Antibody for specifically recognizing K88 methylated FTH1 protein as well as preparation method and application of antibody

Abstract

The invention discloses a human FTH1 protein K88 methylation specific antibody as well as a preparation method and application thereof. The amino acid sequence of the antigen synthetic peptide is as shown in SEQ ID NO: 1, and lysine at the 88th site of FTH1 protein is modified by single methylation. The antibody is prepared by immunizing animals with the antigen synthetic peptide subjected to site specific methylation modification. The methylated antigen synthetic peptide is low in cost and high in immunogenicity, and the method for preparing an antibody from the antigen synthetic peptide is efficient and low in cost; the anti-FTH1K88me1 antibody disclosed by the invention can be used for specific detection of single methylation modification of the 88th lysine of FTH1 in peptide fragments, proteins, cells and tissues, and has an important value in methylation function research of the FTH1.

Description

Technical Field

[0001] The present invention relates to the biomedical technology field of antibody development, and in particular to an antibody specifically recognizing K88 monomethylated FTH1 protein, a preparation method and an application thereof. Background Art

[0002] Death resistance is a key characteristic of tumor cells, and novel cell death mechanisms may become new targets for tumor therapy. Ferroptosis is a novel cell death mechanism in which free intracellular divalent iron participates in the Fenton reaction, triggering lipid peroxidation and promoting ferroptosis. Ferritin heavy chain (FTH1) is a major component of ferritin, a protein involved in intracellular iron storage. It can oxidize highly reactive divalent iron ions into trivalent iron, while promoting their mineralization and storage in ferritin cages, inhibiting the release of free iron and thus the occurrence of ferroptosis. Therefore, FTH1, as a key factor regulating ferroptosis in tumor cells, is an important target for inducing ferroptosis in tumor cells and thus preventing and treating tumors.

[0003] The function of FTH1 is regulated at multiple levels, with post-translational modifications (PTMs) being a key component. For example, O-GlcNAcylation can occur at position S179 of the FTH1 molecule. Inhibition of FTH1 O-GlcNAcylation activates ferritin autophagy, releasing stored iron and inducing ferroptosis. Protein lysine (K) methylation is a key form of post-translational modification, including monomethylation (Kme1), dimethylation (Kme2), and trimethylation (Kme3). Protein lysine methylation can exert biological functions by regulating protein activity, protein-protein interactions, and protein degradation. Our study, using mass spectrometry analysis of lung cancer tissues, demonstrated that FTH1 can undergo monomethylation at lysine 88 (K88), and the degree of monomethylation of K88 in FTH1 is higher in lung cancer tissue than in adjacent tissues. Considering that FTH1 is distributed both extracellularly and intracellularly and has normal physiological functions, antibodies targeting the monomethylation of FTH1 molecule K88 theoretically have safer and more precise application potential in the diagnosis and prevention of lung cancer than antibodies that simply target the FTH1 molecule.

[0004] The role of monomethylation at K88 of FTH1 in the ferroptosis sensitivity of tumor cells urgently requires investigation. Conventional mass spectrometry-based assays for identifying methylation at the FTH1 protein site are time-consuming, unstable, and require high instrumentation. Currently, there are no antibodies specifically recognizing monomethylation at K88 of FTH1 on the market, making it difficult to perform specific antibody-based techniques such as immunoblotting, immunofluorescence, and immunohistochemistry for detecting monomethylation at the protein, cellular, and tissue levels. Furthermore, since FTH1 is assembled into cage-like structures within cells, some of its antibody epitopes are not fully exposed on the surface. Furthermore, the methylation modification itself has a low molecular weight. Therefore, selecting appropriate antigenic synthetic peptides and developing specific antibodies that can be used in immunofluorescence, flow cytometry, and other techniques to effectively distinguish methylated and unmethylated K88 FTH1 molecules is challenging. Summary of the Invention

[0005] The purpose of the present invention is to overcome the technical difficulties in preparing site-specific modified antibodies and provide an antibody that specifically recognizes K88 methylated FTH1 protein, a preparation method and application.

[0006] The technical solution adopted by the present invention includes:

[0007] A synthetic antigen peptide methylated at K88 site of human FTH1 protein, the amino acid sequence of the synthetic antigen peptide is shown in SEQ ID NO: 1, and the lysine at position 88 of the FTH1 protein in the peptide segment is monomethylated.

[0008] The invention relates to the use of the synthetic peptide of the human FTH1 protein K88 site methylation antigen in the preparation of a reagent and / or a kit for detecting FTH1 protein K88 methylation.

[0009] An antibody that specifically recognizes the monomethylated FTH1 protein at K88 is prepared by immunizing animals with the synthetic peptide of the methylated antigen at K88 of the human FTH1 protein of the present invention.

[0010] The invention provides an application of the antibody that specifically recognizes the monomethylated FTH1 protein at K88 in the preparation of a reagent and / or a kit for detecting the methylation of FTH1 protein at K88.

[0011] The invention provides an application of the antibody that specifically recognizes the monomethylated FTH1 protein at position K88 in the preparation of a reagent and / or a kit for detecting cancer.

[0012] The method for preparing the antibody that specifically recognizes the monomethylated FTH1 protein at position K88 of the present invention comprises the following steps:

[0013] 1) coupling the synthetic peptide of the human FTH1 protein K88 methylated antigen of the present invention with a carrier protein to obtain the antigen;

[0014] 2) immunizing animals with the antigen described in step 1) and collecting antiserum;

[0015] 3) The antiserum described in step 2) is subjected to antigen-based affinity purification and counter-selection, and after titer and specificity identification, an antibody that specifically recognizes the monomethylated FTH1 protein at position K88 is obtained.

[0016] Specifically, the step 3) includes:

[0017] Chromatography is performed on the antiserum using the affinity column coupled with the synthetic peptide of the human FTH1 protein K88 methylation antigen according to claim 1, and the eluted components are then subjected to an affinity column of the non-modified antigen peptide to remove non-specific components bound to the non-modified peptide;

[0018] The flow-through antibodies were used to determine whether they specifically recognized K88-methylated FTH1 protein by ELISA and dot blot hybridization.

[0019] Specifically, the sequence of the non-methylated modified antigen synthetic peptide is LQDIKKPDCDDWES.

[0020] Optionally, the carrier protein in step 1) is KLH.

[0021] Specifically, the animal in step 2) is a New Zealand white rabbit.

[0022] Step 2) The specific immunization method is: the first immunization is combined with three booster immunizations; the first immunization uses a complete adjuvant, and the booster immunization uses an incomplete adjuvant, and the volume ratio of adjuvant to antigen is 1:1; the amount of antigen in the booster immunization is half of that in the first immunization; all antigens are injected subcutaneously at multiple points.

[0023] Compared with the prior art, this application has the following beneficial effects:

[0024] ① The synthetic peptide of the FTH1 protein K88 site antigen in the present invention is designed based on clinical mass spectrometry and the three-dimensional structure of the protein to obtain an antigen with maximum immunogenicity and thus prepare antibodies that can recognize endogenous protein modifications in cells and tissues;

[0025] ② The immunization, purification and identification technical processes for identifying modified antigens in the present invention are easy to implement and simple to operate, and can ensure the specificity of the modified antibodies. The above two points are of great reference value for the design of experimental protocols for site-specific protein-modified antibodies. On the other hand, the antibodies prepared by the present invention can efficiently and specifically identify K88-methylated FTH1 proteins at the peptide, protein, cell and tissue levels, providing a more convenient and inexpensive FTH1 methylation research method compared to mass spectrometry detection, which is of great significance for advancing the study of the mechanism by which FTH1 methylation modification regulates the sensitivity of cancer cells to ferroptosis. Based on this antibody, preparations or drugs for the diagnosis, prognosis and treatment of tumors such as lung cancer can also be developed, which has good translational potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following detailed description, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0027] Figure 1 The secondary mass spectrometry spectrum of the identification of monomethylation modification at position K88 of FTH1 in the tissue sample of a lung cancer patient in Example 1 of the present invention;

[0028] Figure 2 The position of the methylated modified antigenic peptide in the three-dimensional structure of the FTH1 molecule in Example 1 of the present invention. The synthetic peptide is displayed as a ball-and-stick model, and the entire peptide is fully exposed on the protein surface;

[0029] Figure 3 Example 3 of the present invention uses ELISA to identify FTH1 K88 methylation antibodies. The antibody binds to the modified antigen peptide and has weak binding to the unmodified peptide segment;

[0030] Figure 4 Example 3 of the present invention uses a dot blot assay to identify FTH1 K88 methylation antibodies. The antibody binds to the modified antigen peptide and does not react with the unmodified peptide.

[0031] Figure 5 The antibody that specifically recognizes methylation at the K88 site of FTH1 was used in immunoblotting experiments according to Example 4 of the present invention. The antibody was able to recognize methylation at the K88 site of FTH1 endogenously expressed in cells;

[0032] Figure 6 Example 4 of the present invention shows the results of an antibody that specifically recognizes methylation at the FTH1 K88 site and is applied to tissue immunofluorescence experiments. The antibody can recognize methylation at the FTH1 K88 site in tissues and can distinguish elevated modification in cancer tissues;

[0033] Figure 7The results of immunohistochemistry experiments using an antibody that specifically recognizes methylation at the K88 site of FTH1 according to Example 4 of the present invention show that the antibody can recognize methylation at the K88 site of FTH1 in tissues and can distinguish elevated modification in cancer tissues. DETAILED DESCRIPTION

[0034] In order to facilitate understanding of the purpose, significance and advantages of the technical solutions of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and examples. In the following description, the experimental methods are conventional methods unless otherwise specified, and the reagents and consumables used can be obtained through commercial channels unless otherwise specified. The present invention can be implemented in many different forms and is not limited to the embodiments described herein.

[0035] The present invention provides a synthetic antigen peptide methylated at the K88 site of human FTH1 protein based on clinical sample mass spectrometry detection and protein three-dimensional structure design. The amino acid sequence of the synthetic antigen peptide is LQDIKK(me1)PDCDDWES, wherein K(me1) indicates that the lysine is monomethylated, as specifically shown in SEQ ID NO: 1.

[0036] The present invention provides an antibody capable of specifically recognizing the monomethylation of the K88 site of human FTH1 protein. The antibody is prepared by immunizing an animal with the methylated antigen synthetic peptide.

[0037] The present invention provides a method for preparing an antibody that specifically recognizes monomethylation at the K88 site of human FTH1 protein, comprising the following steps:

[0038] 1) coupling the methylated antigen synthetic peptide with a carrier protein to obtain an antigen;

[0039] 2) immunizing animals with the antigen from step 1) and collecting antiserum;

[0040] 3) The antiserum described in step 2) is subjected to antigen-based affinity purification and counter-selection, and after titer and specificity identification, an antibody that specifically recognizes the methylation of FTH1 protein K88 is obtained.

[0041] The carrier protein in step 1) is KLH.

[0042] The immunized animals in step 2) are New Zealand white rabbits.

[0043] Step 2) The specific immunization method is: the first immunization is combined with three booster immunizations; the first immunization uses a complete adjuvant, and the booster immunization uses an incomplete adjuvant, and the volume ratio of adjuvant to antigen is 1:1; the amount of antigen in the booster immunization is half of that in the first immunization; all antigens are injected subcutaneously at multiple points in the neck and back, which are relatively loose areas.

[0044] Step 3) The specific method for purifying and identifying the antibody is as follows: affinity chromatography is first performed using a methylated antigen synthetic peptide, and the eluted fraction containing the target antibody is then subjected to an affinity column with an unmodified antigen peptide to remove nonspecific components; the binding of the antibody to the modified peptide and the unmodified peptide is compared by ELISA and dot blot hybridization to determine the antibody's potency and ability to specifically recognize FTH1 protein K88 methylation.

[0045] The sequence of the non-modified antigen peptide in step 3) is LQDIKKPDCDDWES, and no methyl group is added at the 88th lysine site.

[0046] The present invention provides the use of a synthetic peptide of a methylated antigen at the K88 site of an FTH1 protein in the preparation of a preparation for detecting the methylation of the K88 site of the FTH1 protein.

[0047] The present invention also provides use of an antibody that specifically recognizes methylation at K88 site of FTH1 protein in preparing a preparation for detecting methylation at K88 site of FTH1 protein.

[0048] Based on this antibody, preparations or drugs for the diagnosis, prognosis and treatment of tumors such as lung cancer can also be developed, which has good translational potential. For example, antibodies that specifically recognize monomethylated FTH1 protein at position K88 can be used in the preparation of reagents and / or kits for detecting cancer, especially lung cancer.

[0049] Example 1 Design and synthesis of methylated modified antigen peptides

[0050] The present invention uses mass spectrometry to detect clinical samples of lung cancer and screens out the presence of K88 monomethylation modification in FTH1 protein, such as Figure 1 In order to prepare a specific antibody that can be used in immunofluorescence, flow cytometry and other techniques and can effectively distinguish between methylated and unmethylated K88 FTH1 molecules, the region containing the K88 site in the FTH1 three-dimensional structure and fully exposed on the surface of the ferritin molecule was selected as the sequence of the antigen synthetic peptide, as shown in FIG. Figure 2 The amino acid sequence of the methylated antigen peptide used for animal immunization was ultimately determined to be LQDIKK(me)PDCDDWES, where K(me) represents the addition of a monomethylated lysine at position 88 of the FTH1 protein. The corresponding unmodified peptide sequence used for antibody screening is LQDIKKPDCDDWES. Both peptides were synthesized using chemical synthesis and purified by HPLC (high-performance liquid chromatography) to a purity exceeding 95%.

[0051] Example 2 Antibodies that specifically recognize FTH1 protein K88 methylation and their preparation method

[0052] The present application provides an antibody that specifically recognizes FTH1 protein K88 methylation and a preparation method thereof, which specifically comprises the following steps:

[0053] 1. Preparation of antigen for immunization by coupling antigen peptide with KLH: Weigh 4 mg of the antigen peptide synthesized in Example 1, dissolve it in 50 μL of DMSO, add 200 μL of 1×PBS, mix quickly, and then immediately add activated KLH according to the ratio of antigen peptide: KLH = 1 mg: 680 μg. Incubate in a 4°C refrigerator overnight or at room temperature for 2 hours. Place the cross-linked KLH-antigen peptide complex in a dialysis bag and dialyze it in 4 L of 1×PBS at 4°C overnight under magnetic stirring. Remove the dialyzed KLH-antigen peptide into a clean 1.5 mL centrifuge tube, divide it into portions according to the immunization dose, and store in a -20°C refrigerator until use.

[0054] 2. Animal immunization: (1) First immunization: Take 3-4 New Zealand white rabbits that have been raised for 2 weeks, and give 0.1-1.0 mg of antigen (the specific amount depends on the type and weight of the rabbit) for the first immunization, 0.5 ml per rabbit. Complete adjuvant is used for the first immunization, with the adjuvant and antigen in a volume ratio of 1:1. After the antigen and adjuvant are fully emulsified, multiple subcutaneous injections are performed, with 0.2 ml per point. (2) Second immunization: After 14 days, the second immunization is performed, the amount of antigen is halved based on the first immunization, and the antigen is emulsified with Freund's incomplete adjuvant. The injection method is the same as the first immunization. (3) Third immunization: After 7 days, the third immunization is performed, the amount of antigen is halved based on the first immunization, and the antigen is emulsified with Freund's incomplete adjuvant. The injection method is the same as the first immunization. (4) Booster immunization: After 7 days, the booster immunization is performed, the amount of antigen is halved based on the first immunization, and the antigen is emulsified with Freund's incomplete adjuvant. The injection method is the same as the first immunization. (5) Blood collection: After 7 days, whole blood was collected and the serum obtained by standing was added with sodium thimerosal solution with a final concentration of 0.02% for antibacterial preservation.

[0055] 3. Antibody purification: (1) Preparation of modified antigen peptide and non-modified peptide affinity column: The modified antigen peptide containing FTH1 K88 methylation and the corresponding non-modified peptide in Example 1 were coupled to agarose filler (CNBr-activated Sepharose 4FF, Shanghai Biotechnology) according to the standard process of commercial filler to prepare a specific peptide affinity chromatography column. (2) Purification of antibodies that specifically recognize FTH1 protein K88 methylation: The rabbit serum obtained after immunization was first applied to the modified antigen peptide affinity column equilibrated with PBS buffer, washed with PBS buffer for about 5 column volumes, and then eluted with glycine solution (pH 2.7, 0.2M). The eluted fraction was collected and immediately neutralized to pH 7 with 1M sodium bicarbonate. The eluted antibody was further passed through the non-modified peptide affinity column, and the column was repeated once to fully remove the antibody that can bind to the non-methylated modified peptide. After collecting the flow-through fraction, the antibody that specifically recognizes FTH1 K88 methylation was obtained. The antibody was dialyzed into 0.01 M PBS (pH 7.4) buffer and concentrated using an ultrafiltration tube. After the concentration was measured, it was quickly frozen in liquid nitrogen for storage.

[0056] Example 3 Identification of Antibodies Specific for K88 Methylated FTH1 Protein

[0057] Identification of FTH1 K88 methylation antibodies using ELISA

[0058] 1. The FTH1 K88 methylated antibody obtained after purification in Example 2 was tested for potency and specificity. The methylated modified antigen peptide determined in Example 1 and the non-modified antigen peptide as a control were cross-linked with BSA and coated into the reaction wells of the polystyrene plate. After washing once with PBST, they were blocked with 1% BSA. The purified antibody to be tested in Example 2 was added with gradient dilutions and incubated for 1 hour and then washed three times with PBST. HRP-labeled anti-rabbit secondary antibody was added and incubated. After washing three times with PBST, TMB substrate was added for color development and the plate was read. The results of ELISA titer detection of methylated antibodies are shown in the figure. Figure 3 As shown, the antibody to be tested can well distinguish methylated and non-methylated peptides in the range of 1:256000 to 1:1000, demonstrating its high titer and high specificity.

[0059] 2. Identification of FTH1 K88 methylation antibodies using dot blot assay

[0060] 100 ng of the methylated modified antigen peptide determined in Example 1 and the non-modified antigen peptide solution as a control were spotted on the NC membrane, air-dried, and blocked with 5% skim milk. The purified antibody to be tested in Example 2 was added to the NC membrane after gradient dilution and incubated overnight. The NC membrane was washed 3 times with TBST, and the anti-rabbit secondary antibody was added to the NC membrane and incubated. After washing the NC membrane with TBST, chemiluminescence was performed. The results are shown in Figure 2. Figure 4 As shown, the tested antibody specifically recognized the FTH1K88 methylated peptide in the range of 1:200 to 1:51200, but had no reaction with the non-modified peptide, demonstrating the high specificity of the antibody.

[0061] Example 4 Application of antibodies that specifically recognize K88-methylated FTH1 protein

[0062] 1. Antibodies that specifically recognize FTH1 K88 methylation can be used in Western blot experiments

[0063] The human FTH1 wild-type eukaryotic expression plasmid (Flag-FTH1WT, K88 site can be methylated) and K88 methylation site mutant plasmid (Flag-FTH1 K88R, K88 site cannot be methylated) synthesized by Beijing Qingke Biotechnology were transiently transfected into FTH1 knockout H460 lung cancer cells according to conventional methods. After 48 hours, the cells were collected and lysed, and the lysate protein was subjected to SDS-PAGE electrophoresis. After transfer to a membrane, the Flag antibody (1:2000, Wuhan Tri-Tac), β-actin antibody (1:2000, Wuhan Tri-Tac) and the anti-FTH1K88 methylation antibody (1:3000 dilution) prepared in Example 2 were used for immunoblotting detection. The results are shown in Figure 2. Figure 5 As shown, the antibody of the present invention can specifically recognize the methylation modification of the FTH1 K88 site expressed in cells, but has no reaction with the FTH1 K88R mutant that cannot undergo methylation modification.

[0064] 2. Antibodies that specifically recognize FTH1 K88 methylation can be used in tissue immunofluorescence experiments

[0065] The frozen sections of lung adenocarcinoma and adjacent tissues were fixed with 4% paraformaldehyde (PFA) at room temperature for 30 minutes and rinsed three times with PBS after fixation. The sections were permeabilized by incubating with 0.2% Triton X-100 in PBS solution at room temperature for 10 minutes. After permeabilization, rinsed three times with PBS. The samples were incubated with blocking solution (10% FBS in PBS solution) for 30 minutes to reduce nonspecific binding. The FTH1 K88 site methylation antibody prepared in Example 2 was diluted 1:500 with blocking solution, added to the tissue sections, and incubated overnight at 4°C. After incubation, rinsed three times with PBS and anti-rabbit green fluorescent secondary antibody (dilution ratio 1:500) was added to the sections and incubated in the dark at room temperature for 1 hour. Rinse three times with PBS in the dark. The cell nuclei were stained with DAPI dye and incubated at room temperature for 10 minutes. Rinse with PBS after staining. The coverslip was sealed on the slide with a sealing agent and imaged using a fluorescence microscope. The results are shown in Figure 2. Figure 6The antibody of the present invention can specifically recognize the methylation modification of the FTH1K88 site expressed in the background cells of the tissue sample, and proves that the methylation level of FTH1 K88 is increased in cancer tissues.

[0066] 3. Antibodies that specifically recognize FTH1 K88 methylation can be used in immunohistochemistry experiments

[0067] Lung adenocarcinoma tissue and adjacent tissue sections were baked at 60°C for 12 hours to remove paraffin. After dewaxing and antigen retrieval according to the conventional experimental procedures, about 1.5 mL of endogenous peroxidase blocking solution (Biyuntian) was dripped onto the tissue for 10 minutes. The sections were placed in a dye vat with PBS buffer and rinsed under tap water for 5 minutes to remove the blocking solution. The sections were placed in a dye vat with PBS buffer and soaked for 5 minutes, then placed in a humidified chamber and blocked with 10% goat serum at room temperature for 40 minutes. The goat serum was discarded, and 1.5 mL of the FTH1 K88 site methylation antibody (1:200 dilution) prepared in Example 2 was added dropwise, and incubated overnight at 4°C. After being rinsed once in a dye vat with PBS buffer, the sections were poured into PBS buffer and washed for 5 minutes, and repeated 3 times. The sections were placed in a humidified chamber, and HRP-labeled anti-rabbit secondary antibody was added dropwise, and incubated at room temperature for 20 minutes. The sections were washed for 5 minutes with PBS buffer, and repeated 3 times. Place the slices on filter paper, add freshly prepared DAB color developing solution, and quickly place them under a microscope in a dark environment for observation. When the protein expression site turns brownish yellow, rinse the slices with tap water to stop coloring. Add hematoxylin staining solution to the slices, rinse lightly with tap water after 7 minutes at room temperature. Place the slices in 1% HCL-75% ethanol for differentiation for 10 seconds, quickly place them in a tap water dyeing tank, and rinse with tap water for 5 minutes. Replace the tap water in the dyeing tank with PBS buffer for anti-blueing for 5 minutes, and rinse with tap water for 2 minutes. Dehydrate and make transparent: 75% ethanol for 2 minutes, 85% ethanol for 2 minutes, 90% ethanol for 2 minutes, 95% ethanol for 2 minutes, 95% ethanol for 5 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, and xylene for 30 minutes. Add an appropriate amount of neutral gum to the center of the slice, press it with a coverslip until no bubbles are generated, and then observe it under a microscope. The results are as follows Figure 7 The antibody of the present invention can detect FTH1 K88 methylation modification in cancer and adjacent tissues, and proves that the level of FTH1-K88 monomethylation modification in tumor tissue is higher than that in adjacent tissues.

[0068] In summary, the FTH1 K88 methylated antigen peptide of the present invention has a short sequence, is easy to synthesize, and has strong immunogenicity. The method for preparing antibodies using this antigen is simple and low-cost. The antibodies prepared using this antigen have high titer and specificity. Using immunoblotting, immunofluorescence, and immunohistochemistry, they can effectively distinguish whether the FTH1 K88 site in cells and tissues is methylated. This has important significance in the functional and clinical translational research related to FTH1.

[0069] The above are only specific embodiments of the present invention, but the present invention is not limited to the above embodiments. Without departing from the design concept and principle of the present invention, various modifications, improvements and changes of the present invention should be included in the scope of protection of the present invention.

Claims

1. A synthetic peptide of a methylated antigen at K88 site of human FTH1 protein, characterized in that: The amino acid sequence of the antigen synthetic peptide is shown in SEQ ID NO: 1, and the lysine at position 88 of the FTH1 protein in the peptide segment is modified by monomethylation.

2. Use of the synthetic peptide of the human FTH1 protein K88 methylation antigen according to claim 1 in the preparation of a reagent and / or a kit for detecting FTH1 protein K88 methylation.

3. An antibody that specifically recognizes K88 monomethylated FTH1 protein, characterized in that: The method is prepared by immunizing animals with the synthetic peptide of the human FTH1 protein K88 site methylation antigen according to claim 1.

4. Use of the antibody that specifically recognizes FTH1 protein monomethylated at position K88 according to claim 3 in the preparation of a reagent and / or kit for detecting FTH1 protein K88 methylation.

5. Use of the antibody that specifically recognizes the monomethylated FTH1 protein at position K88 according to claim 3 in the preparation of a reagent and / or kit for detecting cancer.

6. The method for preparing the antibody that specifically recognizes the monomethylated FTH1 protein at position K88 according to claim 3, characterized in that: The following steps are involved: 1) coupling the synthetic peptide of the human FTH1 protein K88 methylated antigen according to claim 1 with a carrier protein to obtain the antigen; 2) immunizing animals with the antigen described in step 1) and collecting antiserum; 3) The antiserum described in step 2) is subjected to antigen-based affinity purification and counter-selection, and after titer and specificity identification, an antibody that specifically recognizes the monomethylated FTH1 protein at position K88 is obtained.

7. The method for preparing an antibody that specifically recognizes FTH1 protein monomethylated at position K88 according to claim 6, characterized in that: The step 3) specifically includes: Chromatography is performed on the antiserum using the affinity column coupled with the synthetic peptide of the human FTH1 protein K88 methylation antigen according to claim 1, and the eluted components are then subjected to an affinity column of the non-modified antigen peptide to remove non-specific components bound to the non-modified peptide; The flow-through antibodies were used to determine whether they specifically recognized K88-methylated FTH1 protein by ELISA and dot blot hybridization.

8. The method for preparing an antibody that specifically recognizes FTH1 protein monomethylated at position K88 according to claim 7, characterized in that: The sequence of the non-methylated modified antigen synthetic peptide is LQDIKKPDCDDWES.

9. The method for preparing an antibody that specifically recognizes FTH1 protein monomethylated at position K88 according to claim 6, characterized in that: The carrier protein in step 1) is KLH.

10. The method for preparing an antibody that specifically recognizes FTH1 protein monomethylated at position K88 according to claim 6, characterized in that: The animal in step 2) is a New Zealand white rabbit; Step 2) The specific immunization method is: the first immunization is combined with three booster immunizations; the first immunization uses a complete adjuvant, and the booster immunization uses an incomplete adjuvant, and the volume ratio of adjuvant to antigen is 1:1; the amount of antigen in the booster immunization is half of that in the first immunization; all antigens are injected subcutaneously at multiple points.

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