A double-antibody sandwich ELISA kit for detecting serum PTGS1 protein

Abstract

This invention relates to a double-antibody sandwich ELISA kit for detecting serum PTGS1 protein, comprising: a PTGS1 capture antibody, an unlabeled PTGS1 detection antibody, an enzyme-labeled IgG secondary antibody, a solid-phase carrier, an enzyme substrate, a blocking buffer, a washing buffer, and a stop solution; the IgG secondary antibody specifically binds to the PTGS1 detection antibody; the PTGS1 capture antibody is a rabbit monoclonal antibody that specifically binds to the linear epitope A of human PTGS1 protein, the amino acid sequence of which is SEQ ID NO:1, corresponding to amino acids 271-282 of human PTGS1 protein; the PTGS1 detection antibody is a mouse monoclonal antibody that specifically binds to the B epitope of human PTGS1 protein; the amino acid sequences of the B epitope at the A epitope do not overlap and do not constitute steric hindrance. This kit avoids the conserved homologous region between PTGS1 and its homologous protein PTGS2, ensuring the accuracy of the capture antibody recognition, and uses an indirect detection mode with enzyme-labeled goat anti-mouse IgG secondary antibody to achieve signal cascade amplification, significantly improving detection sensitivity.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and immunoassay, specifically to a double-antibody sandwich ELISA kit for detecting the content of prostaglandin intraperoxidase 1 (PTGS1) in serum. Background Technology

[0002] Ovarian cancer, the deadliest gynecological malignancy, sees approximately 70% of patients diagnosed at an advanced stage (stage III / IV), with a five-year survival rate of less than 30%. Current clinical practice faces two major bottlenecks that urgently need to be overcome:

[0003] 1. Diagnostic Bottleneck: Currently, the clinically relied-upon serum biomarker CA125 has unsatisfactory sensitivity and specificity. In early-stage ovarian cancer, its sensitivity is only 50%-60%. Furthermore, various benign diseases such as endometriosis can also cause elevated CA125 levels, leading to a high false-positive rate. Imaging examinations also have limited ability to identify early, small lesions. Therefore, there is an urgent need to discover and validate novel, highly specific serum biomarkers that can be used in conjunction with CA125 for combined assessment, thereby constructing a more effective early screening strategy.

[0004] 2. Bottlenecks in Biochemical Recurrence Monitoring: Up to 80% of patients who achieve clinical remission after initial surgery and chemotherapy experience recurrence. Existing monitoring methods suffer from significant "early warning delays," specifically: ① CA125 monitoring: According to the Gynecologic Cancer Intervention Group (GCIG) criteria, CA125 levels typically need to rise continuously to more than twice the upper limit of normal to indicate recurrence, by which time the tumor burden is often already substantial. ② Imaging monitoring: CT and PET-CT scans can only detect lesions after they are visible to the naked eye, missing the optimal window for early intervention. This monitoring lag severely restricts the timing of secondary treatment and impacts patient survival prognosis.

[0005] 3. PTGS1, as a highly promising new target, has been shown in recent years to play a crucial role in the development of ovarian cancer, including: ① PTGS1 is specifically and highly expressed in ovarian cancer, exhibiting persistently high expression in ovarian cancer tissues and closely related to tumor cell proliferation, apoptosis resistance, and angiogenesis. ② As a typical secreted protein, ovarian cancer cells can actively secrete PTGS1, releasing it into the extracellular environment, making it potentially present in a stable and detectable form in the peripheral blood of patients. ③ PTGS1 has strong early warning potential; its important role in tumorigenesis and the dynamic changes in its serum levels may reflect the microscopic activities of the disease earlier than downstream effector molecules such as CA125. Therefore, non-invasive serum PTGS1 detection can achieve early warning of ovarian cancer. However, due to the current lack of attention to serum PTGS1 in the industry, a reliable method for accurate and specific quantification of serum PTGS1 has long been lacking, severely hindering its clinical application.

[0006] Therefore, developing a kit that can quantitatively detect PTGS1 in serum with high sensitivity and specificity is of urgent clinical need and significant application value for solving the challenges of early diagnosis and recurrence monitoring of ovarian cancer. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a highly sensitive and specific double-antibody sandwich ELISA kit for the quantitative detection of PTGS1 protein in human serum and to apply it to the risk identification of ovarian cancer.

[0009] (II) Technical Solution

[0010] In a first aspect, the present invention provides a double-antibody sandwich ELISA kit for detecting serum PTGS1 protein, comprising: a PTGS1 capture antibody, an unlabeled PTGS1 detection antibody, an enzyme-labeled IgG secondary antibody, a solid-phase carrier, an enzyme substrate, a blocking buffer, a washing buffer, and a stop solution; wherein the IgG secondary antibody is capable of specifically binding to the PTGS1 detection antibody;

[0011] The PTGS1 capture antibody is a rabbit monoclonal antibody that specifically binds to the linear epitope A of the human PTGS1 protein. The amino acid sequence of the linear epitope A is SEQ ID NO:1, which corresponds to amino acids 271-282 of the human PTGS1 protein. The PTGS1 detection antibody is a mouse monoclonal antibody that specifically binds to the epitope B of the human PTGS1 protein. The amino acid sequences of the epitope B at the epitope A do not overlap and do not constitute steric hindrance.

[0012] The PTGS1 detection antibody can be any commercially available PTGS1 monoclonal antibody that meets the requirement of "not overlapping with the A epitope recognized by the capture antibody and not constituting steric hindrance", or it can be a monoclonal antibody that the inventor has screened to meet the requirement, such as a monoclonal antibody that specifically binds to multiple mixed epitopes that do not overlap with the linear A epitope of the PTGS1 protein.

[0013] Preferably, the kit also includes a PTGS1 protein standard.

[0014] Preferably, the PTGS1 capture antibody has three heavy chain complementarity-determining regions of the amino acid sequences shown in SEQ ID NO. 2-4, and three light chain complementarity-determining regions of the amino acid sequences shown in SEQ ID NO. 5-7.

[0015] Preferably, the PTGS1 capture 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.

[0016] Preferably, the PTGS1 capture 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.

[0017] Preferably, the B epitope comprises multiple mixed epitopes (the B epitope is a spatial epitope) based on the full length of the human PTGS1 protein and not overlapping with the linear A epitope; the PTGS1 detection antibody has three heavy chain complementarity-determining regions of the amino acid sequence shown in SEQ ID NO. 12-14, and three light chain complementarity-determining regions of the amino acid sequence shown in SEQ ID NO. 15-17.

[0018] Preferably, the PTGS1 detection antibody has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO.18 and a light chain variable region of the amino acid sequence shown in SEQ ID NO.19.

[0019] Preferably, the PTGS1 detection antibody has a heavy chain of the amino acid sequence shown in SEQ ID NO.20 and a light chain of the amino acid sequence shown in SEQ ID NO.21.

[0020] Preferably, the solid-phase carrier is an enzyme-labeled plate, the enzyme-labeled IgG secondary antibody is HRP-labeled or ALP-labeled goat anti-mouse IgG secondary antibody, and the enzyme substrate is TMB, OPD, or a chemiluminescent substrate.

[0021] Preferably, the blocking solution is BSA (to reduce nonspecific binding), the washing solution is PBS-Tween (to wash away nonspecific adsorption), and the terminating solution is sulfuric acid (to terminate the colorimetric reaction).

[0022] Preferably, the binding affinity Kd of the PTGS1 capturing antibody to the linear epitope A of human PTGS1 protein is ≤1×10⁻⁶. - 9 The concentration of the antibody was mol / L, and the cross-reactivity with PTGS1 protein was ≤5% (using human PTGS2 protein as a control); the binding affinity (Kd) of the PTGS1 detection antibody to the B epitope of human PTGS1 protein was ≤1×10⁻⁶. -9 The concentration is mol / L, and the epitopes recognized by the detection antibody and the capture antibody do not overlap, and they can bind to human PTGS1 protein simultaneously.

[0023] Preferably, the PTGS1 capture antibody is a monoclonal antibody prepared using single B cell technology with amino acids 271-282 of human PTGS1 protein as the antigen, and the detection antibody is a monoclonal antibody prepared using single B cell technology with the full-length sequence of human PTGS1 protein as the antigen, with an antibody purity ≥95%.

[0024] ELISA experiments have verified that the PTGS1 capture antibody and PTGS1 detection antibody can simultaneously recognize different epitopes of the PTGS1 protein without competition, forming an effective double-antibody sandwich complex. Based on the above scheme, this invention utilizes a highly specific capture antibody that recognizes PTGS1, paired with detection antibodies that recognize multiple different epitopes on the full-length PTGS1 protein, and amplifies the signal using a highly sensitive enzyme-labeled secondary antibody to assemble a highly specific and highly sensitive indirect double-antibody sandwich ELISA kit, enabling highly sensitive and accurate quantitative detection of trace amounts of PTGS1 protein in human serum.

[0025] In a second aspect, the present invention provides a method for quantitatively detecting PTGS1 protein in serum, comprising:

[0026] (1) Coating: Dilute PTGS1 capture antibody to working concentration (e.g., 5 μg / mL), add to multi-well microplate, and incubate overnight at 4°C to allow the antibody to bind tightly to the microplate;

[0027] (2) Blocking: Discard the coating solution and wash the plate 3 times with washing solution; then add blocking solution to each well and incubate at 37°C for 1 hour to block non-specific binding sites;

[0028] (3) Primary antibody incubation: After blocking, wash the plate 3 times; add the serum sample to be tested and the PTGS1 protein standard of gradient concentration to the well of the ELISA plate respectively, and incubate at 37°C for 1 hour to allow the PTGS1 protein in the serum sample to be tested and the standard to bind with the capture antibody.

[0029] (4) Secondary antibody incubation: Wash the plate 3 times, add working concentration of PTGS1 detection antibody, incubate at 37°C for 1 hour to form a “capture antibody-PTGS1-detection antibody” sandwich complex; wash the plate 3 times again, add HRP-labeled goat anti-rabbit IgG secondary antibody, incubate at 37°C for 45 minutes;

[0030] (5) Color development and detection: After thoroughly washing the plate 3 times, add TMB solution as a colorimetric reagent to each well and incubate at room temperature in the dark for 10-15 minutes; add stop solution to terminate the reaction and immediately use an ELISA reader to measure the absorbance value of each well at a wavelength of 450 nm; plot a standard curve based on the concentration of PTGS1 protein standard - OD value, and calculate the concentration of PTGS1 protein in the serum sample to be tested using the standard curve equation.

[0031] Thirdly, the present invention provides the application of a double-antibody sandwich ELISA kit in the preparation of products for the auxiliary diagnosis and biochemical recurrence monitoring of ovarian cancer.

[0032] Since the serum PTGS1 level in ovarian cancer patients is significantly higher than that in healthy controls and patients with benign gynecological diseases, serum PTGS1 can be used as a biomarker for diagnosing ovarian cancer. The double-antibody sandwich ELISA kit can accurately quantify the serum PTGS1 protein level, thus aiding in the diagnosis of ovarian cancer.

[0033] (III) Beneficial Effects

[0034] The double-antibody sandwich ELISA kit for detecting serum PTGS1 protein of the present invention has the following technical advantages:

[0035] (1) High specificity and high sensitivity: The PTGS1 capture antibody of the present invention specifically recognizes the specific linear epitope corresponding to amino acids 271-282 (271-282AA) of the PTGS1 protein. This antigenic epitope was first discovered and identified by the applicant. Its sequence design avoids the conserved homologous region between PTGS1 and its homologous protein PTGS2, ensuring the accuracy of antibody recognition from the target selection level, effectively avoiding cross-reaction of homologous proteins, and significantly improving detection specificity.

[0036] Furthermore, this invention uses the full-length PTGS1 protein as an immunogen and obtains a mouse monoclonal antibody that specifically recognizes the full-length human PTGS1 protein (i.e., the PTGS1 detection antibody). This detection antibody can specifically bind to multiple mixed epitopes on the PTGS1 protein that have no sequence overlap or steric hindrance with the linear epitope A (271-282AA), and synergistically form a stable and efficient "sandwich" structure with the capture antibody.

[0037] The dual-antibody sandwich ELISA kit constructed based on this antibody combination uses an indirect detection mode with enzyme-labeled goat anti-mouse IgG secondary antibody. Through enzyme catalysis, the signal is amplified in a cascade, which significantly improves the detection sensitivity and can accurately capture trace amounts of PTGS1 protein in serum. Compared with existing technologies, it has higher detection sensitivity and a lower detection limit.

[0038] (2) Reasonable design to avoid competition: The PTGS1 capture antibody and the detection antibody are from different species (mouse and rabbit), and the PTGS1 capture antibody binds to a central linear epitope, while the PTGS1 detection antibody is a mixed epitope targeting the full-length protein. This design fundamentally avoids direct competition and ensures the efficient formation of the sandwich complex.

[0039] (3) Outstanding clinical application value: The kit provides a brand-new serological marker detection scheme for the diagnosis of ovarian cancer, which can provide clinicians with an earlier intervention window. The method of quantitative detection of serum PTGS1 using this kit is optimized based on the traditional double antibody sandwich method. The process is mature, reproducible, and easy to promote and use in clinical laboratory laboratories. It can achieve non-invasive detection and diagnosis of ovarian cancer and has the advantages of convenience and ease of promotion: only a small amount of venous blood is needed in the diagnosis of ovarian cancer. The operation process is standardized and applicable to the laboratory departments of hospitals at all levels. It has good clinical applicability and promotion prospects and is expected to become a routine monitoring tool in the whole-process management of ovarian cancer. Attached Figure Description

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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) (A1-5) and ovarian cancer tissue (B6-10).

[0046] Figure 7 This is the PTGS1 standard curve of the double antibody sandwich ELISA kit in this invention.

[0047] Figure 8 The results show the serum PTGS1 levels in healthy controls and ovarian cancer patients in Example 6 of this invention.

[0048] Figure 9 The results show the serum PTGS1 levels detected in patients with ovarian cysts and ovarian cancer in Example 6 of this invention.

[0049] (1) Examples 1-3 are from previous cases, mainly to demonstrate that the capture antibody has high specificity. (2) Example 4 provides a double sandwich kit based on the capture antibody and existing commercial antibodies (this is also innovative, based on the capture antibody). (3) Examples 5-6 are the contents of this disclosure, a novel double sandwich kit composed of newly screened detection antibodies and the capture antibody. Detailed Implementation

[0050] 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.

[0051] 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, the current standard practice is to use the complete PTGS1 protein or large fragments of it as immunogens. However, PTGS1 shares high sequence homology with its homologous protein PTGS2, and existing antibodies do not avoid these homologous regions, easily leading to problems such as "high risk of cross-reactivity, insufficient specificity" and "ignored epitope stability, resulting in poor detection reliability." For example, Boster Biologics uses the E318-L599 fragment of PTGS1 as an immunogen, and Proteintech uses the 250-599AA fragment; neither of these existing technologies excludes 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.

[0052] The core principle of this invention lies in ensuring superior antibody performance from the source through rational immunogen design. Traditional antibody preparation strategies neglect the stability of the antigenic epitope itself, leading to 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 (as shown in the amino acid sequence of SEQ ID NO.1). 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 resulting antibody exhibits endogenous high specificity and stability; simultaneously, its low mutation rate ensures the stability and reliability of detection results in a large patient population, guaranteeing 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.

[0053] The following detailed description is provided in conjunction with specific examples. Example 1

[0054] 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:

[0055] (1) Sequence conservation and mutation rate analysis

[0056] 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.

[0057] (2) Transcript expression analysis

[0058] 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.

[0059] (3) Determination and preparation of immunogen sequences

[0060] 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.

[0061] Finally, the DNA sequence encoding the fragment (nucleotide sequence as shown in SEQ ID NO.22) 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

[0062] 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:

[0063] (1) Animal immunization

[0064] 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 rabbits. Booster immunizations were performed every two weeks using the same dose of immunogen emulsified with incomplete Freund's adjuvant, for a total of three immunizations. On day 3 after the final immunization, blood was collected intravenously, and serum titers were determined by indirect ELISA. Rabbits with the highest titers were selected for subsequent experiments.

[0065] (2) Antigen-specific B cell sorting

[0066] Rabbits 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 (i.e., the 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.

[0067] (3) Antibody gene amplification and cloning

[0068] 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).

[0069] The primer mixture for amplifying the gene encoding the variable region of the rabbit antibody heavy chain contains:

[0070] Forward primer, MuIgGVH-F, see the nucleotide sequence shown in SEQ ID NO.23;

[0071] The reverse primer, MuIgGVH-R, is shown in the nucleotide sequence of SEQ ID NO.24.

[0072] The primer mixture for amplifying the variable region gene of the mouse antibody light chain VL (κ chain) contains:

[0073] Forward primer, MuIgKVL-F, see the nucleotide sequence shown in SEQ ID NO.25;

[0074] The reverse primer, MuIgKVL-R, is shown in the nucleotide sequence of SEQ ID NO.26.

[0075] After purification, the PCR products are cloned into mammalian dual expression vectors (such as pTT5 vector) containing the constant regions of the rabbit IgG1 heavy chain and the light chain, respectively, by restriction endonuclease method or homologous recombination method (such as Gibson Assembly), to construct expression plasmids for the heavy chain and light chain.

[0076] (4) Antibody expression and purification

[0077] 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.

[0078] (5) Antibody sequence identification:

[0079] 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. Example 3

[0080] 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:

[0081] (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.

[0082] (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.

[0083] (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.

[0084] (4) Endogenous enzyme blockade: Add 3% H2O2 solution and incubate at room temperature for 10 minutes to block endogenous peroxidase activity.

[0085] (5) Blocking: Add 5% BSA in PBS solution and block at room temperature for 30 minutes.

[0086] (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.

[0087] (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.

[0088] (8) Color development and counterstaining: DAB color development kit was used for color development, and hematoxylin was used for counterstaining of cell nuclei.

[0089] (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.

[0090] like Figure 5 The 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 areas 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 6 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.

[0091] In summary, this invention, through rational design and screening of stable neoantigen epitopes of PTGS1, and combined with advanced single-cell B-cell technology, successfully prepared a highly specific and high-affinity monoclonal antibody. This antibody exhibits excellent performance in the IHC diagnosis of ovarian cancer, significantly and clearly distinguishing cancerous tissue from normal tissue with low background and a high signal-to-noise ratio. Example 4

[0092] This embodiment uses the anti-PTGS1 monoclonal antibody obtained in Example 2 as the PTGS1 capture antibody to construct a double-antibody sandwich ELISA kit, which includes:

[0093] The package includes PTGS1 capture antibody, unlabeled PTGS1 detection antibody, HRP-labeled goat anti-mouse IgG secondary antibody, PTGS1 protein standard, ELISA plate, blocking buffer, washing buffer, enzyme substrate, and stop solution. The HRP-labeled goat anti-mouse IgG secondary antibody specifically binds to the PTGS1 detection antibody. Additionally, it may include coating buffer, sample diluent (serum diluent), antibody diluent, and washing diluent.

[0094] It should be noted that the PTGS1 capture antibody and the PTGS1 detection antibody are a pair of paired antibodies that can recognize different sites of the PTGS1 protein antigen. The PTGS1 capture antibody is the monoclonal antibody obtained in Example 2. Other auxiliary reagents such as coating solution, washing solution, blocking solution, chromogenic substrate (enzyme substrate), and stop solution can be obtained directly by purchasing.

[0095] In this embodiment, the PTGS1 detection antibody can be a pre-existing monoclonal antibody against PTGS1, such as the PTGS1 monoclonal antibody from Boster Biologics (product number PB9002, link). https: / / boster.com / index / products / productsDetail?goods_sn=PB9002#cpjj Proteintech's PTGS1 monoclonal antibody (product number: 67346-2-PBS, link) https: / / www.ptgcn.com / products / PTGS1-Antibody-67346-2- PBS.htm ).

[0096] This embodiment provides a double-antibody sandwich ELISA method for detecting PTGS1 protein in serum samples, which includes the following steps:

[0097] (1) Coat the PTGS1 capture antibody (5 μg / mL) with coating buffer. Add 100 mL to each well of a 96-well microplate and coat overnight at 4°C to ensure tight binding with the microplate and form PTGS1 capture antibody coated on the microplate. After coating, wash the microplate three times with washing buffer, add 300 μL of blocking buffer to each well for blocking, and incubate at 37°C for 2 hours.

[0098] (2) After blocking, add the serum sample to be tested and PTGS1 protein standard (serially diluted to 1.56 ng / mL, three replicates for each sample) to the wells of the ELISA plate, 100 μL / well, and incubate at 37°C for 1 hour. Then add PTGS1 detection antibody, 100 μL / well, and incubate at 37°C for 1 hour.

[0099] (3) Then add horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody, 100 μL / well, and incubate at 37°C for 45 minutes.

[0100] (4) Finally, add 100 μL of the chromogenic substrate TMB solution to the formed complex and incubate at room temperature for 15 minutes. Under the action of horseradish peroxidase, the chromogenic agent will change color. Add 100 μL of the stop solution to terminate the reaction. Determine the presence and concentration of PTGS1 protein in the protein extract solution according to the OD value. Example 5

[0101] This embodiment is based on Example 4, and further uses the full-length PTGS1 protein as an immunogen. After screening, a mouse monoclonal antibody that specifically recognizes the full-length human PTGS1 protein (i.e., the PTGS1 detection antibody) is obtained. The PTGS1 detection antibody screened in this embodiment and the PTGS1 capture antibody screened in Example 2 are used to construct a novel double antibody sandwich ELISA kit.

[0102] The screening method for PTGS1 detection antibodies is as follows:

[0103] (1) Determination and preparation of immunogen sequences

[0104] Based on the above analysis, this invention selected the full-length 599-amino acid sequence of the human PTGS1 protein (its amino acid sequence is shown in SEQ ID NO.27) as the immunogen. Finally, the DNA sequence encoding this fragment (nucleotide sequence is shown in SEQ ID NO.28) was synthesized and cloned into the pGEX-6P-1 prokaryotic expression vector, transformed into *Escherichia coli* BL21(DE3) strain, and the GST-tagged fusion protein was expressed using IPTG. The protein was then purified using GST affinity chromatography to obtain high-purity recombinant PTGS1 immunogen protein.

[0105] (2) Animal immunization

[0106] The recombinant PTGS1 protein (GST tag removed) prepared 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.

[0107] (3) Antigen-specific B cell sorting

[0108] 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.27) 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.

[0109] (4) Antibody gene amplification and cloning

[0110] 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).

[0111] The primer mixture for amplifying the gene encoding the variable region of the mouse antibody heavy chain contains:

[0112] Forward primer, MuIgGVH-F, see the nucleotide sequence shown in SEQ ID NO.29;

[0113] The reverse primer, MuIgGVH-R, is shown in the nucleotide sequence of SEQ ID NO.30.

[0114] The primer mixture for amplifying the variable region gene of the mouse antibody light chain VL (κ chain) contains:

[0115] Forward primer, MuIgKVL-F, see the nucleotide sequence shown in SEQ ID NO.31;

[0116] The reverse primer, MuIgKVL-R, is shown in the nucleotide sequence of SEQ ID NO.32.

[0117] 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.

[0118] (5) Antibody expression and purification

[0119] 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.

[0120] (6) Antibody sequence identification:

[0121] 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. 18, containing three heavy chain complementarity-determining regions: CDR1 (SEQ ID NO. 12), CDR2 (SEQ ID NO. 13), and CDR3 (SEQ ID NO. 14). The amino acid sequence of its light chain variable region (VL) is shown in SEQ ID NO. 19, containing three light chain complementarity-determining regions: CDR4 (SEQ ID NO. 15), CDR5 (SEQ ID NO. 16), and CDR6 (SEQ ID NO. 17). The heavy chain amino acid sequence of a specific monoclonal antibody is shown in SEQ ID NO. 20, and the light chain amino acid sequence is shown in SEQ ID NO. 21.

[0122] The detection antibody can specifically bind to multiple mixed epitopes on the PTGS1 protein that have no sequence overlap or steric hindrance with the linear epitope A (271-282AA), forming a stable and efficient "sandwich" structure in synergy with the capture antibody.

[0123] The SEQ ID NO.1-SEQ ID NO.26 mentioned above are shown in Table 1.

[0124] Table 1:

[0125] Example 6

[0126] Using the double-antibody sandwich ELISA kit constructed in Example 5, the content of PTGS1 in serum samples from patients with ovarian cysts, patients with ovarian cancer, and healthy individuals was detected according to the double-antibody sandwich ELISA detection method for PTGS1 protein in serum samples provided in Example 4.

[0127] Peripheral venous blood was collected from healthy controls, patients with ovarian cysts, and patients with ovarian cancer. The blood was centrifuged at 3000 rpm for 15 minutes, and the supernatant serum was transferred to EP tubes. 100 μL / well of the sample to be tested was added to the antibody-coated microplate in the kit. The plate was incubated at 37°C for 1 hour. Washing buffer was added to the microplate at a rate of 300 μL / well, and the plate was washed three times. The detection antibody was diluted to the working concentration (1:5000), and 100 μL / well was added to the microplate. The plate was incubated at 37°C for 1 hour. Washing buffer was added to the microplate at a rate of 300 μL / well, and the plate was washed three times. The liquid in the wells was discarded again. HRP-labeled goat anti-mouse IgG secondary antibody was diluted to the working concentration (1:10000), and 100 μL / well was added to the microplate. The plate was incubated at 37°C for 45 minutes. Add washing buffer to the microplate at a rate of 300 μL / well and wash the microplate three times. Add 100 μL of TMB substrate to each reaction well and incubate at 37°C for 15 minutes. Then add 100 μL of stop solution to stop the reaction and measure the OD value at 450 nm using a microplate reader.

[0128] Using a double-antibody sandwich ELISA kit, and following the double-antibody sandwich ELISA method for detecting PTGS1 protein in serum samples as described in Example 4, a standard curve was constructed by plotting the concentration of PTGS1 protein standards on the ordinate and OD values ​​on the abscissa. The results are as follows: Figure 7 As shown in the figure, the numerical simulation yielded the following relationship between the concentration y of the PTGS1 protein standard and the OD value x: Y = -0.000259X² + 0.4119X - 0.3626, R = 0.9928 (R² ≥ 0.990). The kit exhibits good linearity in the detection of PTGS1 protein in serum within the range of 0-200 ng / mL, with a correlation coefficient R² ≥ 0.99, enabling accurate quantification within this concentration range. Therefore, the PTGS1 content in the protein extract solution can be calculated based on the OD value obtained from the detection.

[0129] Test results as follows Figure 8-9 As shown in the figure. Serum PTGS1 levels in healthy controls and ovarian cancer patients are as follows. Figure 8 As shown, the serum PTGS1 test results of patients with ovarian cysts and ovarian cancer are as follows: Figure 9 As shown. From Figure 8 and Figure 9The study showed that the mean serum PTGS1 level in ovarian cancer patients was 25.94 ng / ml; the mean serum PTGS1 level in healthy controls was 3.947 ng / ml; and the mean serum PTGS1 level in patients with ovarian cysts was 5.23 ng / ml. Compared with the mean serum PTGS1 levels (ng / ml) in healthy controls and patients with ovarian cysts, the PTGS1 levels (ng / ml) in ovarian cancer patients were significantly increased. This indicates that PTGS1 can be a candidate biomarker for monitoring ovarian cancer (positive or negative diagnosis), disease course and progression, and recurrence risk.

[0130] In conclusion, the occurrence and development of ovarian cancer are closely related to the serum PTGS1 level, making PTGS1 a candidate biomarker for monitoring ovarian cancer (positive or negative diagnosis), disease course and progression, and recurrence risk.

[0131] 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 double antibody sandwich ELISA kit for detecting serum PTGS1 protein, characterized by, It includes: PTGS1 capture antibody, unlabeled PTGS1 detection antibody, enzyme-labeled IgG secondary antibody, solid-phase carrier, enzyme substrate, blocking buffer, washing buffer, and stop solution; the IgG secondary antibody can specifically bind to the PTGS1 detection antibody; The PTGS1 capture antibody is a rabbit monoclonal antibody that specifically binds to the linear epitope A of the human PTGS1 protein. The amino acid sequence of the linear epitope A is SEQ ID NO:1, which corresponds to amino acids 271-282 of the human PTGS1 protein. The PTGS1 detection antibody is a mouse monoclonal antibody that specifically binds to the epitope B of the human PTGS1 protein. The amino acid sequences of the epitope B and the epitope A do not overlap and do not constitute steric hindrance. The PTGS1 capture antibody has three heavy chain complementarity-determining regions as shown in SEQ ID NO.2-4 and three light chain complementarity-determining regions as shown in SEQ ID NO.5-7.

2. The double antibody sandwich ELISA kit according to claim 1, characterized in that, The kit also includes PTGS1 protein standards.

3. The double-antibody sandwich ELISA kit according to claim 1, characterized in that, The PTGS1 capture 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.

4. The double-antibody sandwich ELISA kit according to claim 1, characterized in that, The PTGS1 capture 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.

5. The double-antibody sandwich ELISA kit according to claim 1 or 2, characterized in that, The B epitope comprises multiple mixed epitopes based on the full length of the human PTGS1 protein and not overlapping with the linear A epitope; the PTGS1 detection antibody has three heavy chain complementarity-determining regions of the amino acid sequence shown in SEQ ID NO. 12-14, and three light chain complementarity-determining regions of the amino acid sequence shown in SEQ ID NO. 15-17.

6. The double-antibody sandwich ELISA kit according to claim 5, characterized in that, The PTGS1 detection antibody has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO.18 and a light chain variable region of the amino acid sequence shown in SEQ ID NO.

19.

7. The double-antibody sandwich ELISA kit according to claim 5, characterized in that, The PTGS1 detection antibody has a heavy chain of the amino acid sequence shown in SEQ ID NO.20 and a light chain of the amino acid sequence shown in SEQ ID NO.

21.

8. The double-antibody sandwich ELISA kit according to claim 1 or 2, characterized in that, The solid-phase carrier is an ELISA plate, and the enzyme-labeled IgG secondary antibody is an HRP-labeled or ALP-labeled goat anti-mouse IgG secondary antibody; the enzyme substrate is TMB, OPD, or a chemiluminescent substrate.

9. The use of the double-antibody sandwich ELISA kit according to any one of claims 1-8 in the preparation of products for the auxiliary diagnosis and biochemical recurrence monitoring of ovarian cancer.

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