Monoclonal antibodies against GAPDH carboxyethylation modification and uses thereof

Abstract

The application belongs to the technical field of molecular immunology, and particularly relates to a kind of anti-GAPDH carboxyethylation modified monoclonal antibody and its application, the amino acid sequence of HCDR1~HCDR3 of the monoclonal antibody is sequentially shown as SEQ ID NO.4~SEQ ID NO.6;The amino acid sequence of LCDR1~LCDR3 is sequentially shown as SEQ ID NO.7~SEQ ID NO.9.The application successfully prepared the monoclonal antibody of specific recognition GAPDH Cys247 carboxyethylation modification, the antibody has high affinity and high specificity, can effectively distinguish the carboxyethylation similar to lactoylation modification.The antibody can be applied to immunoblotting, immunofluorescence, immunoprecipitation and other various detection scenes, and can be used for preparing inflammation diagnostic kit and screening anti-inflammatory drugs, has the dual value of basic research and clinical application.

Description

Technical Field

[0001] This invention belongs to the field of molecular immunology technology, specifically relating to a monoclonal antibody against GAPDH carboxyethylated modification and its application. Background Technology

[0002] Macrophages are core cells of the innate immune system, playing a crucial regulatory role in inflammatory responses. Studies have shown that the immune function of macrophages is closely related to their metabolic state. Pro-inflammatory macrophages (such as those stimulated by lipopolysaccharide (LPS)) primarily rely on glycolysis to meet their energy needs for rapid proliferation and synthesis of inflammatory factors; while anti-inflammatory macrophages tend to utilize oxidative phosphorylation or fatty acid oxidation as their main energy source. Therefore, regulating the metabolic shift of macrophages from pro-inflammatory glycolysis to anti-inflammatory mitochondrial oxidation has become a potential strategy for treating inflammatory diseases such as sepsis, pneumonia, ankylosing spondylitis, and rheumatoid arthritis.

[0003] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key rate-limiting enzyme in the glycolysis pathway, catalyzing the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate and simultaneously reducing NAD⁺ to NADH. In activated macrophages, GAPDH expression is significantly upregulated, and glycolytic activity is enhanced. Therefore, GAPDH is considered a potential target for regulating macrophage inflammatory responses.

[0004] Endogenous metabolites can regulate protein function and thus affect immune responses by covalently modifying cysteine ​​residues in proteins. In recent years, various post-translational modifications of GAPDH have been reported: dimethyl fumarate can alkylate GAPDH cysteine, inhibiting glycolysis and exerting anti-inflammatory effects. 4-Octylitacin can alkylate GAPDH cysteine, reducing its enzymatic activity. Malonylation can promote the dissociation of GAPDH from TNF-α mRNA, thus promoting inflammation. However, all of the above modifications involve exogenous compounds or known modification types, and none involve carboxyethylation, a novel post-translational modification.

[0005] 3-Hydroxypropionic acid (3-HPA) is a gut microbiota-derived metabolite. Previous studies have shown that it can induce cysteine ​​carboxyethylation, generating neoantigens and participating in the development of HLA-restricted autoimmune diseases such as ankylosing spondylitis. However, whether 3-HPA regulates macrophage function through carboxyethylation modification and its role in inflammatory diseases have not been previously reported.

[0006] Although it is known that GAPDH can undergo various post-translational modifications, existing technologies have the following significant drawbacks: First, there is a lack of site-specific antibodies against GAPDH carboxyethylation modification. While antibodies targeting the C96 site carboxyethylation modification of the ITGA2B protein exist, they target entirely different protein sites, and the immunogen designs are based on the ITGA2B sequence, failing to recognize the GAPDH protein. Previously, there was no information regarding whether GAPDH undergoes carboxyethylation modification, at which site it occurs, or the impact of this modification on GAPDH function and macrophage metabolic regulation.

[0007] Second, it is impossible to distinguish between carboxyethylation and lactacylation modifications, which have highly similar structures. Carboxyethylation and lactacylation have the same mass shift (+72.021 Da), making it difficult to differentiate between these two modifications using conventional mass spectrometry. Existing antibody technologies have not fully validated cross-reactivity with lactacylation, leading to uncertainty in detection specificity and severely limiting the accurate detection and functional studies of GAPDH carboxyethylation modifications.

[0008] Therefore, developing a high-affinity monoclonal antibody that can specifically recognize GAPDH Cys247 carboxyethylated modification and using it to study macrophage metabolic regulation and inflammatory response has important basic research value and clinical translation prospects. Summary of the Invention

[0009] The purpose of this invention is to provide a monoclonal antibody against GAPDH carboxyethylation modification, which solves the problems existing in the prior art.

[0010] The technical solution adopted in this invention is: This invention provides a monoclonal antibody against GAPDH carboxyethylation modification, wherein the monoclonal antibody comprises HCDR1~HCDR3 and LCDR1~LCDR3; The amino acid sequences of HCDR1 to HCDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively. The amino acid sequences of LCDR1 to LCDR3 are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively.

[0011] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2; The amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown in SEQ ID NO.3.

[0012] Preferably, the monoclonal antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0013] A second aspect of the present invention provides a nucleic acid molecule that encodes the monoclonal antibody.

[0014] A third aspect of the present invention provides an application of the monoclonal antibody and / or the nucleic acid molecule described herein, wherein the application refers to any one of the following: 1) Preparation of detection reagents for GAPDH Cys247 carboxylation; 2) Prepare diagnostic reagents for inflammatory diseases.

[0015] Preferably, the inflammatory disease includes any one of sepsis, pneumonia, ankylosing spondylitis, and rheumatoid arthritis.

[0016] Preferably, the detection reagent further includes an enzyme-labeled secondary antibody or a fluorescently labeled secondary antibody.

[0017] Preferably, the enzyme-labeled secondary antibody includes a horseradish peroxidase-labeled secondary antibody or an alkaline phosphatase-labeled secondary antibody; Fluorescently labeled secondary antibodies include FITC-labeled secondary antibodies, TRITC-labeled secondary antibodies, or Cy3-labeled secondary antibodies.

[0018] Preferably, the detection reagent is suitable for immunoblotting, immunofluorescence and / or immunoprecipitation detection.

[0019] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a monoclonal antibody against GAPDH carboxyethylation modification, comprising HCDR1-HCDR3 and LCDR1-LCDR3; the amino acid sequences of HCDR1-HCDR3 are shown in SEQ ID NO.4-SEQ ID NO.6; and the amino acid sequences of LCDR1-LCDR3 are shown in SEQ ID NO.7-SEQ ID NO.9. This invention successfully prepared a monoclonal antibody (GAPDH-ce247) that specifically recognizes GAPDH Cys247 carboxyethylation modification. This antibody exhibits high affinity and high specificity, effectively distinguishing between structurally similar carboxyethylation and lactylation modifications, filling the technological gap of lacking site-specific detection tools for this modification. Using this antibody, this invention reveals for the first time that 3-HPA-induced GAPDH Cys247 carboxyethylation promotes GAPDH degradation through the K48 ubiquitin-proteasome pathway, achieving dual inhibition of GAPDH function through both protein abundance and enzyme activity, and shifting macrophage metabolism from glycolysis to mitochondrial oxidation, elucidating the core role of this modification in anti-inflammatory regulation. This antibody can be applied to various detection scenarios such as immunoblotting, immunofluorescence, and immunoprecipitation, and can also be used to prepare inflammation diagnostic kits and screen anti-inflammatory drugs, thus having dual value in basic research and clinical applications. Attached Figure Description

[0020] Figure 1For the specific detection of GAPDH-ce247 antibody; A: The affinity of GAPDH-ce247 for unmodified GAPDH peptides was analyzed by SPR. B: The affinity of GAPDH-ce247 for carboxyethylated GAPDH peptides was analyzed by SPR. C: ELISA binding curves of GAPDH-ce247 with modified GAPDH peptides and unmodified GAPDH peptides. Data are expressed as mean ± SD. Each group has n=3. Two-way ANOVA was used to determine statistical significance. **P<0.01. D: Chemical structure of cysteine ​​carboxyethylated modification; E: Chemical structure of cysteine ​​lactylated modification; F: Unmodified GAPDH peptides, carboxyethylated peptides, and lactylated peptides were detected by dot blot assay using GAPDH-ce247 carboxyethylated antibody.

[0021] Figure 2 For the application of GAPDH-ce247 carboxyethylated antibody; A: Detection of carboxyethylated molecules of GAPDH-ce247 in 293T cells by Western blotting; B: Proteins interacting with GAPDH-ce247 carboxyethylated molecules in 293T cells were detected by protein immunoprecipitation. C: Localization of GAPDH-ce247 carboxyethylated molecules in THP-1 cells was detected by immunofluorescence. D: Detection of GAPDH-ce247 carboxyethylated molecules in mouse peritoneal macrophages by Western blotting. Detailed Implementation

[0022] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0023] The inventive concept of this invention is as follows: In the prior art, the closest technical solutions to this invention mainly fall into two categories: The first category is antibodies targeting the carboxyethylation modification of the ITGA2BC96 site. In this study, an antibody capable of specifically recognizing the carboxyethylation modification of cysteine ​​at position 96 of integrin αIIb (ITGA2B) was prepared. This antibody uses a long peptide containing the carboxyethylated C96 site as an immunogen. Through rigorous screening, site-specific antibodies were obtained and successfully used to detect the carboxyethylation level of ITGA2B in the peripheral blood of patients with ankylosing spondylitis, revealing the role of this modification in autoimmune diseases. However, this antibody targets the ITGA2B protein and cannot be used to detect the carboxyethylation status of GAPDH, and cross-reactivity with structurally similar modifications (such as lactylation) has not been verified. The second category is GAPDH site-specific post-translational modification antibodies, such as antibodies targeting the alkylation modification of GAPDH Cys150. However, there are currently no site-specific antibodies targeting the carboxyethylation modification of GAPDH Cys247.

[0024] The aforementioned shortcomings arise because: while ITGA2B C96 carboxyethylation antibodies are site-specific antibodies, they target entirely different protein sites. Immunogens designed based on the ITGA2B sequence cannot recognize GAPDH proteins. Furthermore, the development of site-specific antibodies targeting GAPDH Cys247 carboxyethylation modification faces unique technical challenges. Specifically, it requires designing long peptide immunogens containing Cys247 and its flanking sequences, and establishing a screening strategy capable of specifically selecting carboxyethylation-positive clones from structurally similar lactylation modifications. These technical challenges have prevented the development of antibody tools that specifically recognize GAPDH Cys247 carboxyethylation without cross-reacting with lactylation. Therefore, while existing technologies include site-specific antibodies against ITGA2B carboxyethylation, the lack of antibodies targeting the GAPDH Cys247 site severely restricts the development of research on the function of GAPDH carboxyethylation in macrophage metabolic regulation and inflammatory responses.

[0025] Based on this, the technical problem that this invention aims to solve is: 1. To provide an antibody capable of specifically recognizing carboxyethylation modification at the Cys247 site of GAPDH. While existing technologies have developed antibodies targeting the carboxyethylation site of ITGA2B C96, these antibodies target entirely different protein sites and cannot be used to detect carboxyethylation of GAPDH. The primary technical problem of this invention is to provide a monoclonal antibody that specifically recognizes carboxyethylation modification of cysteine ​​at position 247 of the GAPDH protein, filling the technological gap of lacking site-specific detection tools for this modification.

[0026] 2. This invention provides an antibody capable of distinguishing between GAPDH carboxyethylation and lactacylation, two structurally similar modifications. Because carboxyethylation and lactacylation have the same mass shift (+72.021 Da), conventional mass spectrometry analysis struggles to differentiate between these two modifications. Existing antibody technologies have not adequately validated cross-reactivity with lactacylation, leading to uncertainty in detection specificity. Therefore, the technical problem this invention aims to solve is to provide an antibody that specifically recognizes carboxyethylation modifications without cross-reacting with structurally similar lactacylation modifications, ensuring the accuracy and reliability of detection results.

[0027] 3. To elucidate the biological functions of GAPDH Cys247 carboxyethylation modification in macrophage metabolic regulation and inflammatory responses. Due to the lack of specific detection tools, the occurrence of GAPDH Cys247 carboxyethylation modification in macrophages, its effects on GAPDH protein stability and enzyme activity, and its regulatory role in macrophage metabolic reprogramming and inflammatory responses have not been elucidated in existing technologies. Therefore, the technical problem to be solved by this invention is to clarify how 3-HPA-induced GAPDH Cys247 carboxyethylation modification regulates macrophages and to verify its potential application in anti-inflammatory therapy.

[0028] In summary, the core technical problem to be solved by this invention is to prepare an antibody that specifically recognizes GAPDH carboxyethylated modification and does not cross-react with lactylation, and to reveal the functional mechanism of GAPDH carboxyethylated modification in macrophage metabolic reprogramming and inflammatory regulation based on this antibody, thereby providing the application of this antibody in diagnosis and treatment.

[0029] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0030] Example 1: A monoclonal antibody against GAPDH carboxyethylated modification, the preparation method of which is as follows: 1. Design of carboxyethylated modified antigen peptides.

[0031] Based on the carboxyethylation modification site information of GAPDH protein obtained by mass spectrometry identification, peptides were designed and synthesized according to the flanking sequence of cysteine ​​residue 247 (Cys247) in the GAPDH protein. GAPDH modified antigen peptide design: A 15-amino acid immunogenic peptide, GAPDH ceC247, was designed: Val-Val-Asp-Leu-Thr-Cys(carboxyethyl)-Arg-Leu-Glu-Lys-Pro-Ala-Lys-Thr-Cys. A corresponding unmodified control peptide, GAPDH C247, was also designed for specificity screening. BSA and KLH were conjugated to the above two peptides to construct GAPDH C247-BSA, GAPDH ceC247-BSA, and GAPDHceC247-KLH, respectively. The carboxyethylated modified peptide was conjugated to BSA as the detection antigen to avoid cross-reactivity with the carrier.

[0032] The sequence of the immunogenic peptide is shown in SEQ ID NO.1.

[0033] SEQ ID NO.1: VVDLTCceRLEKPAKYC, where ce represents carboxyethylation modification.

[0034] 2. Immunize mice with modified peptides.

[0035] Five 8-week-old female BALB / c mice were used, and GAPDH ceC247-KLH protein was used as the immunogen. For the first immunization, the immunogen was thoroughly emulsified with an equal volume of Freund's complete adjuvant and administered via subcutaneous injection at multiple sites on the back of each mouse, with a dose of 50 μg per mouse. A second immunization was administered on day 14 after the first immunization, with the immunogen emulsified with an equal volume of Freund's incomplete adjuvant and administered intraperitoneally, with a dose of 50 μg per mouse. A third immunization was administered on day 28, using the same method as the second immunization. A booster immunization was administered on day 42, without adjuvant, via intraperitoneal injection, with a dose of 100 μg per mouse.

[0036] 3. Detection of immune serum titer.

[0037] Seven days after the third immunization, blood was collected from the tail vein of mice, and serum was separated. The binding titers of serum to modified GAPDH ceC247 antigen and unmodified GAPDH C247 antigen were detected using an indirect ELISA method. Mice with a titer greater than 1:12800 for the modified antigen and a titer less than 1:800 for the unmodified antigen were selected as cell fusion donors, and spleen cell fusion was performed on day 3 after the booster immunization.

[0038] 4. Cell fusion and hybridoma screening.

[0039] Mouse spleens were aseptically harvested, and spleen cell suspensions were prepared. These suspensions were then mixed with Sp2 / 0 myeloma cells in logarithmic growth phase at a ratio of 5:1 and fused using polyethylene glycol 1500. The fused cell suspensions were seeded into 96-well culture plates and cultured in HAT selective medium for selection. On day 10 post-fusion, the cell culture supernatant from each well was collected, and the binding activity against modified GAPDH ceC247 and unmodified GAPDH was simultaneously detected using an indirect ELISA method. Hybridoma wells showing strong positive reactions to the modified antigen (OD450 value > 1.5) and negative reactions to the unmodified antigen (OD450 value < 0.2) were selected. Subcloning was performed using limiting dilution, repeated three times, until a 100% positive monoclonal hybridoma cell line with stable antibody secretion was obtained.

[0040] 5. Preservation of hybridoma cell lines.

[0041] The hybridoma cell lines that stably secrete anti-GAPDH carboxyethylated monoclonal antibodies were expanded and cryopreserved for seed preservation.

[0042] 6. Preparation and purification of monoclonal antibodies.

[0043] Hybridoma cell lines were collected and purified using a Protein G affinity chromatography column. The antibody was eluted with 0.1 M glycine-hydrochloric acid buffer (pH 2.7), and the elution peak was collected and immediately neutralized with 1 M Tris-HCl (pH 8.5). The replacement buffer was then phosphate buffer (pH 7.4). SDS-PAGE analysis confirmed that the purity of the obtained monoclonal antibody was greater than 95%, with a concentration of 1 mg / mL, thus yielding high-purity anti-GAPDH carboxyethylated modified monoclonal antibody Anti-ce247, also known as GAPDH-ce247.

[0044] The GAPDH-ce247 monoclonal antibody was sequenced to determine its antibody sequence information, as shown in Tables 1 and 2 below.

[0045] Table 1. Amino acid sequence information of GAPDH-ce247 monoclonal antibody Table 2 Nucleotide sequence information of GAPDH-ce247 monoclonal antibody Example 2: Application of the monoclonal antibody against GAPDH carboxyethylation modification, as detailed below: 1. Experimental methods.

[0046] 1.1 SPR affinity verification of the specificity of GAPDH-ce247.

[0047] The binding affinity of anti-GAPDH-ceC247 carboxyethylated monoclonal antibody to different antigens was determined using surface plasmon resonance (SPR) technology. The experiments were performed on a Biacore T200 biomolecular interaction analyzer. First, the anti-GAPDH-ceC247 carboxyethylated monoclonal antibody was directly immobilized on the surface of a CM5 sensor chip using an amino-coupling method. The specific procedure was as follows: the antibody was diluted to 20 μg / mL with 10 mM sodium acetate buffer (pH 5.0) and injected at a flow rate of 10 μL / min. After activating the chip surface with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), coupling was performed for 420 s, with the final antibody immobilization amount controlled at 5000 RU. After coupling, unreacted activation sites were blocked with 1 M ethanolamine hydrochloride (pH 8.5). The following two peptides were used as analytes: (1) recombinant GAPDH peptide modified with carboxyethylation of Cys247 (GAPDH ceC247); (2) unmodified GAPDH peptide. The above analytes were serially diluted with HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4). The analytes were sequentially flowed through the chip channels immobilized with antibodies at a flow rate of 30 μL / min, with a binding time of 180 s and a dissociation time of 600 s. After each injection, the chip surface was regenerated with 10 mM glycine-hydrochloric acid buffer (pH 2.0) at a flow rate of 30 μL / min for 30 s. The experimental data were fitted using Biacore T200 Evaluation Software with a 1:1 Langmuir binding model to calculate the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD).

[0048] 1.2 ELISA was used to verify the specificity of the GAPDH ceC247 carboxyethylated antibody.

[0049] The specific recognition ability of the anti-GAPDH ceC247 carboxyethylated monoclonal antibody for the modified peptide was verified by ELISA. Two peptides were coated onto 96-well microplates as follows: The two peptides were diluted to 2 μg / mL with phosphate-buffered saline (PBS, pH 7.4), and 100 μL was added to each well. Coating was carried out overnight at 4°C. The coating solution was discarded the next day, and the plates were washed three times with phosphate-buffered saline (PBST) containing 0.05% Tween-20 for 3 min each time. 200 μL of blocking buffer containing 5% bovine serum albumin was added to each well, and blocking was carried out at 37°C for 2 h. After blocking, the blocking buffer was discarded, and the plates were washed three times with PBST. The anti-GAPDH ceC247 carboxyethylated monoclonal antibody prepared in Example 1 was serially diluted with PBST containing 1% bovine serum albumin, and 100 μL was added to each well. Incubation was carried out at 37°C for 1 h. Negative control wells (without primary antibody, only diluent) and blank control wells were also included. After incubation, wash three times with PBST, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (diluted 1:5000 with PBST containing 1% bovine serum albumin) to each well, and incubate at 37°C for 1 h. After washing, add 100 μL of TMB chromogenic solution to each well, incubate in the dark for 15 min, and observe the color change. After the color development is terminated, add 50 μL of 2M sulfuric acid to each well to stop the reaction, and measure the absorbance (OD450) of each well at 450 nm using a microplate reader. Plot the binding curve with the antibody dilution ratio on the x-axis and the OD450 value on the y-axis.

[0050] 1.3. Verification of the specificity of GAPDH ceC247 carboxyethylated antibody by protein dot blot.

[0051] The binding specificity of the anti-GAPDH ceC247 carboxyethylated monoclonal antibody to different antigens was evaluated using Western blotting. The following samples were prepared: (1) GAPDH Cys247 carboxyethylated modified peptide; (2) GAPDH Cys247 lactated modified peptide (GAPDH-lac); and (3) unmodified GAPDH peptide. Each sample was diluted to the same concentration with phosphate buffer, and 2 μL was sequentially spotted onto a nitrocellulose membrane and dried at room temperature for 30 min. The membrane was then placed in TBST blocking buffer containing 5% skim milk powder and blocked at room temperature for 1 h. Subsequently, the anti-GAPDH ceC247 carboxyethylated monoclonal antibody prepared in Example 1 was used as the primary antibody (dilution ratio 1:1000), and incubated at 37°C for 1 h. The membrane was washed three times with TBST for 10 min each time, and HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) was added, followed by incubation at room temperature for 1 h. After washing again, ECL chemiluminescence was used for development, and images were acquired using a chemiluminescence imaging system.

[0052] 1.4. Specific detection of intracellular GAPDH ceC247 modification level by Western blotting.

[0053] Collect the treated cells, add RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, and lyse thoroughly on ice for 30 min, intermittently vortexing to ensure complete lysis; centrifuge at 12000g for 15 min at 4℃, collect the supernatant, and quantify protein using the BCA method; mix an equal volume of total protein sample with 5×SDS loading buffer, and boil in a metal bath for 10 min to fully denature the protein; after separating the protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transfer the protein bands to a PVDF membrane using a wet transfer method; after transfer, block with TBST buffer containing 5% skim milk powder at room temperature for 1 h to block non-specific binding sites; discard the blocking buffer after blocking, and add an appropriately diluted primary antibody (anti-GAPDH). The membrane was incubated overnight at 4°C with ceC247 antibody and anti-GAPDH antibody. The next day, the membrane was washed three times with TBST buffer for 10 min each time, followed by the addition of the corresponding horseradish peroxidase (HRP)-labeled secondary antibody and incubated at room temperature for 1 h. The membrane was then washed four times with TBST for 10 min each time to remove unbound secondary antibody. Finally, ECL chemiluminescence reagent was added, and the band signal was acquired using a chemiluminescence imaging system to analyze the modification level of GAPDH ceC247 and the expression level of GAPDH in the cells.

[0054] 1.5 Immunofluorescence detection of intracellular GAPDH ceC247 molecular localization.

[0055] Cells in the logarithmic growth phase were seeded into confocal culture dishes and cultured routinely until 80% confluence was achieved. The culture medium was discarded, and the cells were gently washed three times for 3 minutes each time with pre-cooled phosphate-buffered saline (PBS, pH 7.4). Then, 4% paraformaldehyde fixative was added, and the cells were fixed at room temperature in the dark for 15 minutes. The cells were washed three times with PBS to remove any residual fixative. PBS permeation buffer containing 0.2% Triton X-100 was added, and the cells were incubated at room temperature for 10 minutes to increase cell membrane permeability. After thorough washing with PBS, the cells were blocked with PBS containing 5% bovine serum albumin (BSA) at room temperature for 1 hour to reduce non-specific binding. The blocking buffer was discarded, and a mixture of primary antibodies (anti-GAPDH antibody and anti-GAPDH ceC247 carboxyethylated modified antibody) appropriately diluted with the blocking buffer was added separately and incubated overnight at 4°C in a humidified chamber. The next day, the cells were washed with PBST (PBS containing 0.05%...). Cells were washed four times with Tween-20 for 5 min each time to thoroughly remove unbound primary antibodies. Then, the corresponding species-labeled secondary antibody (diluted in the dark) was added and incubated at room temperature in the dark for 1 h. After washing four times with PBST, the nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI) at room temperature in the dark for 5 min. After rinsing with PBS, the cells were mounted with anti-fluorescence quenching mounting medium. Finally, images were acquired using a laser confocal scanning microscope at the corresponding excitation wavelength to observe the subcellular localization and co-localization distribution of GAPDH and GAPDH ceC247 molecules in the cells.

[0056] 1.6 Immunoprecipitation detection of intracellular GAPDH-ceC247 molecular interaction proteins.

[0057] Cells in the logarithmic growth phase were collected, the culture medium was discarded, and the cells were gently washed twice with pre-chilled PBS. IP lysis buffer containing protease inhibitors and phosphatase inhibitors was added, and the cells were lysed on ice for 30 min with intermittent gentle vortexing. The cells were then centrifuged at 14000g for 15 min at 4°C, and the supernatant was used as the total protein sample. Protein concentration was determined using a BCA kit to ensure consistent loading. An equal volume of protein lysis buffer was incubated overnight at 4°C with Protein A / G agarose beads pre-conjugated with GAPDH ceC247 carboxyethylated modified antibody to specifically capture the target protein and its interacting proteins. After incubation, agarose beads were washed with pre-cooled IP lysis buffer to remove non-specifically bound proteins. After cooling on ice, the supernatant was collected by short-term centrifugation. Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membranes by wet transfer, blocked with 5% skim milk powder at room temperature for 1 h, incubated overnight at 4°C with the corresponding primary antibody, and incubated with HRP-labeled secondary antibody at room temperature for 1 h. After thorough washing with TBST, the membranes were developed using an ECL chemiluminescence kit, and signals were acquired using a chemiluminescence imaging system to analyze the endogenous interacting proteins of GAPDH ceC247 in cells.

[0058] 1.7 Assessment of GAPDH carboxyethylation status in vivo and in vitro.

[0059] Peritoneal macrophages were isolated from mice and seeded into cell culture plates. The plates were incubated at 37°C with 5% CO2 for 2 hours. Non-adherent cells were discarded, and the plates were replaced with complete culture medium for another 24 hours. Cells were gently washed three times with pre-cooled phosphate-buffered saline (PBS, pH 7.4) for 3 minutes each time. RIPA lysis buffer containing protease inhibitors, phosphatase inhibitors, and proteasome inhibitors was added, and the cells were lysed thoroughly on ice for 30 minutes, with intermittent vortexing to ensure complete lysis. The cells were centrifuged at 12000g for 15 minutes at 4°C, and the supernatant was collected as the total protein sample. Protein concentration was determined using a BCA protein quantification kit, ensuring consistent loading amounts across groups. An equal volume of total protein was mixed with 5×SDS loading buffer and boiled in a metal bath for 10 minutes to fully denature the proteins. After separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), protein bands were transferred to a PVDF membrane using a wet transfer method. After transfer, the membrane was rinsed with a solution containing 5% PBS. BSA was blocked with TBST buffer at room temperature for 1 h to block non-specific binding sites; the blocking buffer was discarded, and anti-GAPDH ceC247 carboxyethylation modified specific antibody diluted with the blocking buffer was added, and incubated overnight at 4°C; the next day, the membrane was washed 3 times with TBST buffer for 10 min each time, and then horseradish peroxidase (HRP) labeled secondary antibody was added, and incubated on a shaker at room temperature for 1 h; then washed thoroughly with TBST 4 times for 10 min each time to remove unbound secondary antibody; finally, ECL chemiluminescence staining solution was added, and the band signal was acquired using a chemiluminescence imaging system to detect and compare the differences in GAPDH ceC247 carboxyethylation modification levels in peritoneal macrophages of mice in each group.

[0060] 2. Results.

[0061] 2.1 Specificity verification of GAPDH-ce247.

[0062] The present invention verifies the specificity of GAPDH-ce247 antibody through a variety of methods, including: (1) surface plasmon resonance detection of the affinity of GAPDH-ce247 antibody for carboxyethylated peptides; (2) ELISA detection of the binding of GAPDH-ce247 antibody to carboxyethylated modified peptides; and (3) cross-reaction of GAPDH-ce247 antibody with structurally similar lactylated modified peptides.

[0063] 2.1.1 Surface plasmon resonance assay to detect the affinity of GAPDH-ce247 for carboxyethylated peptides.

[0064] Surface plasmon resonance experiments showed that the binding affinity of the GAPDH-ce247 antibody to unmodified GAPDH was weak (KD = 1.535 × 10⁻⁶). -5 M)( Figure 1 The A antibody only produced a significant response at high antigen concentrations; while the binding affinity of the GAPDH-ce247 antibody to carboxyethylated GAPDH was significantly enhanced (KD=2.404×10⁻⁶). -8 M)( Figure 1 (B), demonstrating its high specific binding activity to carboxyethylated modified GAPDH.

[0065] 2.1.2 ELISA detection of the binding of GAPDH-ce247 antibody to carboxyethylated modified peptides.

[0066] Within a dilution gradient range of 1:2000 to 1:256000, the GAPDH-ce247 antibody produced a strong recognition signal only for modified peptides, and no significant signal for unmodified GAPDH peptides. ELISA experiments further confirmed that GAPDH-ce247 specifically recognizes carboxyethylated modified GAPDH peptides, and the recognition activity decreases gradient with increasing antibody dilution, while almost no binding occurs to unmodified GAPDH peptides. The difference between the two groups was statistically significant. Figure 1 (C).

[0067] 2.1.3 Cross-reaction of GAPDH-ce247 antibody with structurally similar lactylated modified peptides.

[0068] Although carboxyethylation and lactation (lac) of cysteine ​​have the same molecular weight, their chemical structures differ. Carboxyethylation involves a cysteine ​​thiol group linked to a carboxyethyl group. Figure 1 The D), lactoylation modification to a cysteine ​​thiol-linked lactyl structure ( Figure 1 The carbon chain length and functional group arrangement of the side chains of the two are different (E).

[0069] Equal amounts of unmodified GAPDH peptides, carboxyethylated GAPDH-ce (234-260), and lactated GAPDH lac were spotted onto the antibody. After incubation with GAPDH-ce247, only the GAPDH-ce group showed a clear hybridization signal; no visible signal was observed in the unmodified GAPDH group or the GAPDH lac group. Dot blot results showed that the GAPDH-ce247 antibody specifically recognizes the carboxyethylated GAPDH ce peptide, and the signal intensity increases gradient with the amount of peptide loaded (10 ng~500 ng). Furthermore, no cross-reactivity was observed with the unmodified GAPDH peptide or the lactated GAPDH lac peptide. Figure 1(F).

[0070] The above experimental results fully demonstrate that the GAPDH-ce247 antibody of the present invention has high affinity, modification site specificity, and high detection sensitivity, and can be stably used for the qualitative and quantitative detection of carboxyethylated modified GAPDH, providing a core tool for basic research, disease diagnosis and targeted drug development of this modified protein.

[0071] 2.2 Application of GAPDH-ce247 antibody.

[0072] 2.2.1 Protein detection and localization in basic research.

[0073] Experiment 1: In immunoblotting, the level of 3-HPA-induced GAPDH carboxyethylation in cell and tissue samples was detected using the GAPDH-ce247 antibody.

[0074] Western blot analysis of the effect of 3-HPA treatment on GAPDH ceC247 modification levels in 293T cells. Cells were pretreated with MG132 and divided into 3-HPA treatment and control groups. Protein samples were analyzed using GAPDH-ceC247 antibody, total GAPDH antibody, and Tubulin antibody. The GAPDH-ceC247 modification band was located at 35 kDa. Results showed that carboxyethylated GAPDH protein bands were specifically recognized at approximately 35 kDa–40 kDa, and that 3-HPA intervention significantly upregulated the expression level of carboxyethylated GAPDH protein in addition to treatment with the proteasome inhibitor MG132. Figure 2 A).

[0075] Experiment 2: In the immunoprecipitation experiment, proteins that interact with carboxyethylated GAPDH were detected, providing support for the study of the molecular mechanism of GAPDH carboxyethylation.

[0076] This experiment was used to detect the interaction between GAPDH-ce247 and Myc-labeled Ub molecules. Both groups of cells were treated with the protease inhibitor MG132; one group was also treated with 3-HPA, while the other group was not. GAPDH-ce247 and its interacting protein complex were enriched by immunoprecipitation using a GAPDH-ce247 antibody. Subsequently, the expression of Myc-labeled substrate (IB: Myc) and GAPDH ceC247 (IB: ceC247) was detected by Western blotting.

[0077] Further immunoprecipitation experiments showed that GAPDH-ce247 could effectively enrich endogenously carboxyethylated GAPDH protein, and in the presence of MG132, 3-HPA treatment enhanced the ubiquitination level of carboxyethylated GAPDH, confirming that GAPDH-ce247 can be used for immunoprecipitation and ubiquitination modification analysis of carboxyethylated GAPDH. Figure 2 B).

[0078] Experiment 3: In immunofluorescence assay, the subcellular localization of GAPDH carboxyethylation modification within cells was detected.

[0079] DAPI staining of cell nuclei (blue), GAPDH antibody recognizing GAPDH protein (green), and GAPDH-ce247 antibody recognizing GAPDH ceC247 modification signal (red) showed that endogenous GAPDH ceC247 modification is mainly located in the cytoplasm without nonspecific background fluorescence interference. This further demonstrates that the antibody can accurately recognize naturally occurring GAPDH ceC247 modification epitopes in cells and does not produce cross-reactions against unmodified GAPDH or other modified forms.

[0080] Immunofluorescence assays showed that GAPDH-ce247 (red fluorescence) clearly marked the distribution of GAPDHceC247 in the cytoplasm and co-localized with GAPDH (green fluorescence) in the cytoplasm. DAPI (blue fluorescence) labeled the nucleus (…). Figure 2 The subcellular localization of carboxyethylated GAPDH was clarified, and the antibody was demonstrated to be highly efficient for immunofluorescence detection.

[0081] The above experimental results fully demonstrate that the antibody of the present invention has excellent specificity, sensitivity and applicability to multiple scenarios. It can be stably used for various immunoassays such as immunoblotting, immunoprecipitation and immunofluorescence of carboxyethylated modified GAPDH, providing a core tool for basic research related to carboxyethylated modified GAPDH.

[0082] 2.2.2 Assessment and diagnosis of inflammatory status.

[0083] At the cellular level, changes in GAPDH carboxyethylation levels in a macrophage inflammation model were detected using GAPDH-ce247.

[0084] Two groups of mouse peritoneal macrophage samples treated with LPS combined with 3-HPA were selected for the experiment. The levels of GAPDH ceC247 in the mouse peritoneal macrophage samples were detected by Western blotting using GAPDH-ce247 antibody and anti-Tubulin antibody, respectively. The results showed that the GAPDH-ce247 antibody could specifically detect the expression of endogenous carboxyethylated modified GAPDH in primary mouse peritoneal macrophages, validating the applicability of this antibody in primary cell samples. Figure 2 (D).

[0085] Based on the above applications, GAPDH-ce247 can be used to prepare diagnostic kits for inflammatory diseases. By detecting the level of GAPDH carboxylation in biological samples (such as blood and tissue lysates), the inflammatory status can be assessed, providing a new biomarker for the auxiliary diagnosis and disease monitoring of inflammatory-related diseases such as sepsis and autoimmune diseases.

[0086] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against GAPDH carboxyethylated modification, characterized in that, The monoclonal antibody comprises HCDR1~HCDR3 and LCDR1~LCDR3; The amino acid sequences of HCDR1 to HCDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively. The amino acid sequences of LCDR1 to LCDR3 are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively.

2. The monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.2; The amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown in SEQ ID NO.

3.

3. The monoclonal antibody as described in claim 2, characterized in that, The monoclonal antibody also includes a heavy chain constant region and / or a light chain constant region.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody of claim 1.

5. The application of the monoclonal antibody of claim 1 and / or the nucleic acid molecule of claim 4, characterized in that, The application refers to any one of the following: 1) Preparation of detection reagents for GAPDH Cys247 carboxylation; 2) Prepare diagnostic reagents for inflammatory diseases.

6. The application as described in claim 5, characterized in that, The inflammatory diseases include any one of sepsis, pneumonia, ankylosing spondylitis, and rheumatoid arthritis.

7. The application as described in claim 5, characterized in that, The detection reagent also includes enzyme-labeled secondary antibodies or fluorescently labeled secondary antibodies.

8. The application as described in claim 7, characterized in that, The enzyme-labeled secondary antibody includes horseradish peroxidase-labeled secondary antibody or alkaline phosphatase-labeled secondary antibody; Fluorescently labeled secondary antibodies include FITC-labeled secondary antibodies, TRITC-labeled secondary antibodies, or Cy3-labeled secondary antibodies.

9. The application as described in claim 5, characterized in that, The detection reagents are suitable for immunoblotting, immunofluorescence and / or immunoprecipitation detection.

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