Monoclonal antibody targeting ptgs1 protein stable neoantigen epitope and application thereof

Abstract

This invention belongs to the fields of biomedicine and immunology, specifically relating to a monoclonal antibody targeting a stable neoantigen epitope of prostaglandin intraperoxidase 1 (PTGS1) protein and its application. The stable neoantigen epitope is a protein region defined by the amino acid sequence shown in SEQ ID NO.1; the stable neoantigen epitope is located at positions 271-282 of the human PTGS1 protein. This epitope has an extremely low mutation rate in the human population and ovarian cancer tissues, thus ensuring the high specificity and high stability of the antibody. Experiments have demonstrated that the antibody prepared by this invention performs excellently in ovarian cancer IHC applications, clearly and significantly distinguishing cancerous tissue from normal tissue, providing pathologists with a reliable diagnostic basis.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and immunology, specifically relating to a monoclonal antibody that targets a stable neoantigenic epitope of prostaglandin intraperoxidase 1 (PTGS1) protein and its application. Background Technology

[0002] Prostaglandin intraperoxidase 1 (PTGS1), an important cyclooxygenase, has long been considered an enzyme involved in maintaining normal physiological functions. However, recent studies have increasingly revealed that PTGS1 is abnormally highly expressed in various solid tumors, including ovarian cancer and colorectal cancer. By promoting tumor cell proliferation, angiogenesis, and inducing chemotherapy resistance, it has become a highly promising tumor diagnostic biomarker and therapeutic target. Particularly in ovarian cancer, high PTGS1 expression is significantly associated with poor patient prognosis, highlighting its growing value in disease diagnosis.

[0003] Although the clinical diagnostic value of PTGS1 (prostaglandin intraperoxide synthase 1) protein is well-established, and its application potential in the diagnosis and treatment of tumors (such as ovarian cancer) is significant, current antibodies targeting PTGS1 protein face severe technical challenges in development and clinical application, preventing their clinical potential from being effectively translated into practical diagnostic tools. Analysis reveals that the core shortcomings of existing technologies stem from the blind and irrational selection of antigenic epitopes and the imperfections in antibody preparation technology systems, specifically manifested in the following three aspects:

[0004] (i) Blind selection of epitopes leads to defects in the core performance of antibodies.

[0005] In the current market, the vast majority of anti-PTGS1 monoclonal antibodies are prepared using traditional hybridoma technology, resulting in significant blind spots in immunogen selection. Currently, the complete PTGS1 protein or large fragments thereof are commonly used as immunogens, making it impossible to precisely control the specific epitopes targeted by the antibody, directly leading to two key performance issues:

[0006] ① High risk of cross-reactivity and insufficient specificity

[0007] PTGS1 is highly homologous to a certain region of its homologous protein PTGS2, and existing antibody preparations do not avoid this homologous region: for example, Boster Biotech uses the E318-L599 fragment of PTGS1 (product number: PB9002, product link https: / / boster.com / index / products / productsDetail?goods_sn=PB9002#cpjj);

[0008] ProteinTech selected the 250-599AA fragment of PTGS1 (product number: 67346-2-PBS, product link: https: / / www.ptgcn.com / products / PTGS1-Antibody-67346-2-PBS.htm) as an immunogen, without excluding homologous sequences with PTGS2. Such antibodies are prone to cross-reactivity with PTGS2, severely interfering with the specificity of clinical testing and leading to decreased diagnostic accuracy.

[0009] ② Epitope stability is ignored, resulting in poor detection reliability.

[0010] Tumors exhibit extensive heterogeneity (including gene mutations, transcript variations, etc.), and current antibody preparation strategies completely fail to consider the sequence stability of epitope regions within the tumor population. If the epitope targeted by the antibody belongs to a frequently mutated region in the tumor, problems such as large fluctuations in sensitivity, unstable test results, and detection escape (false negatives) will inevitably occur in clinical sample testing, rendering it unreliable as a tumor diagnostic tool.

[0011] (ii) Traditional preparation techniques exacerbate the uncertainty of application performance.

[0012] Traditional hybridoma technology has inherent defects, which further amplify the performance problems caused by improper epitope selection. These include: poor genetic stability of hybridoma cell lines, which are prone to antibody gene loss; long antibody preparation cycle and significant batch-to-batch differences; and even if antibodies with acceptable performance are obtained in the initial screening, it is difficult to achieve continuous and stable large-scale production, which makes it impossible to guarantee the reliability of their clinical application and meet the stringent requirements for reagent stability in tumor diagnosis.

[0013] (iii) The potential of single B cell technology has not been properly developed.

[0014] Single-cell B-cell antibody gene cloning technology, as a new generation of antibody preparation method, has significant advantages: it can directly clone antibody genes from antigen-specific B cells of immunized animals, fundamentally solving the genetic instability problem of hybridoma technology, and preserving the natural diversity of antibodies. However, the advantages of this technology have not yet translated into a performance breakthrough for PTGS1 antibodies. The core bottleneck lies in the irrationality of upstream immunization strategies: existing technologies still use traditional, undesigned immunogens (such as complete PTGS1 protein or random large fragments). Even with the single-cell B-cell technology platform, it can only achieve "faster and more stable antibody production," but cannot guarantee the rationality of the antibody's target epitope from the source—the final product is still an antibody with unknown performance, which cannot meet the demand for "high specificity and high stability" antibodies for the diagnosis of tumors such as ovarian cancer. Currently, the preparation of high-performance antibodies "targeting PTGS1 and specifically designed to overcome the diagnostic challenges of ovarian cancer" using single-cell B-cell technology remains a technological gap.

[0015] In summary, the core technological challenge currently facing the development of PTGS1 antibodies is the lack of a target screening strategy based on rational design and oriented towards clinical diagnostic applications. This field urgently needs a new technological solution that starts from the source (antigen epitope selection), using bioinformatics analysis and other methods to precisely locate "stable neoantigen epitopes" that meet the following criteria: high expression in tumor tissues (such as ovarian cancer tissue), highly conserved sequence (low mutation rate), adaptable to the detection of heterogeneous tumor samples, high distinguishability from homologous proteins (such as PTGS2), and avoidance of cross-reactivity. Guided by these "stable neoantigen epitopes," monoclonal antibodies with high specificity, high stability, and excellent diagnostic performance can be prepared, ultimately applied to the development of ovarian cancer diagnostic reagents, overcoming the performance deficiencies of existing antibodies. Summary of the Invention

[0016] (a) Technical problems to be solved

[0017] To address the problems of poor specificity, low stability, and unsatisfactory application performance of existing PTGS1 antibodies in the diagnosis of ovarian cancer, the main objective of this invention is to provide an antibody that targets a stable neoantigen epitope of the PTGS1 protein. This antibody has high specificity, high affinity, and excellent immunohistochemical diagnostic performance, and can be used to clearly and stably distinguish ovarian cancer tissue from normal tissue. It solves the problems of large sensitivity fluctuations, unstable detection results, and detection escape (false negatives) of existing antibodies used in the diagnosis of ovarian cancer.

[0018] (II) Technical Solution

[0019] In a first aspect, the present invention provides a monoclonal antibody targeting a stable neoantigen epitope of the PTGS1 protein, wherein the stable neoantigen epitope is a protein region defined by the amino acid sequence shown in SEQ ID NO.1; the stable neoantigen epitope is located at positions 271-282 of the human PTGS1 protein.

[0020] Preferably, the method for preparing the monoclonal antibody is as follows:

[0021] (1) The DNA sequence encoding the short peptide shown in SEQ ID NO.1 was cloned into a prokaryotic expression vector, transformed into Escherichia coli strain, induced to express, purified, and recombinant PTGS1 immunogenic protein was obtained. Animals were then immunized with the recombinant PTGS1 immunogenic protein.

[0022] (2) Lymphocytes were isolated from the spleen or peripheral blood of immunized animals and antigen-specific single B cells were sorted by flow cytometry.

[0023] (3) cDNA was synthesized by reverse transcription from the single B cell and the heavy and light chain variable region genes of the antibody were amplified by PCR;

[0024] (4) The amplified heavy chain and light chain variable region genes are cloned into the expression vector, transformed into host cells for expression, and purified to obtain the monoclonal antibody.

[0025] Preferably, the monoclonal antibody has three heavy chain complementarity-determining regions as shown in the amino acid sequences of SEQ ID NO.2-SEQ ID NO.4, and three light chain complementarity-determining regions as shown in the amino acid sequences of SEQ ID NO.5-SEQ ID NO.7.

[0026] Preferably, the monoclonal antibody has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO.8 and a light chain variable region of the amino acid sequence shown in SEQ ID NO.9.

[0027] Preferably, the monoclonal antibody has a heavy chain of the amino acid sequence shown in SEQ ID NO. 10 and a light chain of the amino acid sequence shown in SEQ ID NO. 11.

[0028] Secondly, the present invention provides the application of the monoclonal antibody in the detection of PTGS1 protein.

[0029] Thirdly, the present invention provides a DNA sequence encoding the above-mentioned monoclonal antibody and the use of the DNA sequence in the preparation of the monoclonal antibody.

[0030] Preferably, the DNA sequence encoding the monoclonal antibody contains genes encoding the heavy and light chains of the monoclonal antibody.

[0031] Preferably, the method for preparing the monoclonal antibody is as follows: the coding gene of the heavy chain variable region and the coding gene of the light chain variable region of the monoclonal antibody are co-transfected into host cells using a dual-vector co-transfection system or a single-vector dual-expression system for expression, and then purified to obtain the monoclonal antibody.

[0032] Fourthly, the present invention provides a kit for detecting human PTGS1 protein, which comprises the above-mentioned monoclonal antibody targeting the stable neoantigen epitope of PTGS1 protein.

[0033] Preferably, the kit is an ovarian cancer diagnostic kit.

[0034] Preferably, the diagnostic kit is an immunohistochemical detection kit or an immunoblotting detection kit.

[0035] Preferably, the immunohistochemical detection kit further includes a secondary antibody, chromogenic substrate, buffer solution, and counterstain required for immunohistochemical detection.

[0036] (III) Beneficial Effects

[0037] Compared with the prior art, the present invention has the following significant advantages:

[0038] (1) This invention first proposed and practiced a rational design strategy for “stable neoantigen epitopes” targeting PTGS1, and finally screened out neoantigen epitopes with extremely low mutation rates in the human population and ovarian cancer tissues (located at positions 271-282 of PTGS1). Therefore, the antibody obtained was designed from the beginning to ensure its high specificity and high stability, solving the fundamental defects of existing products.

[0039] (2) The antibody prepared by this invention has excellent performance in the IHC application of ovarian cancer. It can clearly and significantly distinguish cancerous tissue from normal tissue, providing pathologists with a reliable diagnostic basis and showing great potential for clinical application.

[0040] (3) This invention uses a new antigenic epitope with an extremely low mutation rate as an immunogen, combined with single B cell technology, which avoids the instability of traditional hybridoma technology, has a shorter preparation cycle, and can permanently preserve antibody genes, thus ensuring the uniformity of antibody quality and sustainable production. Attached Figure Description

[0041] Figure 1 The expression levels of the PTGS1 transcript (PTGS1-001) in ovarian cancer tissue and normal ovarian tissue were measured using RNA-seq technology.

[0042] Figure 2 The expression levels of the PTGS1 transcript (PTGS1-002) in ovarian cancer tissue and normal ovarian tissue were measured using RNA-seq technology.

[0043] Figure 3 The expression levels of the PTGS1 transcript (PTGS1-001) in ovarian cancer tissues and normal ovarian tissues were determined using the GEPIA 2.0 database.

[0044] Figure 4 The expression levels of the PTGS1 transcript (PTGS1-002) in ovarian cancer tissues and normal ovarian tissues were determined using the GEPIA 2.0 database.

[0045] Figure 5 To detect the expression of PTGS1 protein using the monoclonal antibody provided in this invention, Western blot was used; lane 1 is the marker; lane 2 is the detection of PTGS1 expression by 10-fold dilution of total protein extracted from SKOV3 cells.

[0046] Figure 6The immunohistochemical kit constructed using the monoclonal antibody of this invention was used to detect the staining of PTGS1 in paraffin sections of normal human ovarian tissue (negative control, 1-5#) and ovarian cancer tissue (6-10#).

[0047] Figure 7 for Figure 6 Mean optical density of normal ovarian tissue and ovarian cancer tissue after staining. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods or procedures performed according to the kit instructions.

[0049] The core principle of this invention lies in ensuring the superior performance of the obtained antibodies from the source through rational design of immunogens. Traditional antibody preparation strategies neglect the stability of the antigenic epitope itself, resulting in poor antibody performance when facing tumor heterogeneity and homologous proteins. This invention, through analysis of public genomic and transcriptomic databases, precisely screens a PTGS1-specific sequence that is highly expressed in ovarian cancer and has an extremely low mutation rate in the general population as a "stable neoantigenic epitope." The stable neoantigenic epitope is located at positions 271-282 of the PTGS1 protein, and its sequence is shown in SEQ ID NO.1. Furthermore, this invention uses this antigenic epitope sequence as an immunogen and utilizes highly efficient single-B cell antibody gene cloning technology to directly and rapidly screen for natural antibodies targeting this ideal epitope. The antibodies obtained in this way possess high specificity and high stability that are endogenous; at the same time, their low mutation rate ensures the stability and reliability of detection results in a large patient population, ensuring diagnostic accuracy. Experiments have shown that the antibody of this invention exhibits excellent staining effects in IHC detection of paraffin-embedded ovarian cancer tissue, characterized by low background, high signal-to-noise ratio, and clear contrast between cancerous and normal tissues.

[0050] The following detailed description is provided in conjunction with specific examples. Example 1

[0051] This embodiment relates to the screening and immunogen design of stable neoantigen epitopes of PTGS1. The antigen screening and immunogen design methods are as follows:

[0052] (1) Sequence conservation and mutation rate analysis

[0053] Mutation spectrum analysis of all coding exons of the PTGS1 gene revealed that amino acids 271-282 remained unchanged in more than 99% of ovarian cancer samples, with a population mutation frequency (according to the gnomAD database) of less than 0.1%, indicating that this region has extremely high sequence stability.

[0054] (2) Transcript expression analysis

[0055] RNA-Seq comparison of ovarian cancer tissue and normal tissue (3 cases each) confirmed that this stable region is conserved in all highly expressed PTGS1 transcripts PTGS1-001 (ENST00000223423, see sequence SEQ ID NO.17) and PTGS1-002 (ENST00000362012, see sequence SEQ ID NO.18). Figure 1 and Figure 2 ), and large-scale patient validation was conducted using the GEPIA 2.0 database, confirming that PTGS1 transcripts (PTGS1-001 and PTGS1-002) are both highly expressed in ovarian cancer. Figure 3 and Figure 4 This ensures that the obtained antibodies can recognize the PTGS1 protein, which is highly expressed in the vast majority of ovarian cancers. For example... Figure 1-4 As shown, the stable neoantigen epitopes screened in this invention have extremely low mutation rates in the human population and ovarian cancer tissues.

[0056] (3) Determination and preparation of immunogen sequences

[0057] Based on the above analysis, this invention selects amino acid sequence positions 271-282 of the human PTGS1 protein (its amino acid sequence is shown in SEQ ID NO.1) as the immunogen. This sequence contains the "stable neoantigen epitope" as defined in this invention.

[0058] Finally, the DNA sequence encoding the fragment (nucleotide sequence as shown in SEQ ID NO.12) was synthesized and cloned into the pGEX-6P-1 prokaryotic expression vector, transformed into Escherichia coli BL21(DE3) strain, and the GST tag fusion protein was expressed by IPTG induction and purified by GST affinity chromatography column to obtain high-purity recombinant PTGS1 immunogen protein. Example 2

[0059] This embodiment utilizes the aforementioned stable neoantigen epitopes and combines them with single-B cell technology to prepare anti-PTGS1 monoclonal antibodies. The specific method is as follows:

[0060] (1) Animal immunization

[0061] The recombinant PTGS1 protein (GST tag removed) prepared in Example 1 above was used as the immunogen. Initial immunization: 100 μg of immunogen was thoroughly emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple sites into 6-8 week old BALB / c mice. Booster immunizations were performed every 2 weeks using the same dose of immunogen emulsified with incomplete Freund's adjuvant, for a total of 3 immunizations. On day 3 after the final immunization, blood was collected via the tail vein, and serum titers were determined by indirect ELISA. Mice with the highest titers were selected for subsequent experiments.

[0062] (2) Antigen-specific B cell sorting

[0063] Mice with the highest titers were euthanized, and spleens were aseptically harvested to prepare single-cell suspensions. Spleen cells were co-incubated with biotin-labeled PTGS1 immunogen (amino acid sequence shown in SEQ ID NO.1) and fluorescein-labeled streptavidin to label antigen-specific B cells. Single antigen-positive (PE) cells were sorted by flow cytometry. + ), and are living cells (DAPI) - B cells were directly sorted into 96-well PCR plates pre-added with cell lysis buffer and RNase inhibitor, and immediately stored at -80°C or directly reverse transcribed.

[0064] (3) Antibody gene amplification and cloning

[0065] mRNA from a single B cell was reverse transcribed into cDNA using a commercially available single-cell RNA extraction and reverse transcription kit. Using this cDNA as a template, nested PCR or multiplex PCR was employed to amplify the coding genes for the variable regions of the antibody's heavy chain (VH) and light chain (VL).

[0066] The primer mixture for amplifying the gene encoding the variable region of the mouse antibody heavy chain contains:

[0067] Forward primer, MuIgGVH-F, see the nucleotide sequence shown in SEQ ID NO.13;

[0068] The reverse primer, MuIgGVH-R, is shown in the nucleotide sequence of SEQ ID NO.14.

[0069] The primer mixture for amplifying the variable region gene of the mouse antibody light chain VL (κ chain) contains:

[0070] Forward primer, MuIgKVL-F, see the nucleotide sequence shown in SEQ ID NO.15;

[0071] The reverse primer, MuIgKVL-R, is shown in the nucleotide sequence of SEQ ID NO.16.

[0072] After purification, the PCR products are cloned into mammalian dual expression vectors (such as pTT5 vector) containing the constant regions of mouse IgG1 heavy chain and κ light chain, respectively, by restriction endonuclease method or homologous recombination method (such as Gibson Assembly), to construct expression plasmids for heavy chain and light chain.

[0073] (4) Antibody expression and purification

[0074] The correctly sequenced heavy and light chain plasmids were co-transfected into CHO cells in suspension culture. 72 hours post-transfection, the cell culture supernatant was collected. Antibodies in the supernatant were captured and purified using a Protein A affinity chromatography column. Antibodies were eluted with glycine-HCl buffer (pH 2.5) and immediately neutralized with Tris-HCl buffer (pH 8.0). Finally, the antibody concentration was determined by ultrafiltration to replace the buffer in PBS, aliquoted, and stored at -80°C.

[0075] (5) Antibody sequence identification:

[0076] Sequencing of the plasmids from positive clones yielded a representative antibody of this invention. The amino acid sequence of its heavy chain variable region (VH) is shown in SEQ ID NO. 8, containing three heavy chain complementarity-determining regions: CDR1 (SEQ ID NO. 2), CDR2 (SEQ ID NO. 3), and CDR3 (SEQ ID NO. 4). The amino acid sequence of its light chain variable region (VL) is shown in SEQ ID NO. 9, containing three light chain complementarity-determining regions: CDR4 (SEQ ID NO. 5), CDR5 (SEQ ID NO. 6), and CDR6 (SEQ ID NO. 7). The heavy chain amino acid sequence of a specific monoclonal antibody is shown in SEQ ID NO. 10, and the light chain amino acid sequence is shown in SEQ ID NO. 11.

[0077] The SEQ ID NO.1-SEQ ID NO.16 mentioned above are shown in Table 1.

[0078] Example 3

[0079] This embodiment further verifies the performance of the anti-PTGS1 monoclonal antibody prepared in Example 2 using Western blotting. The experimental method is as follows:

[0080] Total protein was extracted from the human ovarian cancer cell line (OVCAR3). SDS-PAGE electrophoresis was performed, and the sample was transferred to a membrane. The membrane was then incubated with the antibody PTGS1 (1.9 μg / mL) of this invention. Results are as follows: Figure 5As shown, a specific band (approximately 70 kDa) can be detected in SKOV-3 cells. This specific band represents the PTGS1 protein recognized by the anti-PTGS1 monoclonal antibody. Example 4

[0081] In this embodiment, the anti-PTGS1 monoclonal antibody prepared in Example 2 was used for an immunohistochemical (IHC) experiment in ovarian cancer. The experimental method is as follows:

[0082] (1) Preparation of tissue samples: Five ovarian cancer tissue samples and five normal tissue samples were used. All samples were formalin-fixed paraffin-embedded (FFPE) tissues.

[0083] (2) Sectioning and dewaxing: The tissue microarray was sectioned to a thickness of 4 μm and baked in an oven at 60 °C for 2 hours. Then it was dewaxed with xylene and hydrated with graded ethanol.

[0084] (3) Antigen retrieval: The slides were placed in EDTA buffer at pH 6.0 and thermally induced epitope retrieval was performed in a microwave oven.

[0085] (4) Endogenous enzyme blockade: Add 3% H2O2 solution and incubate at room temperature for 10 minutes to block endogenous peroxidase activity.

[0086] (5) Blocking: Add 5% BSA in PBS solution and block at room temperature for 30 minutes.

[0087] (6) Primary antibody incubation: Add the antibody of the present invention (working concentration 1.9 μg / mL, diluted 1:200 with antibody dilution buffer) and incubate overnight at 4°C. The full length of the heavy chain of the antibody is as shown in SEQ ID NO.10, and the full length of the heavy and light chains is as shown in SEQ ID NO.11.

[0088] (7) Secondary antibody incubation: After washing with PBS, add HRP-labeled goat anti-rabbit IgG secondary antibody and incubate at room temperature for 30 minutes.

[0089] (8) Color development and counterstaining: DAB color development kit was used for color development, and hematoxylin was used for counterstaining of cell nuclei.

[0090] (9) Dehydration, clearing and mounting: The slides were dehydrated by gradient ethanol, cleared by xylene, mounted with neutral resin, and analyzed under an optical microscope.

[0091] like Figure 6The image shows the staining results observed under a microscope. The right side corresponds to the ovarian tissue area, and the left side corresponds to the normal ovarian tissue area. In the five ovarian cancer samples on the right, the antibody showed strong and specific cytoplasmic brownish-yellow staining. In contrast, the five paired normal ovarian tissue samples on the left showed only weak background staining or no staining at all. Furthermore, quantitative analysis of the microscopic images of the stained sections using ImageJ software directly correlated with the degree of absorption of specific wavelengths of light by the samples, as shown in the results. Figure 7 As shown, the average optical density of the five normal samples was approximately 0.2, while the average optical density of the five ovarian cancer samples was approximately 0.9. This demonstrates that the antibody prepared in this invention performs excellently in IHC applications for ovarian cancer, clearly and significantly distinguishing cancerous tissue from normal tissue, providing pathologists with a reliable diagnostic basis.

[0092] In summary, this invention, through rational design, screened a stable neoantigen epitope of PTGS1 and successfully prepared a highly specific, high-affinity monoclonal antibody using advanced single-cell B-cell technology. This antibody exhibits excellent performance in IHC diagnosis of ovarian cancer, significantly and clearly distinguishing cancerous tissue from normal tissue with low background and high signal-to-noise ratio, providing a novel and reliable detection tool for the pathological diagnosis of ovarian cancer.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody targeting a stable neoantigen epitope of the PTGS1 protein, characterized in that, The stable neoantigen epitope is a protein region defined by the amino acid sequence shown in SEQ ID NO.1; the stable neoantigen epitope is located at positions 271-282 of the human PTGS1 protein; the monoclonal antibody has heavy chain complementarity-determining regions 1-3 as shown in the amino acid sequences shown in SEQ ID NO.2-SEQ ID NO.4, and light chain complementarity-determining regions 1-3 as shown in the amino acid sequences shown in SEQ ID NO.5-SEQ ID NO.7, respectively.

2. The monoclonal antibody according to 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.8, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

9.

3. The monoclonal antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.10, and the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.

11.

4. The use of the monoclonal antibody according to any one of claims 1-3 in the preparation of a kit for detecting PTGS1 protein.

5. The encoding DNA of the monoclonal antibody according to any one of claims 1-3.

6. The encoding DNA of the monoclonal antibody according to claim 5, characterized in that, The coding DNA encoding the monoclonal antibody contains genes encoding the heavy and light chains of the monoclonal antibody; the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.8, and the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown in SEQ ID NO.

9.

7. The method for preparing the monoclonal antibody according to any one of claims 1-3, characterized in that, The method for preparing the monoclonal antibody is as follows: the coding gene of the heavy chain variable region shown in SEQ ID NO.8 and the coding gene of the light chain variable region shown in SEQ ID NO.9 are co-transfected into host cells using a dual-vector co-transfection system or a single-vector dual-expression system for expression, and then purified to obtain the monoclonal antibody.

8. A kit for detecting human PTGS1 protein, characterized in that, It comprises a monoclonal antibody targeting a stable neoantigen epitope of the PTGS1 protein as described in any one of claims 1-3, and the kit is an immunohistochemical detection kit or an immunoblotting detection kit.

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