1 Peptide information

[0032] Note: IDH2 K155ac is the acetylation modification of IDH2 K155.

[0033] (2) Immunogen mass spectrometry detection: The results of immunogen mass spectrometry detection are shown in Figures 1 to 3 , Figures 1 to 3 The vertical axis represents the intensity of the ion peak, and the horizontal axis represents the ratio of mass to charge. Figures 1 to 3 The results show that the sequences of the three peptides are accurate. The difference between the measured mass and the theoretical mass of the three peptides is within 10ppm, and the sequences are correct.

[0034] (3) Animal husbandry: 6 healthy New Zealand rabbits.

[0035] (4) Peptide coupling: Two modified peptides were coupled to KLH (hemocyanin) for rabbit immunization.

[0036] 2. Rabbit immunization and serum screening (1) Prepare immune materials: dilute the immunogen with saline and then mix it with Freund's adjuvant in a 1:1 volume ratio. The antigen and adjuvant are completely mixed to form a stable emulsion. Use a syringe to extract the antigen mixture and inject the antigen into two points under the skin of the rabbit's shoulders and two points in the muscles of both hind legs. Each area is about 1 / 4 volume of the immunogen.

[0037] (2) Immunization: Each rabbit was immunized 4 times, on the 1st day, 21st day, 28th day, and 35th day respectively.

[0038] (3) Blood collection: Blood collection for the first time: On the 45th day, 30 ml of whole blood was collected and centrifuged. The supernatant was collected and sent to the laboratory for serum screening test; Blood collection for the 2nd, 3rd and 4th time: Draw whole blood three times on the 50th, 65th and 70th days, 20 mL each time, collect the supernatant after centrifugation and send it to the laboratory for serum screening test.

[0039] 3. Serum Screening After multiple immunizations, a small amount of serum was collected from 6 SPF experimental New Zealand white rabbits for ELISA testing to preliminarily evaluate the titer and specificity of the antiserum.

[0040] (1) Serum screening ELISA test: Rabbits immunized with IDH2 peptide A were labeled R1, R2, and R3, and rabbits immunized with IDH2 peptide B were labeled R4, R5, and R6. The results of ELISA test on the serum of 6 rabbits are shown in Figure 4 and 5 ,Depend on Figure 4 and 5 The titer of the identified modified peptides is basically around 1:16K (OD>1).

[0041] (2) Serum screening WB test: The immunized rabbit serum was used to detect endogenous IDH2 protein by WB. The serum dilution ratio of WB was 1:100, and the incubation time was 2h; the secondary antibody dilution ratio was 1:10k, and the incubation time was 45min; the exposure time was 30s, and the detection material was MCF-7. The WB results are shown in Figure 6 , Figure 6 The results showed that R3 and R4 could detect a band with a theoretical molecular weight of about 43 kDa of the target protein. Based on the results of ELISA and WB, the sera of rabbits R3 and R4 were selected for purification, and antibodies Ab3 and Ab4 were obtained after purification.

[0042] 4. Antibody Purification (1) Prepare the protein A affinity column: Select 10 ml of protein A filler, mix and stir equal volumes of filler and PBS (phosphate) buffer, then evacuate to remove bubbles in the filler. Slowly add the treated protein A filler into the glass column to prepare the chromatography column. Avoid column drying during this process. After filling, balance the column with 10 times the volume of pre-cooled PBS buffer solution.

[0043] (2) Protein A affinity chromatography: After filtering the serum with a filter, load it onto a well-balanced protein A affinity column. To detect the binding efficiency of the antiserum to the filler, retain the load effluent. Then wash the column with PBS buffer solution, elute with 150mM glycine buffer, collect the eluate, and add neutralization buffer solution to adjust the pH to 7 to obtain crude IgG.

[0044] (3) Enrichment of target antibodies: Load the crude IgG onto the well-equilibrated IDH2K 155ac modified peptide A affinity chromatography column to specifically enrich the target antibodies.

[0045] (4) Removal of non-specific antibodies: The target antibody obtained in the previous step is loaded onto an affinity chromatography column of the IDH2K155 non-modified peptide, and the flow-through (FT3) is directly collected to remove non-specific antibody components.

[0046] (5) Antibody preservation: Determine the protein content of the effluent. Add 10% glycerol to preserve the antibody, and store the purified antibody in aliquots at -20°C.

[0047] 5. Antibody quality control The antibodies were subjected to ELISA, Dot blot, WB, and IHC.

[0048] 5.1 ELISA test (1) Antigen coating: dilute the antigen (antigen-modified peptide and control peptide) with coating solution, add 50 μg / well of the ELISA plate in turn, and keep in refrigerator at 4°C overnight.

[0049] (2) Washing: Take out the ELISA plate coated the day before and add 1× TBST to wash three times.

[0050] (3) Blocking: Add 1% BSA (bovine serum albumin) blocking solution to the cleaned ELISA plate, incubate at 37°C for 1 hour, and then wash 1-3 times.

[0051] (4) Primary antibody incubation: dilute the antibody in a 3-fold gradient starting from 1:1K. Adjust the dilution volume according to the actual situation. Add to the ELISA plate in sequence, incubate at 37℃ for 1.5h, and then wash 1-3 times.

[0052] (5) Secondary antibody incubation: Dilute the secondary antibody to 1:10K with 1% BSA blocking solution, incubate at room temperature or 37°C for 45 min, and then wash twice.

[0053] (6) Color development: Add TMB colorimetric solution and then add 1 M sulfuric acid to terminate the display reaction 8 minutes later. Read the data using an ELISA reader.

[0054] ELISA test results are shown in Figure 7 .Depend on Figure 7 It can be seen that the recognition ability of the two antibodies for at least one modified peptide is not less than 1:50K dilution, and they do not recognize the non-modified peptide. The dilution of the ELISA positive (OD450>1.0) antibody is greater than 54000, and the signal is more than 10 times stronger than that of the negative non-modified peptide.

[0055] 5.2 Dot blot detection (1) Spotting: Spot the uncross-linked antigen peptide onto the PVDF (polyvinylidene fluoride) membrane in a gradient of 1 ng, 4 ng, 16 ng, and 64 ng.

[0056] (2) Blocking: After the membrane surface is dry, add blocking solution and block at room temperature for 60 minutes.

[0057] (3) Washing: Wash with 1× TBST for 10 min.

[0058] (4) Primary antibody incubation: dilute the antibody with 5% skim milk powder at a dilution ratio of 1:2K. Incubate at room temperature for 2 h, then wash three times with 1× TBST, each time for 15 min.

[0059] (5) Secondary antibody incubation: Select the corresponding rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:10K, incubate at room temperature for 45 min, and then wash three times with 1× TBST, each time for 5 min.

[0060] (6) Add a color developing substrate to the washed membrane and then expose it to light.

[0061] Dot blot test results are shown in Figure 8 , Figure 8 This indicates that the positive detection limit of Dot blot is 4ng, and the signal is more than 10 times stronger than that of negative non-modified peptides. Ab3 and Ab4 have almost no binding to non-modified peptides, and have strong binding to positive peptides, especially IDH2 peptide A.

[0062] 5.3 WB detection (1) Cell lysis: Select cells or tissues for testing according to experimental requirements. Select different lysis methods according to different samples to obtain the total protein of the sample, and determine the protein concentration using the BCA (bicinchoninic acid) method.

[0063] (2) Protein electrophoresis and membrane transfer: Protein electrophoresis: According to the molecular weight of the target protein, select the corresponding concentration of separation gel. Add 10% ammonium persulfate and TEMED (tetramethylethylenediamine) at the end of the gel preparation. 4.7ml of separation gel per plate. Load 30μg of sample per well and perform electrophoresis; the electrophoresis conditions are: 80V for concentrated gel and 120V for separation gel.

[0064] Wet transfer: Cool the transfer solution before transfer, and lay the gel, membrane, and filter paper in the transfer solution to form a sandwich structure to avoid bubbles. The transfer current is 300mA.

[0065] (3) Blocking: Add blocking solution to the transferred membrane and block at room temperature for 60 minutes. After incubation, wash with 1× TBST for 10 minutes.

[0066] (4) Primary antibody incubation: Dilute the antibody with primary antibody diluent, incubate at room temperature for 2 h, and then wash three times with 1× TBST, each time for 15 min.

[0067] (5) Secondary antibody incubation: Select the corresponding rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:10K, incubate at room temperature for 45 minutes, and then wash three times with 1× TBST, each time for 15 minutes.

[0068] (6) Add a color developing substrate to the washed membrane and then expose it to light.

[0069] WB detection was performed using U87-MG cells and MCF7 cells, and both antibodies failed to detect a band around 43 kDa.

[0070] 5.4 IHC (1) Dewaxing / rehydrating: Heat in an oven at 63 - 65 degrees Celsius for approximately 1 hour. Use the following procedure for dewaxing / rehydrating: soak in xylene twice (10 minutes each), soak in anhydrous ethanol twice (5 minutes each), soak in 95% ethanol twice (5 minutes each), soak in 85% ethanol (3 minutes), soak in 75% ethanol (3 minutes), and finally wash with distilled water three times.

[0071] (2) Antigen repair: Add 2000 ml of sodium citrate antigen repair solution (pH 6.0) to the pressure cooker, immerse the slide in the antigen repair solution, run the pressure cooker under high pressure for 2 minutes, and after the high pressure ends, naturally cool to room temperature (10-30 minutes).

[0072] (3) Blocking: Wash the slides three times with distilled water; soak the tissues in 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase; wash the slides three times with 1× TBST, 10 min each time.

[0073] (4) Incubation with primary antibody: Dilute the primary antibody with primary antibody diluent according to the instructions. Place the sections in a humidified chamber and add the primary antibody. Then incubate the humidified chamber at room temperature for 2.5 hours and wash the slides three times with 1× TBST, 10 minutes each time.

[0074] (5) Incubation with secondary antibody: Add HRP-Polymer anti-Rabbit secondary antibody, incubate at room temperature for 30 min, and then wash the slides three times with 1× TBST, each time for 10 min.

[0075] (6) Color development: Add freshly prepared DAB substrate to the slices and incubate until brown staining occurs. Rinse the slices with distilled water.

[0076] (7) Restain with hematoxylin for 1 min, rinse the sections with water, and differentiate.

[0077] (8) Rinse with 75% ethanol (3 minutes), 95% ethanol (3 minutes), 2 times with absolute ethanol (3 minutes each), and then 2 times with xylene or tissue clearing agent (5 minutes each). Dry the slide with a hair dryer and fix the cover slip to the slide using mounting medium.

[0078] Ab3 and Ab4 were subjected to IHC analysis, and paraffin-embedded Mouse kidney (mouse kidney tissue extract) tissues were labeled with Ab3 and Ab4 diluted 1:1000, respectively. The IHC test results showed that Ab3 localized mitochondria and had obvious staining. In the tissue samples blocked with modified peptides by Ab3, mitochondrial staining was significantly weakened (nuclei were counterstained with hematoxylin), indicating that Ab3 can specifically bind to IDH2 (K155ac) in tissues, and Ab3 antibody can recognize endogenous IDH2 K155ac through IHC, and localize accurately in tissues.

[0079] KM survival curve analysis was performed based on the survival months and survival status of patients in the IDH2 K155 acetylation-positive group (n=24) and the IDH2 K155 acetylation-negative group (n=21). The analysis results are shown in Fig.10 , Fig.10 The antibody used was Ab3. Fig.10 The results showed that the median overall survival of the positive group was 26.9 months, while that of the negative group was 7.5 months. IDH2 K155 acetylation modification was negatively correlated with the survival rate of IDH wild-type GBM (Log-rank P=0.0088, HR=0.4098 (95% CI, 0.1970-0.8524)).

[0080] The second embodiment of the present invention is: an IDH2 K155 acetylated modified antibody prepared by the preparation method of the IDH2 K155 acetylated modified antibody of the first embodiment.

[0081] Embodiment 3 of the present invention is: use of the IDH2 K155 acetylated modified antibody in the embodiment in the preparation of a kit for the diagnosis and prognosis evaluation of glioblastoma, the kit detects the expression level of IDH2 K155 acetylated modification, and the diagnosis and prognosis evaluation of glioblastoma are performed through the negative correlation between the expression level of IDH2 K155 acetylated modification and the survival rate of IDH wild-type GBM.

[0082] In summary, the IDH2 K155 acetylated modified antibody provided by the present invention and its preparation method and application have the following advantages: 1. The expression level of IDH2 K155 acetylation-modified antibody is negatively correlated with the survival rate of IDH wild-type GBM, which can help evaluate the diagnosis and prognosis of GBM.

[0083] 2. Prepare antibodies, and then perform ELISA, immunohistochemistry, etc. to detect the acetylation level of IDH2 K155 in glioma, shortening the detection time and difficulty.

[0084] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing an IDH2 K155 acetylated antibody, characterized in that: The following steps are involved: IDH2K155 acetylated modified peptide coupled with KLH was used to immunize rabbits to obtain immunized rabbit serum, and then the antibody titer of the antibody in the immunized rabbit serum was determined, and the antibody was purified to obtain IDH2 K155 acetylated modified antibody.

2. The method for preparing an IDH2 K155 acetylated antibody according to claim 1, characterized in that: The antibody titer of the antibodies in the sera of immunized rabbits was determined by enzyme-linked immunosorbent assay.

3. The method for preparing an IDH2 K155 acetylated antibody according to claim 1, characterized in that: The IDH2 K155 acetylation-modified polypeptide includes IDH2 K155ac polypeptide A or IDH2 K155ac polypeptide B.

4. The method for preparing an IDH2 K155 acetylated antibody according to claim 3, characterized in that: The polypeptide sequence of the IDH2 K155ac polypeptide A is CREPIIS-(acetyl)K-NIPR, and the polypeptide sequence of the IDH2 K155ac polypeptide B is REPIIS-(acetyl)K-NIPRLVC.

5. The method for preparing an IDH2 K155 acetylated antibody according to claim 1, characterized in that: The purification process is specifically as follows: S1: After filtering the immunized rabbit serum, load it onto a protein A chromatography column and then elute it; collect the eluate and add a neutralization buffer solution to adjust the pH to 7 to obtain crude IgG; S2: Load the crude IgG onto an antigen polypeptide affinity chromatography column to obtain the target antibody; S3: Load the target antibody onto a non-modified affinity chromatography column and collect the flow-through.

6. The method for preparing an IDH2 K155 acetylated antibody according to claim 5, characterized in that: In the S1, the column is washed with PBS buffer solution and then eluted with glycine buffer.

7. An IDH2 K155 acetylated antibody prepared by the method for preparing an IDH2 K155 acetylated antibody according to any one of claims 1 to 6.

8. Use of the IDH2 K155 acetylated-modified antibody as claimed in claim 7 in the preparation of a kit for diagnosis and prognosis assessment of glioblastoma.

9. The use according to claim 8, characterized in that The kit is used for detecting the expression level of IDH2 K155 acetylation modification by immunohistochemistry.

10. The use according to claim 8, characterized in that: The kit is used to detect the survival rate of IDH wild-type GBM.

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