Anti-acsl4 recombinant monoclonal antibody and preparation and application thereof

Abstract

The application discloses an anti-ACSL4 recombinant monoclonal antibody and a preparation and application thereof, and belongs to the technical field of biotechnology. The anti-ACSL4 recombinant monoclonal antibody takes an artificially synthesized polypeptide as an immunogen, immunizes a New Zealand white rabbit, and obtains a heavy chain variable region and a light chain variable region of the antibody through specific B cell screening and RT-PCR. A plasmid containing the heavy chain variable region and the light chain variable region is obtained through homologous recombination, and the recombinant monoclonal antibody is obtained through transfection expression and purification. The recombinant monoclonal antibody provided in the application has high affinity with ACSL4 protein, can recognize and detect normal cells and tumor cells with high specificity and high sensitivity, and can be applied to the detection and screening fields such as immunohistochemistry, flow cytometry, protein immunoblotting, immunofluorescence chemistry and immunoprecipitation.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to anti-ACSL4 recombinant monoclonal antibodies and their preparation and application. Background Technology

[0002] Acyl-CoA synthase long-chain member 4 (ACSL4) is an enzyme involved in fatty acid metabolism and is considered a specific biomarker and driver of ferroptosis. Upregulation of ACSL4 increases the content of polyunsaturated fatty acids (PUFAs) in phospholipids. The binding of PUFAs to phospholipids is crucial for maintaining membrane fluidity, but this also brings risks. High levels of PUFAs in membrane lipids can make cells more sensitive to ferroptosis because PUFAs are easily oxidized by hydroxyl and peroxide free radicals, leading to the formation of unstable iron pools (Fe2+). 2 + It is produced via the Fenton reaction. The resulting lipid peroxide radicals then oxidize neighboring PUFAs, leading to a chain reaction that, if carried out unchecked, ultimately results in cell death, a specific form of cell death.

[0003] Studies have shown that drug-resistant cancer cells, especially those with a stromal state and prone to metastasis, are highly susceptible to ferroptosis. Furthermore, many organ injuries and degenerative diseases are caused by ferroptosis. ACSL4, as a specific biomarker and driver of ferroptosis, holds great potential in treating drug-resistant cancers, ischemic organ damage, and other degenerative diseases associated with excessive lipid peroxidation. To achieve more precise drug development, ACSL4 has become a focus of attention for pharmaceutical companies, diagnostic companies, and clinicopathologists.

[0004] Currently, commercially available ACSL4 antibody products have limited functionality and cannot meet diverse testing needs. The accuracy of immunocytochemistry, immunohistochemistry, and flow cytometry directly affects the reliability of test results, thus requiring very high sensitivity and specificity from antibodies. With the deepening research into ferroptosis in cancer treatment, organ damage, and degenerative diseases, the demand for monoclonal antibodies targeting the ferroptosis biomarker ACSL4 is increasing.

[0005] Therefore, it is essential to develop a highly sensitive and specific anti-ACSL4 recombinant monoclonal antibody. Summary of the Invention

[0006] 1. Purpose of the invention

[0007] The purpose of this application is to provide a recombinant monoclonal antibody against ACSL4, its preparation, and its application. This recombinant antibody can specifically recognize ACSL4 protein in tissues. Immunohistochemical (IHC) and immunocytochemical (ICC) assays on various tissues have shown that this recombinant antibody can effectively detect the expression of ACSL4 protein on normal epithelial cells and tumor cells. It can be applied in detection and screening fields such as immunohistochemistry, flow cytometry, Western blotting, immunoprecipitation, immunofluorescence, and ELISA.

[0008] 2. Technical Solution

[0009] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0010] This application provides a recombinant monoclonal antibody against ACSL4, which includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5. It can specifically bind to the ACSL4 protein.

[0011] Furthermore, the aforementioned anti-ACSL4 recombinant monoclonal antibody also includes a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO.6 and the light chain constant region includes the amino acid sequence shown in SEQ ID NO.7.

[0012] This application also provides a nucleic acid that encodes the heavy chain variable region and / or light chain variable region of the aforementioned anti-ACSL4 recombinant monoclonal antibody.

[0013] Furthermore, the nucleic acid encoding the heavy chain variable region of the above-mentioned anti-ACSL4 recombinant monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO.2, which encodes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.4 of the above-mentioned anti-ACSL4 recombinant monoclonal antibody.

[0014] Furthermore, the nucleic acid encoding the light chain variable region of the above-mentioned anti-ACSL4 recombinant monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO.3, which is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO.5 of the above-mentioned anti-ACSL4 recombinant monoclonal antibody.

[0015] Furthermore, the nucleic acid encoding the heavy chain constant region of the above-mentioned anti-ACSL4 recombinant monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO. 8, which is used to encode the heavy chain constant region of the amino acid sequence shown in SEQ ID NO. 6 of the above-mentioned anti-ACSL4 recombinant monoclonal antibody.

[0016] Furthermore, the nucleic acid encoding the light chain constant region of the above-mentioned anti-ACSL4 recombinant monoclonal antibody includes the nucleotide sequence shown in SEQ ID NO. 9, which is used to encode the light chain constant region of the amino acid sequence shown in SEQ ID NO. 7 of the above-mentioned anti-ACSL4 recombinant monoclonal antibody.

[0017] This application also provides a recombinant expression vector containing the above-mentioned nucleic acid sequence encoding the anti-ACSL4 recombinant monoclonal antibody, which can express the anti-ACSL4 recombinant monoclonal antibody.

[0018] Furthermore, the recombinant expression vector includes a pcDNA3.4 plasmid containing the nucleic acid encoding the recombinant monoclonal antibody against ACSL4.

[0019] This application also provides a recombinant expression cell, which includes the above-mentioned recombinant expression vector or the above-mentioned nucleic acid, and can be used to express a recombinant monoclonal antibody against ACSL4.

[0020] Furthermore, the recombinant expression cells mentioned above include the 293F cell line.

[0021] This application also provides the application of the above-mentioned nucleic acid in the preparation of recombinant monoclonal antibodies against ACSL4.

[0022] This application also provides the application of the above-mentioned recombinant expression vector in the preparation of recombinant monoclonal antibodies against ACSL4.

[0023] This application also provides the application of the above-mentioned recombinant expression cells in the preparation of recombinant monoclonal antibodies against ACSL4.

[0024] This application also provides a method for preparing an anti-ACSL4 recombinant monoclonal antibody, the method comprising: transfecting cells with the above-mentioned recombinant expression vector to obtain recombinant expression cells; culturing the transfected recombinant expression cells; collecting the supernatant and purifying it to obtain an anti-ACSL4 recombinant monoclonal antibody.

[0025] This application also provides the application of the above-mentioned methods for preparing anti-ACSL4 recombinant monoclonal antibodies, nucleic acids, recombinant expression vectors, recombinant expression cells, or anti-ACSL4 recombinant monoclonal antibodies in the detection of ACSL4 protein molecules or in the preparation of products for detecting ACSL4 protein molecules.

[0026] Furthermore, the above-mentioned detection of ACSL4 protein molecules includes any one or more of the following detection methods: immunohistochemistry, flow cytometry, immunoblotting, immunoprecipitation, immunofluorescence, and ELISA.

[0027] Furthermore, the aforementioned products for detecting ACSL4 protein molecules include any one or more of the following: kits, antibody conjugates, antibody chips, etc.

[0028] This application also provides a kit for detecting ACSL4 protein molecules, the kit comprising the above-mentioned anti-ACSL4 recombinant monoclonal antibody, with the anti-ACSL4 recombinant monoclonal antibody as the primary antibody.

[0029] Furthermore, the above-mentioned kit is a flow cytometry detection kit, which includes the above-mentioned anti-ACSL4 recombinant monoclonal antibody and also includes flow cytometry detection reagents.

[0030] Furthermore, the above-mentioned flow cytometry detection kit also includes reagents such as fluorescently labeled secondary antibody, PBS, bovine serum albumin, Human IgG, and 7-AAD.

[0031] Furthermore, the above-mentioned kit is an immunohistochemical detection kit, comprising: the above-mentioned anti-ACSL4 recombinant monoclonal antibody, and immunohistochemical detection reagents.

[0032] Furthermore, the aforementioned immunohistochemical detection kit includes: HRP-labeled secondary antibody, EDTA retrieval solution, catalase blocking solution, DAB concentrate, DAB buffer, hematoxylin, and blueing solution.

[0033] This application also provides the application of the above-mentioned kit for detecting ACSL4 protein molecules in the detection of ACSL4 protein molecules.

[0034] Furthermore, the above-mentioned flow cytometry detection kits are used for: cell resuscitation, FC receptor blockade, primary antibody incubation, secondary antibody incubation, 7-AAD activity staining, and data collection.

[0035] Furthermore, the applications of the above-mentioned immunohistochemical detection kits include: dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB staining, counterstaining, dehydration, mounting, and microscopic examination.

[0036] 3. Beneficial effects

[0037] Compared with the prior art, the advantages of this application are as follows:

[0038] (1) The anti-ACSL4 recombinant monoclonal antibody provided in this application and its preparation and application, the anti-ACSL4 recombinant monoclonal antibody has high specificity and high sensitivity in binding to the ACSL4 protein molecule, and can specifically recognize and detect the expression of ACSL4 protein on tumor and normal tissues. It shows positive high expression when detecting ACSL4 protein. The antibody can be applied to the fields of immunohistochemistry, flow cytometry, protein immunoblotting, immunofluorescence chemistry, immunoprecipitation, etc., which is conducive to obtaining accurate assessment and detection results.

[0039] (2) The anti-ACSL4 recombinant monoclonal antibody provided in this application and its preparation and application, due to its good specificity and strong positive signal, are beneficial to the accuracy of companion diagnosis for targeted therapy. Attached Figure Description

[0040] Figure 1 This is a serum titer chart of rabbits (K1275).

[0041] Figure 2 It is an anti-ACSL4 recombinant monoclonal antibody detected by SDS-PAGE gel electrophoresis.

[0042] Figure 3 The results are from immunohistochemical detection using recombinant anti-ACSL4 monoclonal antibodies, and are in the following order: human liver cancer tissue, human adrenal gland tissue, human prostate cancer tissue, human ovarian cancer tissue, rat kidney tissue, and human colon cancer tissue.

[0043] Figure 4 The results are from flow cytometry analysis using recombinant monoclonal antibodies against ACSL4.

[0044] Figure 5 This is the result of Western blot detection of anti-ACSL4 recombinant monoclonal antibody protein.

[0045] Figure 6 This is the result of immunoprecipitation assay using recombinant monoclonal antibody against ACSL4.

[0046] Figure 7 This is the result of immunofluorescence detection of recombinant monoclonal antibody against ACSL4. Detailed Implementation

[0047] The present application will be further described below with reference to specific embodiments.

[0048] It should be noted that terms such as "upper", "lower", "left", "right", and "middle" used in this specification are only for clarity of description and are not intended to limit the scope of implementation. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered as within the scope of this application.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0050] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0051] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0052] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0053] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as “less than about 4.5,” which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0054] Example 1

[0055] This embodiment provides the screening and preparation of rabbit anti-human / mouse anti-ACSL4 recombinant monoclonal antibodies, specifically including the following steps:

[0056] Antigen preparation: The amino acid sequence of ACSL4 protein was analyzed. Based on the structure of ACSL4 protein molecule on the cell membrane, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids and secondary structure, a synthetic polypeptide with the amino acid sequence KKNAMAKRIKAKPTSDKPGSPYRS (SEQ ID NO.1) was selected as ACSL4 antigen. This sequence has strong immunogenicity and high hydrophilicity.

[0057] Animal Immunization: The above-mentioned synthetic polypeptide (ACSL4 antigen) was conjugated with KLH and used as an immunogen to immunize three New Zealand white rabbits via multiple dorsal immunization. Specifically, the immunogen was mixed with complete Freund's adjuvant (1:1) and emulsified, and then injected subcutaneously into each of the three rabbits. Two weeks later, the immunogen was emulsified with incomplete Freund's adjuvant (1:1) for a second and third immunization. After all three immunizations, blood was collected and serum titers were determined using a serially diluted indirect ELISA method. The OD value at a serum dilution of 64,000 was selected. 450Rabbits (K1275) with serum titers greater than 0.3 and strong IHC staining signals in different tissues underwent splenectomy; their serum titers were as follows: Figure 1 As shown.

[0058] Monoclonal antibody screening: The removed rabbit spleen was ground up, and a population of B cells capable of efficiently secreting antibodies was obtained through antigen capture, cell sorting, and limiting dilution. The B cell population that secretes specific antibodies against ACSL4 protein was screened by immunohistochemistry and indirect ELISA. The nucleic acids encoding the heavy and light chains of the B cells that secrete specific antibodies were amplified by RT-PCR. The obtained nucleic acids encoding the heavy and light chains were constructed into the pcDNA3.4 vector by homologous recombination, transformed into TOP10 competent cells (DF1010M) and cultured overnight at 37°C. Single colonies were picked and sequenced to obtain the nucleic acid sequences encoding the heavy and light chains. Plasmids linked with different heavy and light chain nucleic acid sequences were paired and transfected into the 293F cell line using a transfection reagent. Cell supernatant was collected, and the collected cell supernatant was verified by ELISA, IHC, and WB. It was confirmed that the antibody expressed by cells containing the plasmid encoding the heavy chain variable region nucleic acid sequence shown in SEQ ID NO.2 and the light chain variable region nucleic acid sequence shown in SEQ ID NO.3 had a good immune response to the antigen ACSL4 protein. This antibody was named SDT-461-1.

[0059] The nucleic acid sequence encoding the heavy chain variable region (named VH-DNA) is as follows:

[0060] GGTCGCCTGGTCACGCCTGGGACACCCTGACACTCACCTGCACAGTCTCTGGATTATCCCTCAGTAGCAATGCAATAAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAAACATCGGATGGATTAGTACTAGTGGTAGCATATACTACGCGAGCTGGGCGAATGGTCGATTCACC ATCTCCAAAACCTCGTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAATGAGAATTTATAATGGGGCCGTCTGGGGCCCAGGCTCTTCGGTTACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCC(SEQ ID NO.2),

[0061] The nucleic acid sequence encoding the light chain variable region (named VL-DNA) is as follows:

[0062] TCTCCCGTGTCTGCAGCTGTGGGAGGCACAGCCAGCATCAGTTGCCAGGCCAGTCAGAGTGTTTATAGTAGCTACTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTAATCCAGAAGGCTTCCACTCTGGCATCTGGGGTCCCATCACGGTTCAC CGGCAGTGGATCTGGGACACAGTTCACTCTCACCATTAGTGACGTGCAATGTGACGATGCTGCCACTTACTACTGTCAATGTAATTTGGGTAGTAGTGGTAGTAATAATTATGGTTATGGTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTT(SEQ ID NO.3);

[0063] Based on the above nucleic acid sequences encoding the heavy chain and light chain, the amino acid sequences of the corresponding antibody's heavy chain variable region (VH) and light chain variable region (VL) are obtained:

[0064] The amino acid sequence of the heavy chain variable region (VH) is as follows:

[0065] GRLVTPGTPLTLTCTVSGLSLSSNAINWVRQAPGKGLENIGWISTSGSIYYASWANGRF TISKTSSTTVDLKITSPTTEDTATYFCARMRIYNGAVWGPGSSVTVSSGQPKAPSVFPLA(SEQ ID NO.4),

[0066] The amino acid sequence of the light chain variable region (VL) is as follows:

[0067] SPVSAAVGGTASISCQASQSVYSSYLSWFQQKPGQPPKLLIQKASTLASGVPSRFTGSG SGTQFTLTISDVQCDDAATYYCQCNLGSSGSNNYGYGFGGGTEVVVKGDPV(SEQ ID NO.5);

[0068] The sequences of the heavy chain constant region (CH) and light chain constant region (CL) of the rabbit monoclonal antibody that specifically binds to the ACSL4 protein are as follows:

[0069] The amino acid sequence of the heavy chain constant region (CH) is as follows:

[0070] METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPGASLTLTCKASGFSISSVYGMCWVRQAPGKGLEWIASIYAGVGASTYYANWAKGRFTISRTSSTTVTLQMTSLTAADTATYFCARGFIGDGYAAGAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTPGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO.6);

[0071] The amino acid sequence of the light chain constant region (CL) is:

[0072] MDVVMTQTPASVEAAVGGTVTIKCQASQNIYSNLAWYQQKPGQRPKLLIYKASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTWYDNTYVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO.7);

[0073] The nucleic acid sequence encoding the heavy chain constant region (CH-DNA) is:

[0074]

[0075] The nucleic acid sequence encoding the light chain constant region (CL-DNA) is as follows:

[0076] (SEQ ID NO.9).

[0077] Expression and purification: The above-verified positive expression vector (the expressed antibody showed a good immune response to the antigen ACSL4 protein) was transfected into a large number of cells (transfection reagent was used to transfect 293F cell line). After culturing at 37°C and 5% CO2 for 3 days, the cell suspension was collected, centrifuged at 1000g, and the supernatant was collected. The supernatant was purified by Protein A affinity chromatography and detected by SDS-PAGE gel electrophoresis to obtain a recombinant monoclonal antibody against ACSL4 with a purity greater than 95%. Figure 2 ).

[0078] Example 2

[0079] This embodiment provides the application of anti-ACSL4 recombinant monoclonal antibody in the detection of ACSL4 protein. The detection is performed using an immunohistochemical method, with the anti-ACSL4 recombinant monoclonal antibody (SDT-461-1) purified in Example 1 as the primary antibody and the Anti-Rabbit and Mouse HRP-DAB IHC detection kit (catalog number: S0C1001) as the secondary antibody. Specifically, it includes:

[0080] Six paraffin-embedded normal or cancer tissue sections were placed in a drying oven and baked at 63℃~65℃ for about 1 hour; dewaxing and hydration: (all operations were completed in a fume hood) the sections were placed in (1) xylene twice, 7 minutes each time; (2) anhydrous ethanol twice, 5 minutes each time; (3) 90% ethanol twice, 5 minutes each time; (4) 75% ethanol once, 3 minutes; (5) 50% ethanol once, 3 minutes; after the operation, the sections were removed and rinsed with distilled water three times, 5 minutes each time.

[0081] Antigen retrieval: Place the tissue sections in a staining chamber containing sodium citrate buffer or EDTA retrieval solution. Add 2L of distilled water to the antigen retrieval pot, and place the staining chamber in an autoclave for high-temperature, high-pressure retrieval for 20 minutes. Then, stop heating, open the lid, remove the container containing the tissue sections, and allow it to cool naturally to room temperature. Rinse thoroughly with distilled water, and wash three times with 1×PBS for 3 minutes each time.

[0082] After antigen retrieval, the slides were placed in 3% H2O2 solution for 10 minutes (operated in a fume hood), and then washed three times with 1×PBS for 5 minutes each time to block endogenous peroxidase.

[0083] Block nonspecific antigens with 5% BSA + 10% sheep serum (prepared using PBST) at room temperature for 30 minutes. After blocking, knock off the blocking solution from the tissue sections, add diluted primary antibody, 80-100 μl per well, adjusting the amount according to the size of the immunohistochemistry pen circle. Place the slides in a humidified chamber and then slowly place them in a refrigerator at 4°C overnight.

[0084] After incubation, remove the humidified chamber and let it stand at room temperature for 10 minutes. Rinse three times with 1×PBST for 5 minutes each time, add secondary antibody, incubate at room temperature for 30 minutes, and wash three times with 1×PBST for 5 minutes each time. Develop the color with DAB chromogenic solution, 100 μl per slice, add freshly prepared chromogenic solution to each slice, develop for 1-2 minutes, observe under a microscope until a brownish-yellow color appears, and then rinse with distilled water to stop the color development. Counterstain with hematoxylin for 2-5 minutes, rinse with distilled water for 5 minutes, decolorize and return to blue in 1×PBS for 30 seconds, and rinse with distilled water for 5 minutes. Dehydrate and dry sequentially with a gradient of ethanol: (1) 50% ethanol, dehydrate for 5 minutes; (2) 75% ethanol, dehydrate for 5 minutes; (3) 90% ethanol, dehydrate for 5 minutes; (4) anhydrous ethanol, dehydrate three times for 5 minutes each time; (5) xylene, dehydrate twice for 5 minutes each time.

[0085] Add 50–100 μl of neutral resin to each tissue section, then slowly add a coverslip. After mounting, allow the slides to air dry overnight. The next day, observe under a microscope and obtain immunohistochemical results using a tissue sectioning instrument.

[0086] Results: The recombinant monoclonal antibody SDT-461-1 against ACSL4 in this application showed strong staining in human liver cancer tissue, human adrenal gland tissue, human prostate cancer tissue, human ovarian cancer tissue, rat kidney tissue, and human colon cancer tissue. Figure 3 This demonstrates that the recombinant monoclonal antibody SDT-461-1 against ACSL4 in this application has a high affinity for the ACSL4 protein.

[0087] Example 3

[0088] This embodiment provides the application of anti-ACSL4 recombinant monoclonal antibody in the detection of ACSL4 protein. The detection method is flow cytometry, using the anti-ACSL4 recombinant monoclonal antibody (SDT-461-1) purified in Example 1 as the primary antibody, specifically including:

[0089] After the pretreatment of hepatocellular carcinoma cells for resuscitation, cell viability was assessed for all cell batches using trypan blue. Cells with a viability greater than 90% were used, and the cells were spaced at 5 × 10⁶ cells per well. 5 ~1×10 6 Aliquot the cells into 96-well plates, centrifuge at 200g for 3 minutes at 4°C, and discard the supernatant.

[0090] Add 100 μl of 10% sheep serum blocking buffer to each well, mix thoroughly by pipetting, and incubate on ice for 60 minutes. During incubation, dilute the primary antibody with washing buffer and store at 4°C for later use. Centrifuge for 3 minutes, discard the supernatant, add 100 μl of diluted primary antibody, mix thoroughly by pipetting, and incubate on ice for 30 minutes. Wash three times with washing buffer, centrifuging for 3 minutes each time, and discard the supernatant. Add 100 μl of diluted A488-labeled sheep anti-rabbit secondary antibody, mix thoroughly by pipetting, and incubate on ice in the dark for 30 minutes. Then wash three times, centrifuging for 3 minutes each time, and discard the supernatant.

[0091] Resuspend each well in 200 μl of 7-AAD premixed solution, incubate on ice in the dark for 5 minutes, and then run on the instrument.

[0092] Results: Flow cytometry staining analysis was performed after adding recombinant anti-ACSL4 monoclonal antibody to liver cancer cells. The fluorescence signal intensity (MFI) comparison between the blank isotype control and the anti-ACSL4 recombinant monoclonal antibody sample was shown in the image. Figure 4 (A and B are negative controls, and C and D are positive controls by flow cytometry.) The recombinant monoclonal antibody SDT-461-1 against ACSL4 showed a strong signal in cultured liver cancer cells, but no staining in the isotype control.

[0093] Example 4

[0094] This embodiment provides the application of anti-ACSL4 recombinant monoclonal antibody in the detection of ACSL4 protein. The detection method is Western blotting, using the anti-ACSL4 recombinant monoclonal antibody (SDT-461-1) purified in Example 1 as the primary antibody, specifically including:

[0095] Dissolve RIPA at room temperature and add an appropriate amount of protease inhibitor (PMSF) at a concentration of 1 mM. Add 200 μL of RIPA to each well of cells to fully wet the cell surface, lyse the cells on ice for 15 minutes, centrifuge at 1000 rpm for 10 minutes at 4°C, and collect the supernatant.

[0096] Set up the electrophoresis tank and load samples using a 4%–20% gradient gel. Add 10 μL of protein sample to each well of the gel, incubate at 100V for 60 minutes. Once clear band separation is observed, stop electrophoresis, peel and cut the gel for transfer to a membrane.

[0097] Cut a PVDF membrane slightly larger than the gel and prepare dedicated filter paper. Activate the PVDF membrane with methanol, and wet the filter paper and gel with transfer buffer. Install the filter paper, gel, membrane, and filter paper onto the sponge pad of the transfer apparatus in the order of negative to positive, removing air bubbles with a plate. Set up the transfer apparatus and perform the transfer under ice bath conditions. Set the voltage and transfer for 60 minutes. After transfer, shake with 15 mL of milk powder blocking buffer at room temperature for 2 hours. Dilute the corresponding antibody with 1×PBS to prepare primary antibody dilution buffer, incubate overnight at 4°C, and then wash the PVDF membrane three times with TBST for 10 minutes each time. Subsequently, prepare the corresponding secondary antibody with TSBT and incubate the PVDF membrane on a shaker at room temperature for 2 hours. Finally, wash the membrane three times with TBST for 10 minutes each time. Finally, prepare the developing solution at a 1:1 ratio, incubate the PVDF membrane for 5 minutes, and then develop and photograph the membrane.

[0098] Results: Five detection materials, including HepG2, HeLa, 293T, NIH / 3T3, and C6, were used for Western blotting detection with the anti-ACSL4 recombinant monoclonal antibody SDT-461-1 as the primary antibody. The results are as follows: Figure 5 As shown, the target protein band can be seen in all five types of detection materials.

[0099] Example 5

[0100] This embodiment provides the application of anti-ACSL4 recombinant monoclonal antibody in the detection of ACSL4 protein. The detection method is immunoprecipitation, using the anti-ACSL4 recombinant monoclonal antibody (SDT-461-1) purified in Example 1 as the primary antibody, specifically including:

[0101] 100 μg of liver cancer cell lysate was incubated overnight at 4°C with gentle shaking, with 1:1000 diluted anti-ACSL4 recombinant monoclonal antibody added. Simultaneously, 100 μl of preotein A / G beads was added to the incubator and the mixture was gently shaken at 4°C for 4 hours. The mixture was then centrifuged at 1000g for 5 minutes at 4°C, and the supernatant was carefully aspirated. The pellet was washed three times with cell lysis buffer (1 ml each time), followed by centrifugation at 1000g for 5 minutes at 4°C, and the supernatant was carefully aspirated.

[0102] After a final wash to remove the supernatant, add 50 μl of 2× electrophoresis loading buffer, boil at 100°C for 5 minutes to denature the proteins and separate them from the protein-A / G beads. Centrifuge, collect the supernatant, and discard the precipitate. The obtained supernatant is immediately subjected to Western blotting analysis.

[0103] Results: Using HepG2 cell lysate as the detection material and the recombinant anti-ACSL4 monoclonal antibody SDT-461-1 as the primary antibody, ACSL4 protein was correctly detected. Figure 6This demonstrates that the recombinant monoclonal antibody SDT-461-1 against ACSL4 in this application has a strong affinity for the ACSL4 protein.

[0104] Example 6

[0105] This embodiment provides the application of anti-ACSL4 recombinant monoclonal antibody in the detection of ACSL4 protein. The detection method is immunofluorescence, using the anti-ACSL4 recombinant monoclonal antibody (SDT-461-1) purified in Example 1 as the primary antibody, specifically including:

[0106] Remove the cell culture dish and wash the cells twice with PBS buffer, 2 minutes each time. Add 500 μl of cell fixation solution (PBS solution containing 4% paraformaldehyde) and incubate at room temperature for 20 minutes. Discard the fixation solution and wash twice with PBS, 5 minutes each time.

[0107] Add 500 μl of PBST (0.5% Triton X-100 in PBS) and incubate at room temperature for 5 minutes via permeation. Wash twice with PBS for 5 minutes each time. Add the primary antibody working solution and incubate at room temperature for 2 hours. The primary antibody used is diluted 1:500.

[0108] After incubation, wash cells twice with PBST for 5 minutes each time, then wash twice more with PBS for 5 minutes each time. Add fluorescent secondary antibody (1:500) and incubate at room temperature in the dark for 1 hour. After incubation, wash three times with PBS for 5 minutes each time. Add DAPI staining solution and incubate at room temperature in the dark for 5 minutes. Wash cells three times with PBS for 5 minutes each time. Remove the cell slides, gently place them on a glass slide containing 90% glycerol, fix and mount the slides, and observe and photograph the results using a laser confocal microscope.

[0109] Results: The recombinant anti-ACSL4 monoclonal antibody SDT-461-1 showed positive staining in HepG2 cells. The cell nucleus was counterstained with DAPI (blue fluorescence), and microtubules were counterstained with red fluorescence. The recombinant anti-ACSL4 monoclonal antibody (green fluorescence) correctly located the ACSL4 protein in the cytoplasm and cell membrane. Figure 7 ).

Claims

1. A recombinant monoclonal antibody against ACSL4, characterized in that, The recombinant monoclonal antibody includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

5.

2. A nucleic acid, characterized in that, The nucleic acid encodes the heavy chain variable region and the light chain variable region of the anti-ACSL4 recombinant monoclonal antibody according to claim 1.

3. A nucleic acid according to claim 2, characterized in that, The nucleic acid includes the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid as described in claim 2 or 3.

5. A recombinant expression cell, characterized in that, The recombinant expression cell comprises the recombinant expression vector of claim 4, or the nucleic acid of claim 2 or 3.

6. The use of the nucleic acid of claim 2 or 3, or the recombinant expression vector of claim 4, or the recombinant expression cell of claim 5 in the preparation of recombinant monoclonal antibody against ACSL4.

7. The method for preparing the anti-ACSL4 recombinant monoclonal antibody according to claim 1, characterized in that, The method includes: transfecting cells with the recombinant expression vector of claim 4 to obtain recombinant expression cells; culturing the transfected recombinant expression cells; collecting the supernatant and purifying it to obtain an anti-ACSL4 recombinant monoclonal antibody.

8. The use of the anti-ACSL4 recombinant monoclonal antibody of claim 1, or the nucleic acid of claim 2 or 3, or the recombinant expression vector of claim 4, or the recombinant expression cell of claim 5 in the preparation of products for detecting ACSL4 protein.

9. The application according to claim 8, characterized in that, The detection of ACSL4 protein includes any one or more of the following methods: immunohistochemistry, flow cytometry, immunoblotting, immunoprecipitation, immunofluorescence, and ELISA.

10. A kit for detecting ACSL4 protein molecules, characterized in that, The kit includes the anti-ACSL4 recombinant monoclonal antibody as described in claim 1.

Images