Monoclonal antibody targeting PTGS1 protein stable neoepitope and application thereof

Abstract

The invention belongs to the field of biological medicine and immunology, and particularly relates to a monoclonal antibody of a stable neoepitope of a targeted prostaglandin endoperoxide synthase 1 (PTGS1) protein and application of the monoclonal antibody. Wherein the stable neoantigen epitope is a protein region defined by an amino acid sequence as shown in SEQ ID NO. 1; the stable neoantigen epitope is located at the 271 site to the 282 site of the human PTGS1 protein. The epitope has extremely low mutation rate in people and ovarian cancer tissues, so that high specificity and high stability of the antibody are ensured. Experiments prove that the antibody prepared by the invention has excellent performance in ovarian cancer IHC application, can clearly and remarkably distinguish cancer tissues from normal tissues, and provides a reliable diagnosis basis for pathologists.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine and immunology, and particularly relates to a stable neoantigen epitope monoclonal antibody targeting prostaglandin endoperoxidase 1 (PTGS1) protein and application thereof. BACKGROUND

[0002] As an important cyclooxygenase, prostaglandin endoperoxidase 1 (PTGS1) has long been considered as an enzyme involved in maintaining the normal physiological functions of the body. However, in recent years, more and more studies have revealed that PTGS1 presents abnormally high expression in various solid tumors such as ovarian cancer, colorectal cancer, etc., and it becomes a highly potential tumor diagnostic marker and therapeutic target by promoting tumor cell proliferation, angiogenesis and inducing chemotherapy resistance. In particular, in ovarian cancer, the high expression of PTGS1 is significantly associated with the poor prognosis of patients, which makes its application value in disease diagnosis increasingly prominent.

[0003] Although the clinical diagnostic value of PTGS1 (prostaglandin endoperoxidase 1) protein has been clear, its application potential in tumor (such as ovarian cancer) diagnosis and treatment is significant. However, the antibodies against PTGS1 protein currently face severe technical challenges in the development and clinical application process, which leads to the fact that its clinical potential cannot be effectively converted into a practical diagnostic tool. Through analysis, the core defects of the existing technology are rooted in the blindness and irrationality of antigen epitope selection, and the imperfection of antibody preparation technology system, which specifically embodies the following three problems: (1) Blindness of epitope selection leads to core performance defects of antibodies In the existing market, most of the anti-PTGS1 monoclonal antibodies rely on traditional hybridoma technology for preparation, and the selection of their immunogens has significant blindness. At present, the complete PTGS1 protein or its large fragments are generally used as immunogens, which cannot precisely control the specific epitope targeted by the antibodies, directly causing two key performance problems: ① High risk of cross-reaction and insufficient specificity PTGS1 and its homologous protein PTGS2 have highly homologous sequences in some regions, and the existing antibody preparation does not avoid this homologous segment: for example, Boster Biological selects the E318-L599 fragment of PTGS1 (product number: PB9002, product link https: / / boster.com / index / products / productsDetail?goods_sn=PB9002#cpjj); 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.

[0004] ② Epitope stability is ignored, resulting in poor detection reliability. 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.

[0005] (ii) Traditional preparation techniques exacerbate the uncertainty of application performance. 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.

[0006] (iii) The potential of single B cell technology has not been properly developed. 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.

[0007] In summary, the core technical dilemma faced by the current PTGS1 antibody development field is the lack of a target screening strategy based on rational design and oriented towards clinical diagnostic applications. There is an urgent need for a new technical solution that starts from the source (antigen epitope selection) and uses bioinformatics analysis to precisely locate a "stable neo-antigen epitope" that meets the conditions of "high expression in tumor tissue (such as ovarian cancer tissue), high sequence conservation (low mutation rate), suitable for detecting tumor heterogeneity, high discrimination from homologous proteins (such as PTGS2), and avoidance of cross-reactions." Then, using this "stable neo-antigen epitope" as a guide, a monoclonal antibody with high specificity, high stability, and excellent diagnostic performance is prepared for the development of an ovarian cancer diagnostic reagent, ultimately solving the performance deficiencies of existing antibodies. SUMMARY

[0008] (I) Technical problems to be solved In view of the poor specificity, low stability, and poor application performance of existing PTGS1 antibodies in the diagnosis of ovarian cancer, the main purpose of the present application is to provide an antibody targeting a stable neo-antigen epitope of PTGS1 protein, which has high specificity, high affinity, and excellent immunohistochemical diagnostic performance, and can clearly and stably distinguish between ovarian cancer tissue and normal tissue. It solves the problems of large sensitivity fluctuations, unstable test results, and test escape (false negatives) in the use of existing antibodies for the diagnosis of ovarian cancer.

[0009] (II) Technical solutions In a first aspect, the present application provides a monoclonal antibody targeting a stable neo-antigen epitope of PTGS1 protein, wherein the stable neo-antigen epitope is a protein region defined by the amino acid sequence of SEQ ID NO. 1; and the stable neo-antigen epitope is located at positions 271-282 of human PTGS1 protein.

[0010] Preferably, the preparation method of the monoclonal antibody is as follows: (1) The DNA sequence encoding the short peptide shown in SEQ ID NO. 1 is cloned into a prokaryotic expression vector, transformed into an E. coli strain, induced for expression, purified, and a recombinant PTGS1 immunogen protein is obtained, which is used to immunize animals; (2) Lymphocytes are isolated from the spleen or peripheral blood of the immunized animals, and antigen-specific single B cells are sorted by flow cytometry; (3) cDNA is synthesized by reverse transcription from the single B cells, and the heavy chain and light chain variable region genes of the antibody are amplified by PCR; (4) The amplified heavy chain and light chain variable region genes are cloned into an expression vector, transformed into host cells for expression, and the monoclonal antibody is purified.

[0011] Preferably, the monoclonal antibody has three heavy chain complementarity determining regions with the amino acid sequences shown in SEQ ID NO. 2-SEQ ID NO. 4, and three light chain complementarity determining regions with the amino acid sequences shown in SEQ ID NO. 5-SEQ ID NO. 7.

[0012] Preferably, the monoclonal antibody has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO. 8, and a light chain variable region with the amino acid sequence shown in SEQ ID NO. 9.

[0013] Preferably, the monoclonal antibody has a heavy chain with the amino acid sequence shown in SEQ ID NO. 10, and a light chain with the amino acid sequence shown in SEQ ID NO. 11.

[0014] In a second aspect, the present application provides use of the monoclonal antibody in detecting PTGS1 protein.

[0015] In a third aspect, the present application provides a DNA sequence encoding the above monoclonal antibody and use of the DNA sequence in preparing the monoclonal antibody.

[0016] Preferably, the DNA sequence encoding the above monoclonal antibody comprises genes encoding the heavy chain and the light chain of the monoclonal antibody.

[0017] Preferably, the preparation method of the monoclonal antibody is as follows: the encoding gene of the heavy chain variable region and the encoding gene of the light chain variable region of the monoclonal antibody are co-transfected into a host cell by using a double-vector co-transfection system or a single-vector double-expression system for expression, and the monoclonal antibody is obtained by purification.

[0018] In a fourth aspect, the present application provides a kit for detecting human PTGS1 protein, which comprises the above monoclonal antibody targeting the stable neo-epitope of PTGS1 protein.

[0019] Preferably, the kit is an ovarian cancer diagnosis kit.

[0020] Preferably, the diagnosis kit is an immunohistochemical detection kit or an immunoblotting detection kit.

[0021] Preferably, the immunohistochemical detection kit further comprises a secondary antibody, a chromogenic substrate, a buffer and a counterstain required for immunohistochemical detection.

[0022] (Three) beneficial effects Compared with the prior art, the present application has the following remarkable advantages: (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.

[0023] (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.

[0024] (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

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Figure 5 To detect the expression of PTGS1 protein using the monoclonal antibody provided in this invention, Western blot was used; lane 1 was the marker; lane 2 was the detection of PTGS1 expression by 10-fold dilution of total protein extracted from SKOV3 cells.

[0030] Figure 6 The 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#).

[0031] Figure 7 forFigure 6 Average optical density after staining of normal ovarian tissue and ovarian cancer tissue. DETAILED DESCRIPTION

[0032] In order to make the purposes, technical solutions and advantages of the present application more clear, the present application is further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. The reagents not described in detail in the present application are conventional reagents and can be obtained from commercial channels; the methods not described in detail are conventional experimental methods or operated according to the kit instructions.

[0033] The core principle of the present application is to ensure the excellent performance of the obtained antibody from the source by rationally designing the immunogen. The traditional antibody preparation strategy ignores the stability of the antigen epitope itself, resulting in poor performance of the antibody when facing tumor heterogeneity and homologous proteins. The present application precisely screens a PTGS1 specific sequence with high expression in ovarian cancer and extremely low mutation rate in the population as a "stable neo-antigen epitope" by analyzing public genome and transcriptome databases. The stable neo-antigen epitope is located at position 271-282 of PTGS1 protein and the sequence is shown as SEQ ID NO. 1. Further, the present application uses the antigen epitope sequence as an immunogen, and uses the efficient single B cell antibody gene cloning technology, which can directly and quickly screen the natural antibody targeting the ideal epitope. The high specificity and high stability of the antibody obtained are endogenous; at the same time, its low mutation rate ensures the stability and reliability of the detection results in a large number of patient groups, ensuring the accuracy of the diagnosis. Experiments prove that the antibody of the present application can present excellent staining effects with low background, high signal-to-noise ratio, and clear contrast between cancer tissue and normal tissue in IHC detection of ovarian cancer paraffin-embedded tissues.

[0034] The following is described in detail with examples. Example 1

[0035] This example relates to the screening of a PTGS1 stable neo-antigen epitope and the design of an immunogen, and the antigen screening and immunogen design methods are as follows: (1) Sequence conservation and mutation rate analysis Through mutation spectrum analysis of all coding exons of PTGS1 gene, it is found that the amino acids at positions 271-282 remain unchanged in more than 99% of ovarian cancer samples, and the mutation frequency in the population (according to gnomAD database) is less than 0.1%, indicating that this region has extremely high sequence stability.

[0036] (2) Transcript expression analysis By RNA-Seq comparison of ovarian cancer tissues and normal tissues (3 cases each), it is confirmed that the 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 verification is performed using the GEPIA2.0 database, confirming that the PTGS1 transcripts (PTGS1-001 and PTGS1-002) are highly expressed in ovarian cancer Figure 3 and Figure 4 ), ensuring that the obtained antibody can recognize the highly expressed PTGS1 protein in most ovarian cancers. As shown in Figures 1-4 , the stable neoantigen epitope screened by the present application has a very low mutation rate in the human population and ovarian cancer tissues.

[0037] (3) Determination and preparation of immunogen sequence Based on the above analysis, the present application selects the 271-282 amino acid sequence of human PTGS1 protein (the amino acid sequence is shown as SEQ ID NO. 1) as the immunogen. The sequence contains the "stable neoantigen epitope" defined by the present application.

[0038] Finally, the DNA sequence encoding the fragment (the nucleotide sequence is shown as SEQ ID NO. 12) is synthesized and cloned into the pGEX-6P-1 prokaryotic expression vector, transformed into the E. coli BL21 (DE3) strain, and the GST-tagged fusion protein is expressed using IPTG induction and purified by GST affinity chromatography column to obtain high-purity recombinant PTGS1 immunogen protein. Example 2

[0039] This embodiment uses the above stable neoantigen epitope and combines single B cell technology to prepare anti-PTGS1 monoclonal antibodies, and the specific method is as follows: (1) Animal immunization The recombinant PTGS1 protein (GST tag has been removed) prepared in the above Example 1 is used as the immunogen. First immunization: 100 μg of immunogen is emulsified with an equal amount of complete Freund's adjuvant and then subcutaneously injected into 6-8 week old BALB / c mice in multiple points. Every 2 weeks, the same dose of immunogen is emulsified with incomplete Freund's adjuvant for booster immunization, a total of 3 times. Three days after the last immunization, blood is collected from the tail vein, and the serum titer is determined by indirect ELISA, and the mouse with the highest titer is selected for subsequent experiments.

[0040] (2) Antigen-specific B cell sorting The mouse with the highest titer was sacrificed, and the spleen was aseptically removed to prepare a single cell suspension. The splenocytes were co-incubated with biotin-labeled PTGS1 immunogen protein (amino acid sequence shown in SEQ ID NO. 1) and fluorescein-labeled streptavidin to label antigen-specific B cells. Single antigen-positive (PE + ) and live cells (DAPI - ) were sorted by flow cytometry and directly sorted into a 96-well PCR plate pre-added with cell lysis solution and RNAase inhibitor, immediately stored at -80°C or directly subjected to reverse transcription.

[0041] (3) Antibody gene amplification and cloning The mRNA in the single B cell was reverse transcribed into cDNA using a commercial single cell RNA extraction and reverse transcription kit. The cDNA was used as a template to amplify the encoding genes of the heavy chain (VH) and light chain (VL) variable regions of the antibody by nest PCR or multiplex PCR, respectively.

[0042] The primer mixture for amplifying the encoding gene of the heavy chain variable region of the mouse antibody comprises: Forward primer, MuIgGVH-F, see the nucleotide sequence shown in SEQ ID NO. 13; Reverse primer, MuIgGVH-R, see the nucleotide sequence shown in SEQ ID NO. 14.

[0043] The primer mixture for amplifying the VL (κ chain) variable region gene of the mouse antibody comprises: Forward primer, MuIgKVL-F, see the nucleotide sequence shown in SEQ ID NO. 15; Reverse primer, MuIgKVL-R, see the nucleotide sequence shown in SEQ ID NO. 16.

[0044] After purification, the PCR products were respectively cloned into a mammalian double expression vector (such as pTT5 vector) containing the constant region of the mouse IgG1 heavy chain and the constant region of the kappa light chain by restriction enzyme method or homologous recombination method (such as Gibson Assembly) to construct the expression plasmids of the heavy chain and the light chain.

[0045] (4) Antibody expression and purification The sequenced heavy chain and light chain plasmids were co-transfected into suspension-cultured CHO cells. 72 hours after transfection, the cell culture supernatant was collected. The antibodies in the supernatant were captured and purified using a Protein A affinity chromatography column. The antibodies were eluted with glycine-HCl buffer at pH 2.5 and immediately neutralized with Tris-HCl buffer (pH 8.0). Finally, the buffer was replaced into PBS by ultrafiltration tube, the antibody concentration was determined, aliquoted and stored at -80°C.

[0046] (5) Antibody sequence identification: By sequencing the plasmid of the positive clone, a representative antibody of the application was obtained, and the heavy chain variable region (VH) amino acid sequence thereof is shown in SEQ ID NO. 8, comprising three heavy chain complementarity determining regions CDR1 (SEQ ID NO. 2), CDR2 (SEQ ID NO. 3), CDR3 (SEQ ID NO. 4); the light chain variable region (VL) amino acid sequence thereof is shown in SEQ ID NO. 9, comprising three light chain complementarity determining regions CDR4 (SEQ ID NO. 5), CDR5 (SEQ ID NO. 6), 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.

[0047] The above SEQ ID NO. 1-SEQ ID NO. 16 are shown in Table 1.

[0048] Example 3

[0049] This example further verifies the performance of the anti-PTGS1 monoclonal antibody prepared in Example 2 by Western Blot technology. The experimental method is as follows: The total protein of human ovarian cancer cell line (OVCAR3) was extracted. SDS-PAGE electrophoresis was performed and the membrane was transferred. The antibody PTGS1 (1.9 μg / mL) of the application was used for incubation, respectively. As shown in Figure 5 , the antibody can detect a specific band (about 70 kDa) in SKOV-3 cells. The specific band is the PTGS1 protein recognized by the anti-PTGS1 monoclonal antibody. Example 4

[0050] This example uses the anti-PTGS1 monoclonal antibody prepared in Example 2 for ovarian cancer immunohistochemistry (IHC) experiment, and the experimental method is as follows: (1) Preparation of tissue samples: 5 cases of ovarian cancer tissues and 5 cases of normal tissues were used, all of which were formalin-fixed paraffin-embedded (FFPE) tissues.

[0051] (2) Sectioning and deparaffination: the tissue chip was sectioned with a thickness of 4 μm, and the section was baked in an oven at 60°C for 2 hours. Then the section was deparaffinated with xylene and gradiently hydrated with ethanol.

[0052] (3) Antigen retrieval: the section was placed in EDTA buffer at pH 6.0, and heat-induced epitope retrieval was performed in a microwave oven.

[0053] (4) Endogenous enzyme blocking: 3% H2O2 solution was added dropwise, and incubation was performed at room temperature for 10 minutes to block the activity of endogenous peroxidase.

[0054] (5) Blocking: 5% BSA solution in PBS was added dropwise, and blocking was performed at room temperature for 30 minutes.

[0055] (6) Incubation of primary antibody: the antibody of the application (working concentration 1.9 μg / mL, diluted 1:200 with antibody diluent) was added dropwise, and incubation was performed at 4°C overnight. The full length of the heavy chain of the antibody is shown in SEQ ID NO. 10, and the full length of the light chain is shown in SEQ ID NO. 11.

[0056] (7) Incubation of secondary antibody: after washing with PBS, HRP-labeled goat anti-rabbit IgG secondary antibody was added dropwise, and incubation was performed at room temperature for 30 minutes.

[0057] (8) Color development and counterstaining: color development was performed using a DAB color development kit, and the nucleus was counterstained with hematoxylin.

[0058] (9) Dehydration, transparency and mounting: dehydration was performed with gradient ethanol, transparency was performed with xylene, and neutral resin mounting was performed, and analysis was performed under a light microscope.

[0059] As shown in Figure 6 , the staining results were observed under a microscope. Among them, the right side corresponds to the ovarian tissue region, and the left side corresponds to the normal ovarian tissue region. In the right side of the 5 ovarian cancer sample tissue regions, the antibody showed strong and specific cytoplasmic brownish yellow staining. In the left side of the 5 paired normal ovarian tissue regions, only weak background staining or no staining was observed. Further, the values obtained by quantitative analysis of the microscopic images of the stained sections by image analysis software (ImageJ) directly correspond to the degree of absorption of a specific wavelength of light by the sample, and the results are shown in Figure 7 : the average optical density of the staining of the 5 normal samples was about 0.2, and the average optical density of the staining of the 5 ovarian cancer samples was about 0.9. It can be seen that the antibody prepared in the application performs excellently in the application of ovarian cancer IHC, and can clearly and significantly distinguish cancer tissues from normal tissues, thereby providing a reliable diagnostic basis for pathologists.

[0060] In summary, by rational design, a "stable neo-antigen epitope" of PTGS1 is screened, and a high-specificity and high-affinity monoclonal antibody is successfully prepared by using advanced single B cell technology. The antibody shows excellent performance in IHC diagnosis of ovarian cancer, can significantly and clearly distinguish cancer tissues from normal tissues, has low background and high signal-to-noise ratio, and provides a new and reliable detection tool for pathological diagnosis of ovarian cancer.

[0061] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements, or the combination of the technical features in the above embodiments without mutual conflict, can be combined in the manner recorded in the embodiments, and these modifications, replacements or combinations do not make the essence of the corresponding technical solution deviate from the scope of the technical solutions of the embodiments of the present application.

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.

2. The monoclonal antibody according to claim 1, characterized in that, The method for preparing the monoclonal antibody is as follows: (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. (2) Lymphocytes were isolated from the spleen or peripheral blood of immunized animals and antigen-specific single B cells were sorted by flow cytometry. (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; (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.

3. The monoclonal antibody according to claim 1, characterized in that, 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.

4. The monoclonal antibody according to claim 1 or 3, characterized in that, 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.

5. The monoclonal antibody according to claim 4, characterized in that, 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.

6. The use of the monoclonal antibody according to any one of claims 1-5 in the detection of PTGS1 protein.

7. A DNA sequence encoding the monoclonal antibody according to any one of claims 1-5.

8. The DNA sequence of the monoclonal antibody according to claim 7, characterized in that, The DNA sequence encoding the monoclonal antibody contains genes encoding the heavy and light chains of the monoclonal antibody.

9. The method for preparing the monoclonal antibody according to any one of claims 1-5, 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.

10. 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 1-5, and the diagnostic kit is an immunohistochemical detection kit or an immunoblotting detection kit.

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