Monoclonal antibody against p53 R248Q as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antibodies, and discloses an anti-p53 R248Q monoclonal antibody as well as a preparation method and application thereof. The invention specifically discloses amino acid sequences of heavy chain and light chain variable regions of the antibody. The invention also discloses nucleic acid molecules for coding the heavy chain and light chain variable regions. Comprising the nucleic acid molecule; comprising the vector; a method for preparing the antibody; the invention also discloses an application of the antibody in preparation of a kit for detecting p53 R248Q mutation. The antibody provided by the invention can realize specific screening of p53 R248Q mutation, solves the problem of few accessible tissue samples of lung adenocarcinoma patients in late stage or drug resistance progress stage, can achieve the advantages of simple and rapid detection, easy interpretation and low cost, and is easy to popularize and apply in resource-deficient regions.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a monoclonal antibody against p53 R248Q, its preparation method, and its application. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related deaths worldwide, with lung adenocarcinoma accounting for more than 50% of non-small cell lung cancer (NSCLC), posing a serious threat to human life and health. With the development of targeted therapy technologies, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have become the first-line treatment for patients with EGFR-mutant lung adenocarcinoma, significantly improving patient survival prognosis. However, targeted therapy resistance remains a core challenge in clinical practice—approximately 20-30% of patients exhibit primary resistance, and even those who initially respond to treatment experience disease progression after a median of 9-15 months, leading to treatment failure.

[0003] Numerous clinical studies have confirmed that co-mutations of the TP53 gene are one of the key driving factors for EGFR-TKI resistance. As an important tumor suppressor gene, the TP53 gene encodes the p53 protein, which inhibits tumor development and progression by regulating pathways such as DNA damage repair and cell cycle arrest. The DNA-binding domain (DBD) of this protein is a high-risk mutation region, with the R248Q missense mutation being a common and high-frequency mutation type in clinical practice, accounting for a significant proportion of TP53 mutations in human cancers. Unlike other mutations, the R248Q mutation not only leads to the loss of the p53 protein's tumor-suppressive function but also promotes malignant tumor progression through a "gain-of-function" mechanism. Specifically, it enhances the tumor's stability, drives tumor cell proliferation, induces metabolic reprogramming, and remodels the immune microenvironment. Ultimately, this results in a significant reduction in progression-free survival (PFS) and overall survival (OS) in patients with EGFR-mutant lung adenocarcinoma, and the drug resistance mechanism is more complex, becoming a significant obstacle to precision medicine in clinical practice.

[0004] Accurate detection of TP53 R248Q mutation status is a crucial prerequisite for developing individualized treatment plans, predicting the efficacy of targeted therapy, and assessing patient prognosis. Currently, commonly used clinical detection methods include next-generation sequencing (NGS) and immunohistochemistry (IHC), but both have significant limitations: Firstly, while NGS can accurately identify R248Q mutation sites and abundance, its application has a high barrier to entry. This technology has stringent requirements for sample quality, requiring a tumor cell ratio of ≥20%. However, patients with advanced lung adenocarcinoma often require only small biopsy samples due to the specific location of the lesions or poor physical tolerance, easily leading to test failure. Secondly, NGS testing requires an investment of millions of yuan in equipment and the construction of a professional molecular pathology laboratory. The costs of reagents, consumables, equipment maintenance, and bioinformatics analysis personnel far exceed the budget of primary healthcare institutions. Furthermore, the complete testing process takes 3-7 days and has a "start-up threshold." Primary hospitals often need to send samples out for testing due to insufficient sample volume, further extending the cycle and increasing additional costs, making it difficult to popularize this technology at the primary level. On the other hand, the normal TP53 gene corresponds to the normal / wild-type p53 protein, which is expressed in the nucleus of all normal cells. Due to its short half-life of only 10-20 minutes, it is difficult to detect by IHC. When the TP53 gene mutates, the expressed protein is a mutant p53 protein, which is often detected by IHC. IHC technology, due to its ease of operation, low cost, and rapid detection (completed in 4-6 hours), has been widely used in pathology departments of hospitals at all levels, becoming the preferred initial screening technique for tumor molecular subtyping. However, currently available commercially available p53 protein IHC antibodies can only preliminarily determine the presence of TP53 gene mutations through modes such as "overexpression, complete deletion, and cytoplasmic expression." They cannot accurately identify specific mutation sites such as R248Q, nor can they distinguish between different mutation types, easily leading to misdiagnosis or missed diagnosis, and failing to meet the clinical need for precise mutation site detection.

[0005] Therefore, it is necessary to develop a highly specific and high-affinity anti-p53 R248Q monoclonal antibody, establish a mature preparation process, and apply it to convenient detection scenarios such as IHC, so as to achieve rapid and accurate screening of TP53 R248Q mutations in lung adenocarcinoma tissue. Summary of the Invention

[0006] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a monoclonal antibody against p53 R248Q, which enables rapid and accurate screening of TP53 R248Q mutations in lung adenocarcinoma tissues in convenient testing scenarios such as IHC. This not only compensates for the deficiencies of current detection technologies and solves the problem of accurate detection in primary healthcare institutions, but also provides key technical support for EGFR-TKI resistance prediction, patient prognosis assessment, and the development of individualized treatment plans, demonstrating significant clinical application value and translational potential.

[0007] This invention also proposes a nucleic acid molecule.

[0008] This invention also proposes a recombinant expression vector.

[0009] This invention also proposes a recombinant cell.

[0010] The present invention also proposes a method for preparing the above-mentioned monoclonal antibody against p53 R248Q.

[0011] The present invention also proposes the application of the above-mentioned anti-p53 R248Q monoclonal antibody in the preparation of a kit for detecting p53 R248Q mutation.

[0012] According to one aspect of the present invention, a monoclonal antibody against p53 R248Q is provided, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:1: MGTGLFWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSGKAMSWVRQAPGKGLEWIGAIDGGSGSTWSANWAKGRFTISKTSTTVHLKITSPTTEDTATYFCAGGYNIWGPGTLVTVSL, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:1. NO:2:MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFMC GSVDCILFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC is shown; Alternatively, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:3: MGTGLYWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSGKAMSWVRQAPGKGLEWIGAIDGGSGSTWSANWAKGRFTISKTSTTVHLKITSPTTEDTATYFCAGGYNIWGPGTLVTVSL, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:3. IDNO:4:MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFM CGSVDCILFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC is shown; Alternatively, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:5: METGLHWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSGKAMSWVRQAPGKGLEWIGAIDGGSGSTWSANWAKGRFTISKTSTTVHLKITSPTTEDTATYFCAGGYNIWGPGTLVTVSL, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:5. IDNO:6:MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFM CGSVDCILFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0013] In some embodiments of the present invention, the concentration of the anti-p53 R248Q monoclonal antibody is ≥1 mg / mL.

[0014] According to a second aspect of the invention, a nucleic acid molecule is provided that encodes the heavy chain variable region and the light chain variable region of the aforementioned anti-p53R248Q monoclonal antibody.

[0015] According to a third aspect of the present invention, a recombinant expression vector is provided, the recombinant expression vector comprising the above-described nucleic acid molecule.

[0016] In some embodiments of the present invention, the recombinant expression vector is a mammalian cell expression vector.

[0017] Specifically, the mammalian cell expression vector is not specifically limited, such as at least one of pTT5, pRCH, and pRCK. pRCH is specifically used to express the antibody heavy chain, and pRCK is specifically used to express the antibody light chain. Both require co-transfection of cells to assemble into a complete antibody. pTT5 can be designed as a dual expression cassette vector to express both the heavy and light chains, or it can be split into two single expression cassette vectors for separate transfection.

[0018] According to a fourth aspect of the present invention, a recombinant cell is provided, the recombinant cell comprising the above-described recombinant expression vector.

[0019] According to a fifth aspect of the present invention, a method for preparing the above-mentioned monoclonal antibody against p53 R248Q is provided, comprising the following steps: S1: Provides a specific polypeptide immunogen and a recombinant protein immunogen targeting the p53 R248Q mutation site, wherein the amino acid sequence of the specific polypeptide immunogen is shown in SEQ ID NO:7: SCMGGMNQRPILTII or SEQ ID NO:8: CMGGMNQRPILTI, and the amino acid sequence of the recombinant protein immunogen is shown in SEQ ID NO:9: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPGSGSGSCMGGMNQRPILTIITLEDGSGMGGMNQRPILTIITLEDGSGNSSCMGGMNQRPILTGSGSGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA; S2: Immunize experimental animals with the specific polypeptide immunogen prepared in step S1 and the recombinant protein immunogen. After immunization, collect peripheral blood from the experimental animals, separate peripheral blood mononuclear cells (PBMCs), and screen for antigen-specific B lymphocytes. S3: Culture the antigen-specific B lymphocytes; screen for antigen-specific B lymphocytes that secrete the target antibody and extract their RNA; amplify the heavy chain variable region gene and light chain variable region gene of the target antibody; and express the target antibody after cloning and transfection of the two genes. S4: Purify the target antibody to obtain the anti-p53 R248Q monoclonal antibody.

[0020] Specifically, the simultaneous use of specific peptide immunogens and recombinant protein immunogens aims to synergistically enhance the immune response and ensure that a high-affinity, high-specificity monoclonal antibody against p53 R248Q can ultimately be obtained. The structural characteristics and complementary immune advantages of the two immunogens not only solve the limitations of a single immunogen, but also precisely match the core requirements for preparing site-specific antibodies. The specific reasons are as follows: (1) The core requirement of p53 R248Q monoclonal antibody is to recognize only the R248Q mutation site (site specificity) + to bind to the target protein efficiently (high affinity). The structural design of the two immunogens corresponds to these two requirements respectively: the specific polypeptide immunogen is a short sequence (15~20 amino acids), which only contains the R248Q mutation site and the surrounding conserved sequence. It has a simple structure and a single epitope, which can accurately induce the immune system to produce specific antibodies against the R248Q mutation site, avoiding the antibody from recognizing other irrelevant regions of the p53 protein; the recombinant protein immunogen is a long sequence (containing 3 tandem R248Q mutation epitopes + vector sequence), which has a complex structure and strong immunogenicity. It can enhance the response strength of the immune system and induce the production of antibodies with higher titers and higher affinity. At the same time, it simulates the spatial conformation of the natural p53 protein to ensure that the antibody can recognize the mutant protein in the natural state. (2) Using only one immunogen presents significant technical limitations and fails to meet the core requirements for antibody preparation: the peptide has a small molecular weight and weak immunogenicity, resulting in low serum antibody titers after immunization alone, making it difficult to screen for high-affinity B lymphocytes. The peptide is a linear epitope and cannot mimic the spatial conformation of the native p53 protein, potentially leading to induced antibodies that can only bind to the denatured peptide and not the native p53 R248Q mutant protein, ultimately affecting IHC detection results. Recombinant proteins contain multiple epitopes, and the immune system produces antibodies targeting multiple epitopes, reducing the proportion of site-specific antibodies and significantly increasing the difficulty of subsequent screening. If the mutant epitope of the recombinant protein is blocked by other sequences, it may not effectively induce specific antibodies against the R248Q site, leading to immunization failure. (3) Antibodies induced by peptide immunogens recognize linear epitopes (amino acid sequences of mutation sites), while antibodies induced by recombinant proteins recognize conformational epitopes (spatial structures of mutation sites). The antibodies selected after combined immunization can recognize both linear epitopes (ensuring site specificity) and conformational epitopes (ensuring the ability to bind to native proteins), perfectly matching the needs of clinical IHC testing (detecting the native conformation of p53 protein in the sample). (4) This invention uses single B cell technology to prepare antibodies. The core of this technology is to screen antigen-specific B cells from PBMCs of experimental animals after immunization and then clone their antibody genes.The combined use of two immunogens can significantly improve the screening efficiency of this technology: the recombinant protein immunogen enhances the overall immune response, resulting in a significant increase in the total number of antigen-specific B cells in PBMCs (improving the screening hit rate); the peptide immunogen enriches B cells that recognize only the R248Q site, reducing interference from B cells targeting other epitopes, making the subsequent flow cytometry and ELISA screening process more efficient and avoiding the screening of a large number of irrelevant antibodies.

[0021] In some embodiments of this invention, the full-length p53 protein sequence from the Uniprot database (P04637) is used, combined with NGS data from over 500 NSCLC samples in a public database, to screen for the R248Q missense mutation site. For each site, a linear B-cell epitope peptide containing the mutation site and approximately 15-20 amino acids flanking it is designed. Two Human p53 R248Q polypeptide immunogenic sequences were designed; to enhance immunogenicity, three copies of the same mutant epitope are tandemly linked using the flexible linker GGSG, and a 6×His tag is introduced at the N-terminus or C-terminus of the sequence to facilitate subsequent purification and detection.

[0022] In some embodiments of the present invention, the nucleic acid sequence corresponding to the recombinant protein immunogen is cloned into a prokaryotic expression vector, transformed into competent cells, induced to express, and then purified to obtain the recombinant protein immunogen.

[0023] Specifically, the choice of the prokaryotic expression vector is not specifically limited, such as pET28a or pET32a vectors.

[0024] Specifically, the induction temperature was 25~28℃, and the time was 6~8h.

[0025] Specifically, purification was performed by nickel column (Ni-NTA) affinity chromatography.

[0026] Specifically, after purification, the process also includes steps to verify the purity and molecular weight of the protein using SDS-PAGE and Western blotting, with a target purity of ≥90%.

[0027] In some embodiments of the present invention, the experimental animal in step S2 is selected from any one of rabbit, mouse, rat, guinea pig, and sheep.

[0028] In some preferred embodiments of the present invention, the experimental animal in step S2 is selected from rabbits.

[0029] In some embodiments of the present invention, the mass ratio of the specific polypeptide immunogen and the recombinant protein immunogen in step S2 is 1:1 to 1:3.

[0030] Specifically, the specific mass of the specific polypeptide immunogen and recombinant protein immunogen is determined based on the weight of the selected experimental animal.

[0031] In some embodiments of the present invention, step S2 involves adding an immune chaperone and adjuvant during the immunization process, administering immunization via subcutaneous multi-point injection, with an immunization cycle of 2-3 months. Serum antibody titers are then measured after immunization, and the titer is set at 6 × 10⁻⁶. 4 The animals were given a shock immunization, and peripheral blood was collected from the experimental animals 7-8 days later.

[0032] Specifically, the mass ratio of the total mass of the specific polypeptide immunogen and the recombinant protein immunogen to the mass of the immune chaperone is 20:1 to 100:1, and the mass ratio of the total mass of the specific polypeptide immunogen and the recombinant protein immunogen to the mass of the adjuvant is 1:1 to 1:1.5.

[0033] Specifically, the immune companion is selected from at least one of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, IL-6, IL-10, CD40L, and Hsp70.

[0034] Specifically, the adjuvant is selected from at least one of Freund's adjuvant, Montanide ISA 50V / 720, MF59, ISCOM Matrix, IL-2, and aluminum hydroxide gel.

[0035] Specifically, the core function of booster immunization (also known as enhanced booster immunization) is to maximize the activation of antigen-specific B lymphocytes, laying the foundation for subsequent screening of high-affinity antibodies. Simply put, after the animal's immune titer reaches the target level, a final high-dose, targeted antigen stimulation causes a large proliferation and differentiation of B cells capable of producing the target antibody into highly active plasma cell precursors, thereby improving the efficiency and quality of subsequent single B cell screening.

[0036] In some embodiments of the present invention, after separating the peripheral blood mononuclear cells in step S2, T lymphocytes and mononuclear cells are removed, and the antigen-specific B lymphocytes are screened using fluorescently labeled immunogens.

[0037] Specifically, the T lymphocytes and the monocytes are removed by secondary screening using at least one of the surface markers of T lymphocytes, namely CD4, CD8, CD14, CD28, and CD80.

[0038] Specifically, the method for screening the antigen-specific B lymphocytes includes: selecting activated B lymphocytes by labeling IgG with a fluorescent dye, and then performing positive screening by flow cytometry (FACS) using a screening reagent formed by incubating a biotin-labeled immunogen with avidin and fluorescein to obtain the antigen-specific B lymphocytes.

[0039] Specifically, the fluorescein is selected from at least one of PE, APC, PerCP, PE-Cy7, and APC-Cy7.

[0040] In some embodiments of the present invention, step S3 involves seeding the antigen-specific B lymphocytes into a cell culture plate containing feeder cells, adding cytokines for co-culture, and using ELISA to screen the antigen-specific B lymphocytes that secrete the target antibody.

[0041] Specifically, when inoculating the antigen-specific B lymphocytes, 1 to 2 cells per well are seeded into the cell culture plate.

[0042] Specifically, the cytokines are selected from at least one of IL-2, IL-4, IL-6, IL-10, TNF-α, TGF-β, and SAC.

[0043] Specifically, the co-culture conditions include: culturing at 36-38℃ and 5-6% CO2 for 9-11 days.

[0044] Specifically, during ELISA screening, an ELISA plate coated with p53 R248Q immunogen and reverse screening antigen is used to detect the culture supernatant of antigen-specific B lymphocytes. Only antigen-specific B lymphocytes that can bind to the p53 R248Q immunogen but not the reverse screening antigen are retained. The reverse screening antigen includes p53 R248W, p53 R248L, and p53WT (wild type). The amino acid sequence of p53 R248W is shown in SEQ ID NO:10:SCMGGMNWRPILTII, the amino acid sequence of p53 R248L is shown in SEQ ID NO:11:SCMGGMNLRPILTII, and the amino acid sequence of p53 WT is shown in SEQ ID NO:12:SCMGGMNRRPILTII.

[0045] Specifically, the purpose of selecting the anti-screening antigen is to ensure that the final antibody has extremely high specificity: (1) During the preparation of monoclonal antibodies, the B lymphocytes produced after animal immunization will secrete a variety of antibodies, including not only the target antibody, but also cross-reactive antibodies, such as antibodies that recognize the conserved sequence around the R248 site, or antibodies that simultaneously recognize other mutants of the R248 family. The logic of the above anti-screening is: use the R248Q immunogen to screen out all antibodies that can bind to the R248Q antigen (including the target antibody and cross-reactive antibodies); use antigens with similar structures but different mutation sites (p53 R248W, p53R248L) and wild-type antigen (p53 WT) to exclude those cross-reactive antibodies that can bind to these non-target antigens, and retain only the specific antibodies that bind only to R248Q and do not bind to other antigens. (2) R248Q, R248W, and R248L are all missense mutations of arginine at position 248 of the TP53 gene, and belong to different mutants at the same site. If the antibody only recognizes Q at position 248, it will bind to R248Q, but not to R248W or R248L, because the amino acid structure at position 248 of the latter two is different and cannot match the antigen binding site of the antibody; if the antibody recognizes the conserved sequence around position 248, it will bind to R248Q, R248W, and R248L simultaneously, and such antibodies will be excluded by the reverse screening step. This design ensures that the binding specificity of the finally obtained antibody depends entirely on the key feature that position 248 is Q, thereby achieving accurate identification of R248Q mutation. (3) In clinical testing scenarios, it is necessary to distinguish between R248Q mutant p53 in tumor cells and wild-type p53 in normal cells, so the antibody must meet the requirement of not binding to wild-type p53. If an antibody cross-reacts with wild-type p53, it can cause normal tissue to be stained as well, resulting in false positives and interfering with clinical judgment. By screening with wild-type p53, such cross-reactive antibodies can be excluded, ensuring that the antibody only recognizes mutant p53, which meets the specificity requirements of clinical testing.

[0046] Specifically, when using ELISA to detect culture supernatant, a result greater than 2.1 times the mean negative result is defined as positive; a negative result is serum from unimmunized experimental animals.

[0047] In some embodiments of the present invention, step S3 involves cloning the amplified heavy chain variable region gene and light chain variable region gene into a mammalian expression vector, transfecting cells, and expressing the target antibody; the molar mass ratio of the heavy chain variable region gene to the light chain variable region gene is 1:2~3.

[0048] In some embodiments of the present invention, the purification step S4 includes: collecting the supernatant of transfected and cultured cells, purifying it by Protein G affinity chromatography, and concentrating it by ultrafiltration centrifugation to obtain the monoclonal antibody against p53 R248Q.

[0049] In some embodiments of the present invention, step S4, after purification, further includes evaluating the antibody purity and polymer content by SDS-PAGE (under both non-reducing and reducing conditions) and high-performance liquid chromatography (SEC-HPLC); the target purity of the antibody is ≥95%, and the target content of the polymer is <5%.

[0050] According to a sixth aspect of the present invention, the application of the above-described anti-p53 R248Q monoclonal antibody in the preparation of a kit for detecting p53R248Q mutation is proposed.

[0051] In some embodiments of the present invention, the detection is performed using immunohistochemistry (IHC), and the sample being detected is a tumor tissue sample.

[0052] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved: 1. The monoclonal antibody prepared in this invention exhibits high specificity against the p53 R248Q mutation site, enabling precise screening for this specific mutation and addressing the core issue that general mutation detection cannot meet the needs of personalized diagnosis and treatment. This invention relies on an immunohistochemistry (IHC) technology platform, which offers significant adaptability. IHC testing is flexible in its sample type requirements, utilizing small biopsy specimens and cytological specimens, without requiring a tumor cell ratio of over 20%. The cost of single-indicator detection is only 1 / 10 to 1 / 20 of NGS, and it eliminates the need for additional large-scale equipment or a professional bioinformatics analysis team. The entire process from staining to interpretation can be completed in 4-6 hours, perfectly aligning with the treatment pace and resource capacity of primary healthcare institutions, thus promoting the widespread adoption of TP53 R248Q mutation detection. Furthermore, the antibody of this invention can rapidly determine whether a patient has the p53 R248Q mutation, providing crucial evidence for clinicians to develop personalized targeted therapy strategies, helping to improve progression-free survival (PFS) and overall survival (OS), and filling the technological gap in precision diagnosis and treatment for such patients.

[0053] 2. This invention employs a single B-cell sorting and direct antibody gene extraction technique, eliminating the cumbersome screening steps of traditional hybridoma technology. It directly obtains B-cell clones secreting the target antibody, shortening the preparation cycle. Through purification and ultrafiltration centrifugation concentration, the final antibody purity is >95%, polymer content <5%, and concentration not less than 1 mg / mL, meeting the high standards required for IHC detection.

[0054] 3. The antibody of this invention is applicable to various common clinical sample types, such as formalin-fixed paraffin-embedded tissues, small biopsy specimens, and cytological specimens. It is particularly useful in cases where tissue samples are scarce in patients with advanced lung cancer, allowing for efficient testing with limited samples and avoiding the pain and burden of repeated sampling for patients. The antibody of this invention specifically recognizes the p53R248Q mutant protein, making it suitable not only for clinical testing but also for basic research in tumor cell lines and animal models. It provides a reliable experimental tool for exploring the mechanisms of tumorigenesis, development, and drug resistance mediated by the p53R248Q mutation, thus promoting research progress in related fields. Attached Figure Description

[0055] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a diagram showing the results of IHC verification of paraffin-embedded lung adenocarcinoma samples using anti-p53 antibody in Example 2 of the present invention; where A~D represent P151H+R248W+F270V mutant LUAD; E~H represent R248W mutant LUAD; I~L represent TP53 wild-type LUAD; M~P represent C105V mutant LUAD; Q~T represent R248Q mutant LUAD; U~X represent R248Q mutant LUAD; the magnification is 200×. Detailed Implementation

[0056] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0057] This invention, based on antigenicity analysis of the common TP53 mutation site (R248Q) and its surrounding region, synthesizes specific peptides containing multiple TP53 mutation types as immunogens. Employing a single-B antibody approach, through steps including animal immunization, single-B cell flow cytometry sorting, antibody gene retrieval, small-scale transfection and expression in cell engineering, antibody performance evaluation, and antibody production and purification, a high-affinity, high-specificity monoclonal antibody against p53 R248Q is ultimately obtained. This antibody can be used to recognize tumor tissues with TP53 gene R248Q mutations, including lung adenocarcinoma tissue, using immunohistochemistry. The following examples illustrate this process in detail. Example 1

[0058] This embodiment prepared a monoclonal antibody against p53 R248Q. The specific process is as follows: (1) According to the protein sequence information published by Uniprot: P04637. TP53_HUMAN, p53 protein is a transcription factor consisting of 11 exons, 10 introns and 393 amino acid residues. The main mutation types of p53 include missense mutations, truncation mutations, in-frame mutations and splicing mutations.

[0059] For the Human TP53 R248Q point mutation, two different amino acid regions were selected as polypeptide immunogenic sequences. These two polypeptide immunogenic sequences are linear B-cell epitope peptides containing the mutation site at position 248 and approximately 15-20 amino acids flanking it. The amino acid sequences of the two polypeptide immunogenics are shown in SEQ ID NO:7: SCMGGMNQRPILTII and SEQ ID NO:8: CMGGMNQRPILTI. Simultaneously, to enhance immunogenicity, a recombinant protein (three copies of the same mutant epitope tandemly linked by the flexible linker GGSSG, with a 6×His tag introduced at the N-terminus or C-terminus) was also designed as an immunogenicity. After codon optimization of its nucleotide sequence, the optimized nucleic acid sequence was artificially synthesized and constructed into the pET28a vector, which was then transformed into *E. coli* BL21(DE3). Protein expression was induced for 6 h at 25°C using 0.5 mM IPTG. A recombinant protein immunogen with a purity of up to 90% was obtained after purification by nickel column affinity chromatography. Based on a structure- and surface-dominant epitope scheme, the amino acid sequence of this recombinant protein immunogen is shown in SEQ ID NO:9: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPGSGSGSCMGGMNQRPILTIITLEDGSGMGGMNQRPILTIITLEDGSGNSSCMGGMNQRPILTGSGSGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA. All the above peptides were conjugated using the protein carrier KLH.

[0060] (2) New Zealand white rabbits (healthy, 8-week-old females, specific pathogen-free, SPF grade) were immunized with two polypeptide immunogens and one recombinant protein immunogen from step (1). The mass ratio of the specific polypeptide immunogen to the recombinant protein immunogen was 1:1. During immunization, 100 μg of immunogen (total mass of polypeptide and recombinant protein immunogen) was thoroughly emulsified with 2 μg of GM-CSF2 (immune chaperone) and 100 μg of Freund's adjuvant, and then administered via subcutaneous injection at multiple sites over a period of 3 months. After immunization, blood was collected from the marginal ear vein of the rabbit using the p53 R248Q immunogen for antibody titer detection (using indirect ELISA). The results showed an antibody titer greater than 6 × 10⁻⁶. 4 The results were verified as qualified. New Zealand white rabbits were then immunized with the above-mentioned immunogen p53 R248Q. Seven days later, 10 mL of blood was collected via the ear artery, and total PBMCs were isolated using a commercial rabbit peripheral lymphocyte isolation kit. T lymphocytes and monocytes were removed by secondary screening using the surface markers CD4, CD8, and CD28 of T lymphocytes. Activated B lymphocytes were then selected using FITC-labeled goat anti-rabbit IgG. The immunogen p53 was then labeled using a commercial biotinylation kit according to the instructions, and conjugated with avidin-fluorescein PE reagent. The mixture was incubated at 37°C for 1 hour (with several agitations during incubation) to obtain the antigen-biotin-avidin screening reagent. The reagent was used according to the cell quantity (1×10⁻⁶). 6 Add 5 μL of the sample to the B lymphocytes and then perform positive screening by flow cytometry to obtain antigen-specific B lymphocytes.

[0061] (3) The sorted antigen-specific B lymphocytes were plated and cultured, and pre-treated mouse myeloma trophoblast cells (5×10⁶ cells / well) were added at a density of 1-2 cells / well. 4Cells were cultured in 96-well plates with IL-2 (50 U / mL), IL-4 (50 ng / mL), and IL-6 (100 ng / mL) cytokines added simultaneously, and co-cultured at 37°C and 5% CO2 for 10 days. Cell supernatants were collected and screened using ELISA plates coated with p53 R248Q immunogen and reverse screening antigens (R248W (amino acid sequence as shown in SEQ ID NO:10: SCMGGMNWRPILTII), R248L (amino acid sequence as shown in SEQ ID NO:11: SCMGGMNLRPILTII), and WT (amino acid sequence as shown in SEQ ID NO:12: SCMGGMNRRPILTII)). Serum from unimmunized New Zealand white rabbits was used as a negative control. Only antigen-specific B lymphocytes that could bind to the R248Q immunogen but not the reverse screening antigen were retained. A positive result was defined as a result greater than 2.1 times the negative mean. The presence of target antibodies in the cell culture supernatant was measured.

[0062] All cells from positive wells were collected and lysed. Total RNA was extracted and reverse transcribed into cDNA. The heavy chain variable region (VH) gene and light chain variable region (VL) gene of the target antibody were amplified by PCR. The PCR product was cloned into a T vector, and at least 10 single clones were selected for sequencing. The correct VH and VL genes were confirmed by sequence alignment. The amino acid sequence of the heavy chain variable region encoded by the VH gene is shown in SEQ ID NO:1: MGTGLFWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSGKAMSWVRQAPGKGLEWIGAIDGGSGSTWSANWAKGRFTISKTSTTVHLKITSPTTEDTATYFCAGGYNIWGPGTLVTVSL. The amino acid sequence of the light chain variable region encoded by the VL gene is shown in SEQ ID NO:1. NO:2: MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFMCGSVDCILFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (The clone number corresponding to this gene pair is 2G7).Alternatively, the amino acid sequence of the heavy chain variable region encoded by the VH gene is shown in SEQ ID NO:3: MGTGLYWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSGKAMSWVRQAPGKGLEWIGAIDGGSGSTWSANWAKGRFTISKTSTTVHLKITSPTTEDTATYFCAGGYNIWGPGTLVTVSL, and the amino acid sequence of the light chain variable region encoded by the VL gene is shown in SEQ ID NO:3. NO:4: MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFMCGSVDCILFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (The clone number corresponding to this gene pair is 5D12).Alternatively, the amino acid sequence of the heavy chain variable region encoded by the VH gene is shown in SEQ ID NO:5: METGLHWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSGKAMSWVRQAPGKGLEWIGAIDGGSGSTWSANWAKGRFTISKTSTTVHLKITSPTTEDTATYFCAGGYNIWGPGTLVTVSL, and the amino acid sequence of the light chain variable region encoded by the VL gene is shown in SEQ ID NO:5. IDNO:6: MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFMCGSVDCILFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (The clone number corresponding to this gene pair is 8G3). The VH and VL gene clones, verified by sequencing, were expressed in a mammalian expression vector using the pTT5 plasmid. Using polyethyleneimine (PEI) transfection, the high-concentration, high-purity antibody heavy and light chain recombinant plasmid obtained after expansion culture was transfected into HEK293F cells according to the PEI transfection reagent instructions. The antibody VH and VL genes were premixed at a molar ratio of 1:2. The plasmid was diluted using HEK293F cell basal medium, and an equal volume of PEI was diluted with the same medium (plasmid:PEI = 1:3, w / w). The mixture was transfected into HEK293F cells cultured in the logarithmic growth phase. Forty-eight hours after transfection, the antibody titer secreted in the supernatant was detected using an indirect ELISA method to identify cell lines with relatively high expression levels.

[0063] (4) The selected cell line was cultured to a volume of 100 mL, and the cell growth state was adjusted to the logarithmic growth phase. Plasmids were transfected, and feeding was performed every other day. The cell supernatant was harvested on day 6 post-transfection. The supernatant was filtered through a 0.22 μm filter and purified by affinity chromatography using a Protein G column. The antibodies obtained from the binding adsorption were eluted with citrate buffer at pH 6.0, and the eluent was collected. The pH was rapidly neutralized to between 7.2 and 7.4 using Tris-HCl solution at pH 8.8. After purification, the antibody purity and polymer content were evaluated by SDS-PAGE and SEC-HPLC. Antibodies with a purity >95% and a polymer content <5% were selected, concentrated by ultrafiltration and centrifugation to a concentration above 1 mg / mL, aliquoted, and stored at -80℃. Example 2

[0064] This embodiment describes the immunohistochemical (IHC) identification of the anti-p53 R248Q monoclonal antibody prepared in Example 1. The specific process is as follows: Seventeen surgical specimens of lung adenocarcinoma (LUAD) that were formalin-fixed paraffin-embedded (FFPE) were selected. Clinical information is shown in Table 1. All specimens underwent NGS testing to confirm the TP53 gene mutation status (including R248Q mutation) in the LUAD specimens, and the tumor cell content was >20% in all specimens.

[0065] Table 1 Case gender Age (years) Specimen source Clinical staging 1 male 58 upper lobe of the right lung T2N0M0 (Phase II) 2 female 85 left lower lobe T2N2M0 (Phase III) 3 female 55 upper lobe of the right lung T1N0M0 (Phase I) 4 male 58 upper lobe of the right lung T1N0M0 (Phase I) 5 male 59 upper lobe of the right lung T1N0M0 (Phase I) 6 male 70 upper lobe of the right lung T1cN0M0 (Phase I) 7 male 67 left upper lobe T1N1M0 (Phase I) 8 female 73 left lower lobe T1N0M0 (Phase I) 9 female 58 left upper lobe T1N2M0 (Phase III) 10 male 58 right lower lobe T2N0M0 (Phase II) 11 female 61 left upper lobe T1N0M0 (Phase I) 12 female 47 Right middle lobe T1N0M0 (Phase I) 13 male 59 upper lobe of the right lung T1N0M0 (Phase I) 14 male 69 Right middle lobe T1N0M1b (Stage IV) 15 male 67 4R group lymph nodes T3N1M1 (Stage IV) 16 male 58 left upper lobe T1N0M0 (Phase I) 17 female 56 left lower lobe T1N1M0 (Phase I) FFPE specimen sections were approximately 4 μm thick and stained using the EnVision two-step method. The control used was a commercially available p53 antibody (clone DO-7), purchased from Dako. All antibodies produced a brownish-yellow stain. Staining was performed using a fully automated immunohistochemical staining system. The interpretation criteria for p53 antibody (DO-7) were as follows: >80% of cell nuclei showed strong staining, indicating abnormal expression (overexpression) / mutant staining pattern (usually missense mutations or in-frame deletions in the TP53 gene); all negative staining indicated abnormal expression (complete deletion) / mutant staining pattern (usually nonsense, frameshift, splicing, or deletion / insertion mutations in the TP53 gene); the simultaneous presence of scattered or patchy nuclei with varying proportions and different staining intensities (negative, weakly positive, moderately positive, strongly positive) indicated normal expression / wild-type staining pattern. The interpretation criteria for preparing anti-p53 R248Q monoclonal antibodies were as follows: >80% of cell nuclei showed strong staining, indicating a positive result, i.e., the presence of an R248Q mutation in the TP53 gene; other staining patterns were considered negative, i.e., the absence of an R248Q mutation. The IHC test results of the above specimens are shown in Table 2 and... Figure 1 As shown.

[0066] Table 2 Case Exons Mutation type Abundance p53 R248Q antibody Control p53 antibody (DO-7) Control antibody staining pattern 1 exon5exon7exon8 p.P151Hp.R248Wp.F270V 2.55%21.15%1.10% All negative Strong, 90% mutant 2 exon7 p.R248W 49.65% Weak, 5% Strong, 70%; Medium, 30% Unable to assess 3 WT WT / All negative In China, 5% wild type 4 exon4 p.C105V 28.50% All negative Strong, 73%; Medium, 25%; Weak, 2% Unable to assess 5 exon10 p.G360A 48.64% All negative Weak, 30% wild type 6 exon5 p.R181P 35.39% Medium, 1%; Weak, 5% Strong, 95%; Medium, 5% mutant 7 exon8 p.R282W 45.41% Weak, 2% Strong, 80%; Medium, 15%; Weak, 5% mutant 8 exon8 p.V274fs32 26.51% All negative All negative mutant 9 exon5 p.K161E 16.43% Medium, 30%; Weak, 20% Strong, 95%; Medium, 5% mutant 10 exon6 p.Y220C 29.92% Medium, 5%; Weak, 10% Strong, 90%; Medium, 7%; Weak, 3% mutant 11 exon5 p.A159V 12.48% All negative Strong, 25%; Medium, 70%; Weak, 5% Unable to assess 12 exon8 p.D281E 1.00% Medium, 10%; Weak, 30% Strong, 98%; Medium, 2% mutant 13 exon5 p.C176F 10.58% Weak, 2% Strong, 93%; Medium, 6%; Weak, 1% mutant 14 exon4exon5 p.A69Tp.C176F 1.14%21.10% Weak, 2% Strong, 90%; Medium, 10% mutant 15 exon7 p.R248Q 40.66% Strong, >90% Strong, 100% mutant 16 exon7 p.R248Q 33.62% Strong, >90% Strong, 100% mutant 17 exon7 p.R248Q 14.82% Strong, >90% Strong, 100% mutant Table 2 and Figure 1 The results showed that the anti-p53 R238Q monoclonal antibody prepared in Example 1 included three clones: 2G7, 5D12, and 8G3, and their staining results were basically consistent. Specifically, the three patients with R248Q mutations (cases 15, 16, and 17) all showed strong positivity in >90% of their cell nuclei, and IHC was positive, consistent with the results of the control p53 antibody (DO-7) and NGS; the six patients (cases 1, 3, 4, 5, 8, and 11) showed all-negative staining (corresponding to...). Figure 1 (The results are as follows: 3 cases (i.e., cases 7, 13, and 14) showed 2% weak positive nuclei, 1 case (i.e., case 2) showed 5% weak positive nuclei, and the remaining 4 cases (i.e., cases 6, 9, 10, and 12) showed varying degrees of positive nuclei, but the intensity was moderate to weak staining, with no strong staining. The above IHC results indicated negative results and the absence of R248Q mutation, which is consistent with the NGS results.)

[0067] Figure 1 The staining results are as follows: A~D, P151H+R248W+F270V mutant LUAD, with staining results of negative, negative, negative, and approximately 90% strong positive, respectively; E~H, R248W mutant LUAD, with staining results of approximately 5% weakly positive, approximately 5% weakly positive, approximately 3% weakly positive, and approximately 60% moderately to strongly positive, respectively; I~L, TP53 wild-type LUAD, with staining results of negative, negative, negative, and approximately 20% weakly to moderately positive, respectively; M~P, C105 V-mutant LUAD, staining results were negative, negative, negative, and approximately 70% moderately to strongly positive; Q~T, R248Q mutant LUAD, staining results were approximately 70% moderately to strongly positive, approximately 50% moderately to strongly positive, approximately 50% moderately to strongly positive, and approximately 100% strongly positive; U~X, R248Q mutant LUAD, staining results were approximately 100% moderately to strongly positive, approximately 100% moderately to strongly positive, approximately 100% moderately to strongly positive, and approximately 100% strongly positive.

[0068] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A monoclonal antibody against p53 R248Q, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1: MGTGLFWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSGKAMSWVRQAPGKGLEWIGAIDGGSGSTWSANWAKGRFTISKTSTTVHLKITSPTTEDTATYFCAGGYNIWGPGTLVTVSL. NO:2:MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFMC GSVDCILFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC is shown; Alternatively, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:3: MGTGLYWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSGKAMSWVRQAPGKGLEWIGAIDGGSGSTWSANWAKGRFTISKTSTTVHLKITSPTTEDTATYFCAGGYNIWGPGTLVTVSL, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

3. NO:4:MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFMC GSVDCILFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC is shown; Alternatively, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:5: METGLHWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSLSGKAMSWVRQAPGKGLEWIGAIDGGSGSTWSANWAKGRFTISKTSTTVHLKITSPTTEDTATYFCAGGYNIWGPGTLVTVSL, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

5. NO:6:MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTINCQASQSVYNNKNLAWYQQKPGQPPKLLIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCQGEFMC GSVDCILFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the heavy chain variable region and the light chain variable region of the monoclonal antibody against p53 R248Q as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that, The recombinant cells comprise the recombinant expression vector of claim 3.

5. The method for preparing the monoclonal antibody against p53 R248Q according to claim 1, characterized in that, Includes the following steps: S1: Provides a specific polypeptide immunogen and a recombinant protein immunogen targeting the p53 R248Q mutation site, wherein the amino acid sequence of the specific polypeptide immunogen is shown in SEQ ID NO:7: SCMGGMNQRPILTII or SEQ ID NO:8: CMGGMNQRPILTI, and the amino acid sequence of the recombinant protein immunogen is shown in SEQ ID NO:9: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPGSGSGSCMGGMNQRPILTIITLEDGSGMGGMNQRPILTIITLEDGSGNSSCMGGMNQRPILTGSGSGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA; S2: Immunize experimental animals with the specific polypeptide immunogen prepared in step S1 and the recombinant protein immunogen. After immunization, collect peripheral blood from the experimental animals, separate peripheral blood mononuclear cells, and screen for antigen-specific B lymphocytes. S3: Culture the antigen-specific B lymphocytes; screen for antigen-specific B lymphocytes that secrete the target antibody and extract their RNA; amplify the heavy chain variable region gene and light chain variable region gene of the target antibody; and express the target antibody after cloning and transfection of the two genes. S4: Purify the target antibody to obtain the anti-p53 R248Q monoclonal antibody.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the specific polypeptide immunogen and the recombinant protein immunogen in step S2 is 1:1 to 1:3; Preferably, in step S2, an immune chaperone and adjuvant are added during the immunization process, and immunization is administered via subcutaneous multi-point injection. The immunization cycle is 2-3 months, and the serum antibody titer is detected after immunization. When the titer reaches 6×10⁻⁶, the antibody titer is determined. 4 The above-mentioned animals were given a shock immunization, and peripheral blood was collected from the experimental animals 7-8 days later. The total mass ratio of the specific polypeptide immunogen and the recombinant protein immunogen to the immune chaperone is 20:1 to 100:1, and the total mass ratio of the specific polypeptide immunogen and the recombinant protein immunogen to the adjuvant is 1:1 to 1:1.

5.

7. The preparation method according to claim 5, characterized in that, Step S3: Seed the antigen-specific B lymphocytes into a cell culture plate containing feeder cells, add cytokines for co-culture, and use ELISA to screen the antigen-specific B lymphocytes that secrete the target antibody; When using ELISA for screening, an ELISA plate coated with p53 R248Q immunogen and reverse screening antigen is used to detect the culture supernatant of antigen-specific B lymphocytes. Only antigen-specific B lymphocytes that can bind to the p53 R248Q immunogen but not the reverse screening antigen are retained. The reverse screening antigen includes p53 R248W, p53 R248L, and p53 WT. The amino acid sequence of p53 R248W is shown in SEQ ID NO:10:SCMGGMNWRPILTII, the amino acid sequence of p53 R248L is shown in SEQ ID NO:11:SCMGGMNLRPILTII, and the amino acid sequence of p53 WT is shown in SEQ ID NO:12:SCMGGMNRRPILTII.

8. The preparation method according to claim 5, characterized in that, Step S3 involves cloning the amplified heavy chain variable region gene and light chain variable region gene into a mammalian expression vector, transfecting cells, and expressing the target antibody; the molar mass ratio of the heavy chain variable region gene to the light chain variable region gene is 1:2~3.

9. The preparation method according to claim 5, characterized in that, The purification steps in step S4 include: collecting the cell supernatant after transfection and culture, purifying it by Protein G affinity chromatography, and concentrating it by ultrafiltration and centrifugation to obtain the anti-p53R248Q monoclonal antibody.

10. The use of the anti-p53 R248Q monoclonal antibody of claim 1 in the preparation of a kit for detecting p53 R248Q mutation.

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