Antibodies to phytocerated isocitrate dehydrogenase 1 and methods of making and uses thereof

Abstract

The present application relates to the antibody of phorbolylated isocitrate dehydrogenase 1 and its preparation method and purpose, and specifically provides a polyclonal antibody, the polyclonal antibody is obtained by immunizing animals with the antigen epitope peptide of IDH1 phorbolylated, the specific site of the antigen epitope peptide is phorbolylated, and high-titer antibodies can be prepared, the phorbolylated modification of IDH1 in clinical specimens can be recognized with high specificity, and the polyclonal antibody can be used for the research of cell / tissue specimen immunoblotting and immunohistochemical method and the diagnosis of IDH1 related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to antibodies against crotonylated isocitrate dehydrogenase 1, their preparation methods, and uses. Background Technology

[0002] Isocitrate dehydrogenase 1 (IDH1) is a key enzyme in the intracellular tricarboxylic acid cycle (TCA cycle). Its main function is to convert isocitrate to α-ketoglutarate and simultaneously produce one molecule of NADH (coenzyme II), participating in the regulation of various post-transcriptional modifications, including alternative splicing. In recent years, post-translational modifications of IDH1 have been found to be closely related to lipid metabolism.

[0003] Crotonylation is a novel modification that can participate in glucose and lipid metabolism by regulating chromatin remodeling, cell cycle, and DNA damage repair. Therefore, the detection of crotonylation levels in IDH1 is of great significance.

[0004] Currently, research methods on crotonylation modification mainly rely on proteomics and immunoprecipitation (e.g., non-patent literature crotonylation on non-histone proteins. Cell Res. 2017 Jul;27(7)). The key reagent in the experimental process is a pan-antibody against crotonylation. Both of these methods are cumbersome and expensive, making them unsuitable for clinical application. Therefore, whether it is possible to provide an antibody that can be applied to the liver or colon and has good specificity in recognizing IDH1 crotonylation modification has become an urgent problem to be solved in this field. Summary of the Invention

[0005] This invention provides a polyclonal antibody obtained through an IDH1 crotonylated epitope peptide. The specific site of the epitope peptide is crotonylated, and the epitope peptide can be used to immunize animals to obtain an antibody with good specificity in recognizing IDH1 crotonylation modification, which can help in experimental research on IDH1 crotonylation and the diagnosis of diseases.

[0006] In a first aspect, the present invention provides a method for preparing an antibody against modified IDH1, the method comprising immunizing an animal with an antigenic epitope peptide of modified IDH1, wherein the antigenic epitope peptide includes modification at at least one of positions 58, 151, 212 or 345 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0007] Preferably, the modification includes crotonylation.

[0008] Preferably, the crotonylation site includes:

[0009] The 58th bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0010] The 151st bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0011] The 212th bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0012] The 345th bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0013] The 58th or 151st bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0014] The 58th and 212th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0015] The 58th or 345th bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0016] The 151st and 212th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0017] The 151st and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0018] The 212th and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0019] The 58th, 151st, and 212th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0020] The 58th, 151st, and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0021] The 151st, 212th, and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0022] The 58th, 212th, or 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2; or

[0023] The 58th, 151st, 212th, and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2.

[0024] Preferably, the preparation method includes the following steps:

[0025] The antigenic epitope peptide was coupled with a carrier protein to obtain a recombinant antigen, which was then used to immunize animals.

[0026] Preferably, the above-mentioned antigenic epitope peptide is coupled with a carrier protein at a mass ratio of 1:1 to 10:1 to obtain a recombinant antigen, which is then used to immunize animals.

[0027] Preferably, the carrier protein includes, but is not limited to, hemocyanin KLH, bovine serum albumin (BSA), or chicken ovalbumin (OVA).

[0028] The methods for immunizing animals include:

[0029] Perform 2-7 immunizations (preferably 5). For each immunization, the adjuvant and the recombinant antigen are mixed at a volume ratio of 1:(0.8-1.2). The concentration of the recombinant antigen is 0.1-5 mg / mL. The immunization is performed by multiple subcutaneous injections, and the amount of immunized antigen is 100-1000 μg (preferably 100-500 μg or 200-1000 μg).

[0030] In one specific embodiment, the method of immunizing the animal includes:

[0031] On day 1, the adjuvant and the recombinant antigen were mixed at a volume ratio of 1:(0.8-1.2), the concentration of the recombinant antigen was 0.1-5 mg / mL, and the mixture was injected subcutaneously at multiple points. The amount of immunized antigen was 200-1000 μg.

[0032] On days 5-14, the adjuvant and the recombinant antigen were mixed at a volume ratio of 1:(0.8-1.2), the concentration of the recombinant antigen was 0.1-5 mg / mL, and the mixture was injected subcutaneously at multiple sites. The amount of immunized antigen was 100-500 μg.

[0033] On days 15-28, the adjuvant and the recombinant antigen were mixed at a volume ratio of 1:(0.8-1.2), with the concentration of the recombinant antigen being 0.1-5 mg / mL. The mixture was administered via subcutaneous injection at multiple sites, with an immunization dose of 100-500 μg.

[0034] On days 29-42, the adjuvant and the recombinant antigen were mixed at a volume ratio of 1:(0.8-1.2), the concentration of the recombinant antigen was 0.1-5 mg / mL, and the mixture was injected subcutaneously at multiple sites, with an immunization dose of 100-500 μg.

[0035] From day 43 to day 70, the adjuvant and the recombinant antigen are mixed at a volume ratio of 1:(0.8-1.2), the concentration of the recombinant antigen is 0.1-5 mg / mL, and the mixture is injected subcutaneously at multiple sites. The amount of immunized antigen is 100-500 μg.

[0036] Animal serum was collected, separated, and purified between days 64 and 84.

[0037] Preferably, the animal is selected from non-human mammals; more preferably, the non-human mammal is selected from rabbits, mice, sheep or horses.

[0038] Preferably, the adjuvant is a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response against the co-inoculated antigen. The adjuvants described herein can be those known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic compounds, quinine, saponins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokine and nucleic acid adjuvants (such as monocyte clone stimulating factor, leukocyte cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid carriers, etc.), and emulsion adjuvants (such as Freund's adjuvant and incomplete Freund's adjuvant). The adjuvant can be a pharmaceutically acceptable adjuvant.

[0039] In a second aspect, the present invention provides an antibody comprising the antibody obtained by the preparation method described above.

[0040] In a third aspect, the present invention provides a nucleic acid molecule that encodes an antibody obtained by the above-described preparation method or the antibody described above.

[0041] In a fourth aspect, the present invention provides an expression cassette, characterized in that the expression cassette expresses an antibody or antibody prepared by the above-described preparation method, or a nucleic acid molecule or antibody prepared by the above-described method.

[0042] In a fifth aspect, the present invention provides a recombinant vector expressing an antibody prepared by the above-described preparation method or the antibody described above, or a nucleic acid molecule described above or an expression cassette described above.

[0043] In a sixth aspect, the present invention provides a cell that expresses an antibody obtained by the preparation method described above or the antibody described above, or an antibody that includes the nucleic acid molecule described above, the expression cassette described above, or the recombinant vector described above.

[0044] A seventh aspect of the present invention provides a method for detecting modified IDH1, characterized in that the method comprises using an antibody obtained by the preparation method described above or an antibody described above.

[0045] The eighth aspect of the present invention provides the use of the antibody obtained by the above-described preparation method, the antibody described above, the nucleic acid molecule described above, the expression cassette described above, the recombinant vector described above, or the cell described above in the preparation of products for the diagnosis and prognosis of non-alcoholic fatty liver disease or metabolic-associated steatohepatitis.

[0046] Preferably, the product includes reagents, test strips, kits, or chips.

[0047] A ninth aspect of the present invention provides a modified IDH1 epitope peptide, said epitope peptide comprising modification at at least one of positions 58, 151, 212 or 345 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0048] Preferably, the modification includes crotonylation.

[0049] Preferably, the crotonylation site includes:

[0050] The 58th bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0051] The 151st bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0052] The 212th bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0053] The 345th bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0054] The 58th or 151st bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0055] The 58th and 212th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0056] The 58th or 345th bit of SEQ ID NO: 1 or SEQ ID NO: 2;

[0057] The 151st and 212th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0058] The 151st and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0059] The 212th and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0060] The 58th, 151st, and 212th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0061] The 58th, 151st, and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0062] The 151st, 212th, and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2;

[0063] The 58th, 212th, or 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2; or

[0064] The 58th, 151st, 212th, and 345th bits of SEQ ID NO: 1 or SEQ ID NO: 2.

[0065] In a tenth aspect, the present invention provides a recombinant antigen comprising the above-described antigenic epitope peptide and carrier protein.

[0066] Preferably, the carrier protein includes, but is not limited to, hemocyanin KLH, bovine serum albumin (BSA), or chicken ovalbumin (OVA).

[0067] In an eleventh aspect, the present invention provides a composition comprising the above-described antigenic epitope peptide and an adjuvant.

[0068] Preferably, the adjuvant is the adjuvant described in the first aspect.

[0069] The terms “comprising” or “including” in this invention are open-ended descriptions that include the specified ingredients or steps described, as well as other specified ingredients or steps that do not materially affect them.

[0070] The beneficial effects of this invention are:

[0071] This invention provides an IDH1 crotonylated epitope peptide, which can be used to prepare high-titer polyclonal antibodies that can specifically recognize crotonylation modifications at positions 58, 151, 212, and 345 of IDH1 amino acids in clinical specimens. It can be used for immunoblotting and immunohistochemical studies of cell / tissue specimens. Attached Figure Description

[0072] Figure 1 Dot blot results.

[0073] Figure 2 ELISA experimental results.

[0074] Figure 3 Western blotting experiments showed the antibody specificity of mouse liver AML12 cells, human embryonic kidney cells HEK293T cells, and human hepatocellular carcinoma HepG2 cells.

[0075] Figure 4 Image showing the results of the IP experiment on HEK293T human embryonic kidney cells.

[0076] Figure 5 Figure: Results of IP assay for mouse liver AML12 cells.

[0077] Figure 6Results of Western blotting experiments on mouse liver AML12 cells and human hepatocellular carcinoma HepG2 cells. Detailed Implementation

[0078] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0079] Unless otherwise specified, all materials, reagents, instruments, etc. used in the following examples are commercially available.

[0080] Example 1: Design and Synthesis of IDH1 Crotonylated Antigenic Epitope Peptide

[0081] Based on the original IDH1 amino acid sequence, IDH1 crotonylated epitope peptides were designed. According to the Mus_musculus_10090 database, the IDH1 amino acid sequence is as follows: MSRKIQGGSVVEMQGDEMTRIIWELIKEKLILPYVELDLHSYDLGIENRDATNDQVTKDAAEAIKKYNVGVKCATITPDEKRVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVTGWVKPIIIGRHAYGDQYRATDFVVPGPGKVEITYTPKDGTQKVTYMVHDFEEGGGVAMGMYNQDKSIED FAHSSFQMALSKGWPLYLSTKNTILKKYDGRFKDIFQEIYDKKYKSQFEAQKICYEHRLIDMVAQAMKSEGGFIWACKNYDGDVQSDSVAQGYGSLGMMTSVLICPDGKTVE AEAAHGTVTRHYRMYQKGQETSTNPIASIFAWSRGLAHRAKLDNNTELSFFAKALEDVCIETIEAGFMTKDLAACIKGLPNVQRSDYLNTFEFMDKLGENLKAKLAQAKL(SEQ IDNO:1).

[0082] The amino acid sequence of Homo sapiens_9606, IDH1 is as follows:

[0083] MSKKISGGSVVEMQGDEMTRIIWELIKEKLIFPYVELDLHSYDLGIENRDATNDQVTKDAAEAIKKHNVGVKCATITPDEKRVEEFKLKQMWKSPNGTIRNILG GTVFREAIICKNIPRLVSGWVKPIIIGRHAYGDQYRATDFVVPGPGKVEMTYTPSDGTQKVTYLVHNFEEEGGGVAMGMYNQDKSIEDFAHSSFQMALSKGWPLYL STKNTILKKYDGRFKDIFQEIYDKQYKSQFEAQKIWYEHRLIDMVAQAMKSEGGFIWACKNYDGDVQSDSVAQGYGSLGMMTSVLVCPDGKTVEAEAAHGTVT RHYRMYQKGQETSTNPIASIFAWTRGLAHRAKLDNNKELAFFANALEEVSIETIEAGFMTKDLAACIKGLPNVQRSDYLNTFEFMDKLGENLKIKLAQAKL(SEQ ID NO: 2).

[0084] Among them, the 58th, 151st, 212th and / or 345th positions of the selected amino acid sequence were modified by crotonylation.

[0085] Finally, the crotonylated antigenic epitope peptide was synthesized by a sequence synthesis company.

[0086] Example 2: Preparation of IDH1 Crotonylated Polyclonal Antibody

[0087] (a) Specifically, it includes the following steps:

[0088] The IDH1 crotonylated antigenic epitope peptide obtained in Example 1 was coupled with a carrier protein (such as hemocyanin KLH) at a mass ratio of 1:1 to 10:1 to obtain a recombinant antigen, which was then used to immunize animals.

[0089] The method for immunizing the animals is as follows:

[0090] On day 1, the complete Freund's adjuvant and the recombinant antigen were mixed at a volume ratio of 1:(0.8-1.2), the concentration of the recombinant antigen was 0.1-5 mg / mL, and the mixture was injected subcutaneously at multiple sites. The amount of immunized antigen was 200-1000 μg.

[0091] On days 5-14, the incomplete Freund's adjuvant and the recombinant antigen were mixed at a volume ratio of 1:(0.8-1.2), with the concentration of the recombinant antigen being 0.1-5 mg / mL. The mixture was administered via subcutaneous injection at multiple sites, with an immunization dose of 100-500 μg.

[0092] On days 15-28, the incomplete Freund's adjuvant and the recombinant antigen were mixed at a volume ratio of 1:(0.8-1.2), with the concentration of the recombinant antigen being 0.1-5 mg / mL. The mixture was administered via subcutaneous injection at multiple sites, with an immunization dose of 100-500 μg.

[0093] On days 29-42, the incomplete Freund's adjuvant and the recombinant antigen were mixed at a volume ratio of 1:(0.8-1.2), with the concentration of the recombinant antigen being 0.1-5 mg / mL. The mixture was administered via subcutaneous injection at multiple sites, with an immunization dose of 100-500 μg.

[0094] From day 43 to 70, the incomplete Freund's adjuvant and the recombinant antigen were mixed at a volume ratio of 1:(0.8-1.2), with the concentration of the recombinant antigen being 0.1-5 mg / mL. The mixture was administered via subcutaneous injection at multiple sites, with an immunization dose of 100-500 μg.

[0095] Animal serum was collected, separated, and purified between days 64 and 84.

[0096] (ii) Validation of the efficacy of the antibody against the obtained crotonylated IDH1.

[0097] In the following experiments of this application, experiments involving mouse cells were performed using polyclonal antibodies obtained by immunization with SEQ ID NO:1, and experiments involving human-related cells were performed using polyclonal antibodies obtained by immunization with SEQ ID NO:2. Given the high homology between human and mouse sequences (especially related sites), the corresponding antibodies can be used for detection in human or mouse-related cells.

[0098] 1. Dot blot experiment

[0099] The experimental steps include:

[0100] (1) Spotting and fixing: Spot the sample to be tested directly onto the solid phase membrane (NC membrane), and fix it by air drying or baking.

[0101] (2) Hybridization and detection: After fixation, the membrane is sealed, hybridization is performed using labeled probes, unbound probes are eluted, and finally the target molecule signal is detected by color development or luminescence.

[0102] Experimental results are as follows Figure 1 As shown, the specificity of these antibodies was validated by dot blot analysis of the corresponding peptides with or without Kcr modification.

[0103] 2. Antibody titer testing

[0104] The experimental steps include:

[0105] 1. Antigen coating

[0106] Dilute the known antigen (the above-mentioned antigenic epitope peptide) to 1–10 μg / mL with coating buffer (such as pH 9.6 carbonate buffer) and add it to a 96-well plate (100 μL / well).

[0107] Incubate overnight at 4°C or for 2 hours at 37°C.

[0108] Wash the plate 3 times (spin dry, soak in PBST washing solution, and pat dry).

[0109] 2. Closed

[0110] Add 200 μL of blocking buffer (5% skim milk powder or PBST with 1% BSA) to each well and block at 37°C for 1 hour.

[0111] Wash the plate 3 times.

[0112] 3. Add the serum to be tested (serially diluted).

[0113] The serum to be tested is serially diluted with diluent (such as PBST of 1% BSA) (e.g., 1:2000, 1:6000, 1:18000, 1:54000, 1:162000, 1:486000, 1:1458000, etc.), and 100 μL is added to each well.

[0114] Negative control: Diluent without serum.

[0115] Positive control: Known high-titer positive serum (antibodies obtained without modification).

[0116] Incubate at 37℃ for 2 hours, then wash the plate 3 times.

[0117] 4. Add enzyme-labeled secondary antibody

[0118] Add HRP-labeled secondary antibody, 100 μL / well, and incubate at 37°C for 45 minutes.

[0119] Wash the plate 5 times (to completely remove the non-conjugated secondary antibody).

[0120] 5. Color Development and Termination

[0121] Add substrate (e.g., TMB, 100 μL / well) and develop color in the dark for 10–15 minutes (the solution turns blue).

[0122] Add the stop solution (50 μL / well, 2M H2SO4), and the solution turns yellow.

[0123] 6. Detection and Potency Determination

[0124] The OD value at 450 nm was read using an ELISA reader.

[0125] Antibody titer calculation: The highest serum dilution factor that is 2.1 times higher than the negative control OD value (or the preset threshold) is used as the antibody titer (e.g., if 1:5000 is still positive, then the titer is 5000).

[0126] like Figure 2 As shown, the antibodies obtained after crotonylation at each site all exhibited higher titers than the unmodified antibodies. The experimental results indicate that the antibodies obtained by crotonylation of IDH1 in both humans and mice obtained high titers.

[0127] 3. IP and Western blotting experiments

[0128] Plasmid construction and cell transfection

[0129] AML12, HepG2, and HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).

[0130] Specific site plasmids were constructed using the pcDNA3.0 vector: WT, K58R, K151R, K212R, and K345R (it should be noted that K58R, K151R, K212R, and K345R are plasmids expressing IDH1 protein with the corresponding site mutated to arginine; the crotonylation modification at the corresponding site is changed in the mutated IDH1 protein).

[0131] HEK293T (human embryonic kidney cells 293) was selected as the tool cell line for plasmid transfection, and the steps are as follows:

[0132] Cells were plated one day prior to transfection. The following day, when the cells reached 80-90% confluency, five plasmids (WT, K58R, K151R, K212R, and K345R) were transfected into the cells using Lipo3000, following the dosage and administration instructions. The transfection system was added to serum- and antibiotic-free DMEM high-glucose cell culture medium and incubated at 37°C, 5% CO2, and saturated humidity for 48 hours before subsequent experiments. AML12 and HepG2 cells were also used as tool cells, and plasmid transfection was performed following the same steps.

[0133] The next step is immunoprecipitation analysis, which includes the following steps:

[0134] The cells obtained above were lysed. Cell lysis buffer was prepared by placing cells in lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA (pH 7.5)] or BC100 buffer [20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100], with a protease inhibitor mixture added. The cells were incubated at 4°C for 20 minutes, followed by centrifugation at 14,000 g for 15 minutes at 4°C. Protein concentration in the cell lysis buffer was determined using a BCA protein quantification kit (Pierce) according to the manufacturer's instructions.

[0135] A 5% (1:20) fraction of cell extract was used as the "Input" (input control). For immunoprecipitation (IP) experiments, 500 μg of protein lysis buffer and 2 μg of specific antibody were incubated at 4°C with continuous rotation for 12 hours; then 60 μL of 50% protein A or G agarose beads were added, and incubation continued for 2 hours. Afterward, the agarose beads were washed five times with lysis buffer, and after each wash, the beads were collected by centrifugation at 500 g for 5 minutes at 4°C.

[0136] The precipitated proteins were eluted by resuspending the beads in 2× SDS-PAGE loading buffer and boiling for 10 minutes. Immunoprecipitates or cell lysate samples were separated by 10% SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane.

[0137] For Western blotting analysis, the membrane was incubated with the corresponding primary antibody for 1 hour at room temperature, or overnight at 4°C, followed by incubation with the secondary antibody. Immunoreaction bands were developed using the manufacturer's recommended method (Santa Cruz Biotechnology) using luminol reagent.

[0138] Experimental results are as follows Figure 3-5 As shown, immunoprecipitation experiments performed in AML12 and HEK293T cells transfected with wild-type IDH1 (WT IDH1) or point mutants IDH1-K58R, IDH1-K151R, IDH1-K212R, and IDH1-K345R with the MYC tag showed that the corresponding IDH1 Kcr (lysine crotonylation modification) antibody could not recognize these mutants after mutating lysine (K) to arginine (R), indicating that the polyclonal antibody obtained in Example 1 has site specificity in different cell lines.

[0139] In addition, polyclonal antibodies were used to detect the Kcr modification at K58, K151, K212, and K345 sites on IDH1 in AML12 and HepG2 cells, respectively. To induce lipid deposition, cells were treated with 0.5 mM palmitic acid (PA) and oleic acid (OA) at a ratio of 1:2 (V / V) PA:OA for 24 h. The control group was treated with 1% BSA for the same 24 h.

[0140] Specific methods include: (1) constructing a hyperlipidemic hepatocyte model

[0141] Plating: 50,000 HepG2 and AML12 cells in logarithmic growth phase were seeded into 24-well plates, with 500 μL of culture medium per well;

[0142] Acidification: After HepG2 and AML12 cells were cultured for 12 hours and observed to have reached 70-80% confluence under a microscope, the medium was replaced with serum-free DMEM (500 μL per well) to starve the cells for 12 hours. After 12 hours, the blank control group was replaced with 500 μL of serum-free medium, while other groups were treated with 500 μL of oleic acid (OA) and 500 μL of palmitic acid (PA) per well. The final concentration of PA+OA in the cells was 0.5 mM. The BSA control group was treated with 1% BSA in the same way. The cells were incubated for 24 hours.

[0143] Staining: After 24 hours of incubation, discard the culture medium, wash once with PBS (pre-cooled at 4°C), add 500 μL of 4% paraformaldehyde fixative to each well and fix overnight at 4°C. Discard the paraformaldehyde fixative, wash once with PBS, rinse with 60% isopropanol for 10 min to enhance cell wall permeability, remove the 60% isopropanol, add 500 μL of 0.3% Oil Red O staining solution to each well, stain at room temperature for 1 h, then rinse once quickly with 60% isopropanol and once with PBS buffer. Then perform nuclear staining, staining with light hematoxylin for 10 min, followed by rinsing twice with PBS.

[0144] Observation: The formation of red lipid droplets in the cells was observed using an inverted microscope. The accumulation of lipid droplets in the PA+OA group was clearly observed, indicating that the model was successfully established.

[0145] (2) Quantitative IP assay to detect IDH1 crotonylation expression in a high-fat hepatocyte model

[0146] Refer to the immunoprecipitation analysis method above.

[0147] The results are as follows Figure 6As shown, compared with bovine serum albumin (BSA)-induced hepatocytes, palmitic acid (PA) and oleic acid (OA)-induced hepatocytes showed significantly reduced Kcr (lysine crotonylation) levels in IDH1.

[0148] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. Use of a modified IDH1 antibody in the preparation of a product for diagnosing non-alcoholic fatty liver disease or metabolic-associated steatohepatitis, wherein the modified IDH1 antibody is obtained by immunizing animals with a modified IDH1 as an antigenic epitope peptide, wherein the antigenic epitope peptide is crotonylated at position 58, 151, 212 or 345 of SEQ ID NO:

2.

2. The use according to claim 1, characterized in that, The modified IDH1 antibody is obtained by conjugating the antigenic epitope peptide to a carrier protein to obtain a recombinant antigen, which is then used to immunize animals.

3. The use according to claim 2, characterized in that, The carrier proteins include hemocyanin KLH, bovine serum albumin (BSA), or chicken ovalbumin (OVA).

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