Annexin A1 specific antibody, use method and diagnostic kit thereof

Abstract

The invention discloses an annexin A1 specific antibody, a use method and a diagnostic kit thereof, and adopts a monoclonal antibody targeting an ANXA1 N-terminal structural domain (amino acid 1-30), which is different from an antibody focusing a C terminal. The antibody improves diagnostic specificity and sensitivity, and can detect ANXA1 in leukemia cells, CTC and tissue samples through flow cytometry, immunohistochemistry and ELISA. The matched diagnostic kit simplifies clinical operation, fills the blank of CTC and leukemia detection, and supports prognosis evaluation of various cancers (such as HCL, breast cancer and cholangiocarcinoma). The requirements of diagnostic tools with high specificity and wide application in the prior art are met, and an innovative solution is provided for early diagnosis and prognosis evaluation of various cancers such as breast cancer, bile duct cancer and lung cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and particularly relates to ANXA1 monoclonal antibodies and applications thereof in cancer diagnosis. Background Art

[0002] Annexin A1 (ANXA1) is a calcium-dependent phospholipid-binding protein that is widely involved in various biological processes, including apoptosis, inflammatory response, and tumorigenesis, development, and metastasis. Studies have shown that the expression level of ANXA1 in different cancers is closely related to disease progression, prognosis, and treatment response. In particular, it is considered a potential diagnostic biomarker in certain cancers (such as breast cancer, bile duct cancer, ovarian cancer, lung cancer, and hairy cell leukemia). The expression pattern of ANXA1 shows significant differences between cancer types. For example, high expression in some cancers is associated with disease aggressiveness and poor prognosis, while in other cancers it is associated with better survival. This tissue specificity makes ANXA1 an important target with both diagnostic and prognostic potential.

[0003] In diagnostic applications, ANXA1 has been shown to be highly specifically expressed in specific cancers. For example, in hairy cell leukemia (HCL), overexpression of ANXA1 is considered a characteristic hallmark of the disease and can be detected by immunohistochemistry or flow cytometry with a specificity and sensitivity approaching 100% (Falini et al., Lancet, 2004). In solid tumors such as breast cancer, cholangiocarcinoma, and lung cancer, ANXA1 is not only expressed in tumor tissues, but also changes in the levels of anti-ANXA1 antibodies can be detected in circulating tumor cells (CTCs) or patient serum, which are correlated with tumor invasiveness or metastatic potential (Gibbs et al., Oncotarget, 2015; An et al., 2015).

[0004] Thorac Cancer 2020, Guan et al., Thorac Cancer 2019; Liang et al., Clin Lab 2018; Huang et al., Tumour Biol 2015). However, existing antibodies targeting ANXA1 are mostly polyclonal or nonspecific monoclonal antibodies, failing to fully utilize specific domains of ANXA1 (such as the N-terminal domain), which play a key role in membrane interactions and cancer subtype specificity. Therefore, the development of a novel ANXA1 monoclonal antibody with strong specificity and broad application to enhance its diagnostic potential in blood and tissue testing is an urgent need in the current technology field.

[0005] Limitations of existing technologies:

[0006] Insufficient specificity: Polyclonal antibodies have poor batch-to-batch consistency and exhibit high levels of nonspecific binding; monoclonal antibodies are not optimized for the functional N-terminal domain.

[0007] Single function: Existing antibodies are mostly limited to tissue expression detection and cannot meet the needs of serum or CTC detection.

[0008] Limited scope of application: Existing tools focus on specific cancers and fail to cover the diverse cancer types where ANXA1 is widely expressed.

[0009] Insufficient clinical translation: Lack of standardized production and validation limits clinical application.

[0010] In the existing technology, the development and application of antibodies against ANXA1 have made certain progress in the diagnosis and research of various cancers, but there are still significant limitations; therefore, it is necessary to propose membrane-specific antibodies, methods of use and diagnostic kits for ANXA1. Summary of the Invention

[0011] In response to the problems existing in the above-mentioned prior art, the present invention provides an Annexin A1-specific antibody, a method of use and a diagnostic kit thereof, with the purpose of solving the above-mentioned technical problems.

[0012] In order to achieve the above-mentioned object, the present invention is implemented by the following technical solutions: the patent of the present invention is an annexin A1-specific antibody, a method of use and a diagnostic kit thereof;

[0013] Specifically, the invention relates to an annexin A1-specific antibody, including a monoclonal antibody, which specifically binds to an epitope within the N-terminal domain of human annexin A1 (ANXA1).

[0014] Furthermore, the epitope includes amino acid residues 1-30 of ANXA1.

[0015] (MAMVSEFLKQAWFIENEEQEYVQTVKSSKG).

[0016] Furthermore, the monoclonal antibody heavy chain sequence is shown in antibody sequence No. 1.

[0017] Furthermore, the monoclonal antibody light chain sequence is shown in antibody sequence No. 2.

[0018] Methods of using an annexin A1-specific antibody to diagnose hairy cell leukemia (

[0019] Hairy cell leukemia (HCL) or acute myeloid leukemia (AML), including:

[0020] a) contacting a blood or bone marrow sample of a subject with the antibody of claim 1;

[0021] b) Detecting the binding of the antibody to cell surface ANXA1 by flow cytometry;

[0022] c) Diagnosis of HCL or AML based on elevated ANXA1 expression.

[0023] Preferably, a method for detecting circulating tumor cells (CTCs) in a cancer subject is used, comprising:

[0024] a) isolating cells from a blood sample of a subject;

[0025] b) contacting the isolated cells with the antibody of claim 1;

[0026] c) detecting ANXA1 on the surface of CTCs by flow cytometry, wherein the cancer is selected from pancreatic cancer, breast cancer or lung cancer. Preferably, the method for diagnosing or prognosing cancer in a subject comprises:

[0027] a) contacting a tissue sample of a subject with the antibody of claim 1;

[0028] b) Detecting the binding of the antibody to ANXA1 by immunohistochemistry;

[0029] c) Determining the presence or progression of cancer based on the ANXA1 expression pattern.

[0030] Annexin A1 specific antibody diagnostic kit, the antibody, a fluorophore-conjugated secondary antibody, and reagents for performing flow cytometry or immunohistochemistry.

[0031] In summary, the present invention provides Annexin A1-specific antibodies, methods of use, and diagnostic kits. This invention utilizes a monoclonal antibody that targets the N-terminal domain (amino acids 1-30) of ANXA1, unlike antibodies that focus on the C-terminus. This antibody has enhanced diagnostic specificity and sensitivity, enabling the detection of ANXA1 in leukemia cells, CTCs, and tissue samples via flow cytometry, immunohistochemistry, and ELISA. The accompanying diagnostic kit simplifies clinical procedures, fills a gap in CTC and leukemia detection, and supports prognostic assessment for a variety of cancers, such as HCL, breast cancer, and cholangiocarcinoma.

[0032] The innovation of this patent lies in:

[0033] 1) Improved specificity: Targeting the N-terminal domain enhances the recognition of cancer cell membrane-bound ANXA1.

[0034] 2) Wide applicability: Applicable to a variety of cancers and sample types (blood, tissue, serum).

[0035] 3) Dual function: supports tissue testing (IHC) and blood testing (flow cytometry, ELISA).

[0036] 4) Mechanistic support: utilizing the role of ANXA1 in the PI3K / AKT, mTOR-S6 and EMT pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of the expression of ANXA1 in HL-60 cells in the present invention;

[0038] Figure 2 is a schematic diagram of a representative image of cell fluorescent immunostaining of the present invention;

[0039] Figure 3 is a schematic diagram of immunohistochemical staining of ovarian cancer tissue of the present invention;

[0040] Figure 4 The standard curve diagram of the present invention successfully detecting the ANXA1 concentration in the cell supernatant. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the accompanying drawings.

[0042] like Figures 1 to 4 As shown:

[0043] The development and application of antibodies targeting ANXA1 have made some progress in the diagnosis and research of various cancers, but significant limitations remain. The following describes the current status in detail, focusing on specific cancer types and existing patented technologies:

[0044] 1. Diagnostic and prognostic value in breast cancer:

[0045] Sobral-Leite et al. (BMCMed, 2015) analyzed tissue samples from 5752 breast cancer patients and found that high ANXA1 expression was associated with triple-negative breast cancer (TNBC), poorly differentiated tumors, and younger patients. Among patients with the HER2+ subtype, high ANXA1 expression was an independent predictor of survival (10-year breast cancer-specific survival HR = 1.70, 95% CI 1.17-2.45), suggesting its association with poor prognosis. Bhardwaj et al. (PLoS One, 2015) reported that ANXA1 expression is high in basal-like breast cancer and is associated with activation of the mTOR-S6 signaling pathway. Patients with high expression have poor overall survival. Huang et al. (Tumour Biol, 2015) found that serum anti-ANXA1 IgG levels were significantly elevated in breast cancer patients (P < 0.0001), with an area under the receiver operating characteristic (ROC) curve of 0.73 (95% CI 0.67-0.78). The sensitivity was 23.2% at a specificity of 90%, indicating the diagnostic potential of serum antibody testing.

[0046] The limitation is that existing studies mostly rely on polyclonal antibodies to detect ANXA1 in tissues or serum, which have limited specificity, and monoclonal antibodies have not been optimized for specific epitopes to improve diagnostic efficiency.

[0047] 2. Diagnostic and prognostic value in cholangiocarcinoma:

[0048] Hongsrichan et al. (World J Gastroenterol, 2013) found through immunohistochemical analysis that ANXA1 was highly expressed in 94.1% (64 / 68) of cholangiocarcinoma (CCA) tissues, whereas ANXA1 was negative in both hepatocellular carcinoma (HCC) and normal liver tissues. ANXA1 expression increased significantly with tumor progression, suggesting its potential as a specific biomarker for CCA. Kotepui et al. (Asian Pac J Cancer Prev, 2022) further demonstrated that high ANXA1 expression was associated with advanced tumor stage, large tumor volume, and lymph node metastasis. Silencing the ANXA1 gene significantly reduced cell proliferation and invasion.

[0049] The limitation is that although existing antibodies can detect ANXA1 expression, there is a lack of antibody design targeting CCA-specific epitopes, and no commercial diagnostic kit has been developed.

[0050] 3. Prognostic value in ovarian cancer:

[0051] Manai et al. (InVivo, 2020) studied 156 epithelial ovarian cancer (EOC) samples and found that 52% of tumor tissues were highly expressed in ANXA1, while only 26% of normal ovarian tissues were positive (P = 0.00794). High ANXA1 expression was associated with longer overall survival and was an independent prognostic factor.

[0052] The limitation is that the role of ANXA1 in EOC is opposite to that in breast cancer and shows tissue specificity, but existing antibodies are not optimized for its prognostic-related properties, which limits its clinical application.

[0053] 4. Diagnostic and prognostic value in lung cancer:

[0054] Elakad et al. (DisMarkers, 2021) found that strong ANXA1 expression in squamous cell lung cancer (SQCLC) was associated with longer survival (P = 0.019). Knockout of ANXA1 promoted cell migration and inhibited proliferation, suggesting a role in suppressing metastasis. Huang et al. (Tumour Biol, 2015) reported elevated serum anti-ANXA1 IgG levels in lung cancer patients, suggesting its diagnostic potential.

[0055] This limitation is that the mechanism of action of ANXA1 in lung cancer has not been fully elucidated and existing antibodies fail to achieve consistent specific detection in different lung cancer subtypes.

[0056] 5. Correlation in other cancers:

[0057] Hairy cell leukemia (HCL): Falini et al. (Lancet, 2004) reported that ANXA1 is specifically expressed in HCL, and immunocytochemistry showed 100% specificity and sensitivity, which can distinguish HCL from other B-cell malignancies. Acute myeloid leukemia (AML): Gaber M et al. (Egyptian Journal of Haematology, 2020) reported that ANXA1 expression in the bone marrow of 60 Egyptian patients with newly diagnosed AML was evaluated by flow cytometry. The average expression at diagnosis was (

[0058] The ANXA1 expression in the 20 patients was significantly higher (57.1±28.0) than in the 20 controls (6.28±5.4, P<0.0001). ANXA1 was positive in 76.7% of patients (≥20%), with a higher complete remission rate (52.2%) than in the negative group (14.3%). Using a commercially available anti-ANXA1 antibody combined with a FITC secondary antibody, the assay was sensitive and specific. ANXA1 positivity was associated with favorable cytogenetics, with expression (67.7±22.9) higher than in the unfavorable group (48.9±29.1, P=0.009), and a longer survival (8.68 vs. 4.16 months, P=0.03). It was positively correlated with MPO and CD64 (P=0.01, 0.03), demonstrating diagnostic and prognostic potential.

[0059] Colorectal cancer: Hagihara et al. (SciRep, 2019) found that ANXA1 promotes cell cluster growth through the PI3K / AKT pathway and is associated with metastatic potential.

[0060] Nasopharyngeal carcinoma: Zeng et al. (MedHypotheses, 2013) proposed that low expression of ANXA1 is associated with radiotherapy resistance and poor prognosis.

[0061] Renal cell carcinoma: Yamanoi et al. (Int J Urol, 2019) found that high ANXA1 expression was associated with malignant potential and poor disease-free survival (P = 0.031).

[0062] Pancreatic cancer: Oshi et al. (Cells, 2021) reported that high expression of ANXA1 was associated with EMT, proliferation, and drug sensitivity, and was associated with poor prognosis.

[0063] Melanoma: Sandri et al. (Cells, 2023) found that ANXA1 secreted by neutrophils promotes lung melanoma metastasis through the FPR pathway.

[0064] This limitation is that most of the above studies used non-specific antibodies and failed to design antibodies targeting specific functional domains of ANXA1 (such as the N-terminus), which limits their wide application in various cancers.

[0065] Antibody preparation method:

[0066] Mice were immunized with a synthetic peptide corresponding to the N-terminal sequence of ANXA1 (amino acid residues 1-40 plus a cystine, sequence MVSEFLKQAWFIENEEQEYVQTVKSSKGGPGSAVSPYPC). This peptide was conjugated to the carrier protein KLH and emulsified in Freund's incomplete adjuvant. Balb / c mice were then immunized. Lymphocytes harvested from spleen tissue were fused with mouse myeloma SP2 / 0-Ag14 cells in a culture medium containing 50% polyethylene glycol. The resulting hybridoma cells were subcloned three times by limiting dilution. Culture supernatants were assayed for anti-ANXA1 antibodies by enzyme-linked immunosorbent assay (ELISA). Hybridomas were screened by ELISA and Western blot to identify clones producing high-affinity, N-terminus-specific antibodies. The resulting hybridoma clone, BY026, was obtained.

[0067] Antibody sequence:

[0068] NO.1Heavy chain sequence: VH Amino acid sequence

[0069]

[0070] NO.2Light chain sequence:VL

[0071] Specific embodiment one:

[0073] Flow cytometry was used to detect the expression of ANXA1 in human leukemia cell line HL-60 cells.

[0074] Experimental materials and preparation

[0075] Flow cytometry was used to examine the expression of ANXA1 in the human leukemia cell line HL-60. Required materials include: the HL-60 cell line (derived from an acute myeloid leukemia, grown in suspension); a purified anti-ANXA1 antibody; a commercially available control antibody; and a fluorescently labeled secondary antibody, donkey anti-mouse IgG-Alexa 488 (from ThermoFisher). Furthermore, the experiment required RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and phosphate-buffered saline (PBS, pH 7.4). A flow cytometer (such as a BD FACSCalibur), a centrifuge, 1.5 mL centrifuge tubes, and pipettes were also required.

[0076] Cell preparation

[0077] First, HL-60 cells were cultured in RPMI-1640 medium containing 10% FBS in a 37°C, 5% CO2 incubator to ensure that the cells were in the logarithmic growth phase (density of approximately 0.5-1×10 6 cells / mL). On the day of the experiment, gently mix the cells and transfer them to a 15 mL centrifuge tube. Centrifuge at 4°C, 300 × g for 5 minutes and discard the supernatant. Resuspend the cell pellet in 1 mL of PBS and gently pipette to disperse into a single-cell suspension. Adjust the concentration to approximately 1 × 10 cells / mL by cell counting. 6 cells / mL, and then dispensed into 1.5 mL centrifuge tubes, each containing 1×10 5 -2×10 5 Prepare cells for subsequent staining.

[0078] Antibody staining

[0079] The staining process consisted of two stages: primary antibody incubation and secondary antibody incubation. For the primary antibody incubation, 50 μL of anti-ANXA1 antibody (diluted 1:50 in RPMI-1640 with 10% FBS) was added to experimental group 1; 50 μL of anti-ANXA1 control antibody (also diluted 1:50) was added to experimental group 2; and 50 μL of RPMI-1640 with 10% FBS (no primary antibody) was added to the control group. After adding the antibodies to the cells, the cells were incubated at 4°C in the dark for 30 minutes. Subsequently, the cells were washed twice with 1 mL of PBS (centrifuged at 300 × g for 5 minutes at 4°C). For the secondary antibody incubation, 50 μL of donkey anti-mouse IgG-Alexa 488 was diluted 1:300 in PBS and added to each tube. After incubation at 4°C in the dark for 30 minutes, the cells were washed again twice with PBS. Finally, the cells were resuspended in 200–300 μL of PBS and transferred to flow cytometry tubes.

[0080] Flow cytometry and data analysis

[0081] Start the flow cytometer, preheat and calibrate it, and set the Alexa 488 fluorescence detection channel (FL1, excitation 488 nm, emission 515-545 nm). First, run the control group (no antibody and secondary antibody only) to adjust the background fluorescence baseline. Then, load the experimental group samples sequentially, collecting 10,000-20,000 events per tube and saving the data in FCS format. For data analysis, use software such as FlowJo to generate a fluorescence intensity histogram. Overlay all samples and use the control group containing only the secondary antibody as the background to assess nonspecific staining. The shift in fluorescence signal in the experimental group compared to the control group reflects the expression level of ANXA1, and the increased intensity indicates the expression of ANXA1 in HL-60 cells.

[0082] Experimental results

[0083] The expression of ANXA1 in HL-60 cells detected by Anti-ANXA1 was 75.9%, which is higher than the detection rate of ANXA1 control antibody purchased from the market. This suggests that Anti-ANXA1 antibody has good affinity and can be used for flow cytometry as a diagnostic tool (such as Figure 1 shown);

[0084] Figure 1 The expression of ANXA1 in HL-60 cells. Auto (no antibodies) is the control group of cells without any reagents added; nd Ab only is the control group with only secondary antibody added; Anti-ANXA1 is the antibody experimental group; ANXA1 control antibody is the antibody control group purchased from the market. Specific embodiment two:

[0086] ANXA1 monoclonal antibody was used for cytofluorescence immunostaining to detect the expression of ANXA1 in cancer cell lines.

[0087] AnnexinA1 (ANXA1) is a calcium-regulated phospholipid-binding protein that is closely associated with the development and progression of cancer. Its expression levels and subcellular localization vary significantly across different tumor types. To further investigate the expression characteristics and biological significance of ANXA1 in tumor cells, we used an ANXA1 monoclonal antibody (mAb) to investigate the expression and distribution of ANXA1 in the human brain tumor cell line SH-SY5Y, the ovarian cancer cell line OVCAR-3, and the mouse melanoma cell line B16 using immunofluorescence staining.

[0088] The experimental process starts with cell culture. After the cells grow to the logarithmic phase in the corresponding culture medium containing 10% fetal bovine serum, about

[0089] 1×105 cells were fixed with 4% paraformaldehyde for 30 minutes to preserve the structure. Subsequently, the fixative was removed by washing three times with PBS. The cells were then permeabilized with PBS containing 1% Triton X-100 and 10% sheep serum for 30 minutes to initially block nonspecific growth sites. Subsequently, cells were further blocked with PBS containing 5% bovine serum albumin and 0.1% Tween 20 for 1 hour to reduce background fluorescence. Anti-ANXA1 monoclonal antibody (diluted to 1:100) was then added to the cells and incubated at 4°C for 4 hours to allow specific binding to ANXA1.

[0090] After washing three times with PBS to remove unbound primary antibody, fluorescently labeled goat anti-mouse IgG-Alexa 594 secondary antibody (1:300 dilution) (Thermofisher Cat#A-11032) was added and incubated in the dark for 1 hour at room temperature to label the location of ANXA1 and emit red fluorescence. After washing three more times with PBS, the nuclei were stained with DAPI (1 μg / mL) for 30 minutes at room temperature to label the nuclei and emit blue fluorescence. Finally, the stained cells were placed on slides and observed using a Nikon fluorescence microscope, capturing the red signal of ANXA1 and the blue signal of the nuclei. A negative control group used only the secondary antibody to assess nonspecific fluorescence.

[0091] The results are as follows Figure 2 The results showed that in the tested cells, ANXA1 was mainly distributed in the cytoplasm or membrane, and the red fluorescence was clearly separated from the DAPI blue signal.

[0092] This experiment demonstrates the potential of ANXA1 monoclonal antibodies in immunofluorescence staining, which can be used to detect the expression and localization of ANXA1 in tumor cell lines, providing a reliable method for studying its function in cancer.

[0093] ANXA1's standardized technology supports the design of diagnostic kits or research tools, especially for the research and application of leukemia and solid tumors. Specific embodiment three:

[0095] Immunohistochemistry was used to detect the expression of ANXA1 in ovarian cancer tissues.

[0096] To validate the potential application of the present method for detecting ANXA1 expression in ovarian cancer tissue, we designed and conducted immunohistochemistry (IHC) experiments to assess ANXA1 expression in epithelial ovarian cancer (EOC) and normal ovarian tissue, and to explore its application in IHC diagnosis. All samples were obtained from clinical pathology archives, approved by the ethics committee, and informed consent was obtained from the patients. The following detailed description of the experimental procedures and analytical methods demonstrates the utility of the present method in cancer diagnosis and prognosis assessment.

[0097] Experimental materials and methods

[0098] 1. Sample Preparation: Fifteen EOC tissue samples (including different histological subtypes, such as serous, mucinous, and endometrioid carcinomas) were collected. All samples were fixed in formalin and embedded in paraffin (FFPE) and prepared into tissue sections (4 μm thickness) to ensure detection consistency. Before IHC staining, tissue sections were dewaxed in xylene, rehydrated with graded ethanol, and subjected to antigen retrieval with citrate buffer (pH 6.0) for 15 minutes to expose ANXA1 antigenic sites.

[0099] 2. Antibodies and staining: Anti-ANXA1 monoclonal antibody was used as the primary antibody for specific detection of ANXA1. The antibody was diluted at a ratio of 1:2000 in phosphate-buffered saline (PBS, pH 7.4) containing 2% bovine serum albumin (BSA). Tissue sections were incubated with the primary antibody for 1 hour at room temperature and then washed three times with PBS for 5 minutes each. Next, a horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody (purchased from a commercial supplier at a dilution ratio of 1:500) was added and incubated at room temperature for 30 minutes. 3,3′-diaminobenzidine (DAB) substrate was used for color development, and the reaction time was controlled within 5 minutes to generate a brown precipitate signal. Subsequently, the cell nuclei were counterstained with hematoxylin (staining for 2 minutes), dehydrated with graded ethanol, and transparentized with xylene before mounting.

[0100] 3. Microscopic Observation and Scoring: Stained tissue sections were independently evaluated using a light microscope (Olympus BX51, 40× magnification). ANXA1 expression was scored by measuring the percentage of tumor cells positively stained, ranging from 0% (no positive cells) to 100% (all cells positive). Positive cells were defined as cells exhibiting a clear brown signal in the cytoplasm or membrane. The score for each sample was based on the average of at least five random fields of view to ensure representative results.

[0101] Experimental results

[0102] like Figure 3 Results showed that among 15 EOC samples, ANXA1 was positive (>55% of tumor cells stained) in 67% (10 / 15) of cases, while negative (≤55% stained) in 33% (5 / 15). In EOC-positive cases, ANXA1 was primarily localized in the cytoplasm and membrane of tumor cells, with some highly expressed samples showing nuclear staining, suggesting a possible association with tumor aggressiveness. Figure 3 Representative ovarian cancer tissues were immunohistochemically stained for ANXA1. According to relevant research reports (Manai M et al., 2020), ANXA1-positive cases have significantly longer OS than negative cases, suggesting that ANXA1 expression levels may serve as a prognostic marker.

[0103] Patent application value

[0104] This experiment successfully detected ANXA1 expression in ovarian cancer tissue using our high-affinity anti-ANXA1 monoclonal antibody using IHC, and achieved differentiation between positive and negative results by setting a threshold. This method is simple to operate, highly reproducible, and can intuitively reflect the spatial distribution and expression level of ANXA1, making it suitable for clinical pathological diagnosis. This invention can be used to develop diagnostic kits based on ANXA1 expression, combining TMA and IHC techniques to provide a reliable tool for the early diagnosis, classification, and prognostic assessment of ovarian cancer. Furthermore, this method can be extended to other ANXA1-related cancers (such as breast and lung cancer), further expanding its clinical application and possessing significant commercial potential. Specific embodiment four:

[0106] The expression of ANXA1 in body fluids and cell supernatants was detected by ELISA.

[0107] To validate the potential application of the present invention's ANXA1 monoclonal antibody for detecting ANXA1 levels in body fluids or cell supernatants using an enzyme-linked immunosorbent assay (ELISA), we selected MCF-7 as a cell line for detecting ANXA1 secretion, as MCF highly expresses ANXA1 (Ang et al., 2009). We conducted an experiment to quantitatively measure ANXA1 levels in cell supernatants using a kit combining an anti-ANXA1 polyclonal antibody as a capture antibody and an anti-ANXA1 monoclonal antibody as a detection antibody. This experiment aimed to demonstrate the high sensitivity and specificity of this method for cancer diagnosis, prognosis, and research. The experimental procedures and analysis of the results are described in detail below.

[0108] Experimental materials and methods

[0109] 1. Sample Collection and Preparation: Cell Supernatant Samples: MCF-7 breast cancer cells (ATCC) were cultured to 80% confluency in DMEM supplemented with 10% fetal bovine serum (FBS). After removing the medium, the cells were incubated in serum-free DMEM for 24 hours. The supernatant was collected, centrifuged at 2000 × g for 10 minutes at 4°C to remove cell debris, and stored at -80°C. Sample Pre-dilution: Cell supernatant was diluted 4-fold (25 μL supernatant + 75 μL diluent) based on preliminary test results to ensure the signal was within the standard curve range. Sample diluent was 1X PBS containing 2% bovine serum albumin (BSA).

[0110] 2. Reagent Preparation: Wash Buffer: Dilute 20X Wash Buffer Concentrate (25 mL) with 380 mL of deionized water to 1X, mix thoroughly, and store at 4°C. 20X Wash Buffer is 20X concentrated PBS containing 1% Tween-20. Biotinylated Antibody: Briefly centrifuge the anti-human Annexin A1 antibody biotin conjugate, add 100 μL of 1X Assay Diluent B to prepare a concentrate, mix gently, and dilute 1:80 (e.g., 50 μL concentrate + 3950 μL of 1X Assay Diluent B). Use immediately. Standards: Briefly centrifuge the lyophilized standard (recombinant human ANXA1) and add 400 μL of AssayDiluent C to prepare a 400 ng / mL stock standard solution. Mix thoroughly and then dilute with AssayDiluent C according to the following concentration gradient (400, 266.7, 177.8, 118.5, 79, 52.7, 0 ng / mL), mixing thoroughly at each step.

[0111] Streptavidin-HRP: Briefly centrifuge Streptavidin-HRP (600X) and dilute 1:600 in 1X Assay Diluent B (e.g., 20 μL concentrate + 11.98 mL diluent) within 15 minutes before use. Assay Diluent B (5x concentrate) is 5x PBS containing 5% BSA and 0.5% tween-20; Assay C is 1X PBS + 1% BSA.

[0112] 3. ELISA Procedure: Remove the 96-well plate pre-coated with ANXA1 antibody from the kit. Determine the number of 8-well strips required based on the sample volume (duplicate wells per sample). Store unused strips at 2-8°C. Capture Antigen: Add 100 μL of each standard solution (0-400 ng / mL) to the standard wells and 100 μL of pre-diluted sample to the sample wells. Cover the plate and incubate at room temperature with shaking for 2.5 hours. Wash: Discard the buffer and wash four times with 300 μL of 1X Wash Buffer, filling each time with a multichannel pipette or automated plate washer. After the final wash, invert and pat dry. Biotinylated Antibody Incubation: Add 100 μL of diluted biotinylated antibody to each well, incubate at room temperature with shaking for 1 hour, and wash four times. Streptavidin-HRP Incubation: Add 100 μL of diluted Streptavidin-HRP solution to each well, incubate at room temperature with shaking for 45 minutes, and wash four times. Color development and termination: Add 100 μL TMB substrate to each well, incubate in the dark with shaking for 30 minutes (the solution turns blue), then add 50 μL Top Solution (the solution turns yellow) and tap to mix.

[0113] 4. Reading and data analysis:

[0114] (1) Within 30 minutes after the reaction was terminated, the absorbance (OD value) at 450 nm was read using a microplate reader (Thermo Fisher Multiskan FC).

[0115] (2) Use four-parameter logistic regression software (such as GraphPad Prism) to generate a standard curve based on the OD value of the standard, calculate the ANXA1 concentration in the sample, and multiply it by the dilution factor to obtain the original concentration.

[0116] Experimental results

[0117] like Figure 4Results showed that the ANXA1 concentration in MCF-7 cell supernatant was 210.3±45.1 ng / mL, reflecting the secretory properties of tumor cells. The standard curve range was 1.6-400 ng / mL, with a limit of detection of 2.5 ng / mL, intra-assay CV% <11%, and inter-assay CV% <12%, demonstrating high reproducibility and stability of the method. The recovery rate of cell supernatant was 77% (range 68-87%), demonstrating good linearity.

[0118] Patent application value

[0119] This study successfully measured ANXA1 concentrations in cell supernatants using an ELISA method using an ANXA1 monoclonal antibody (pre-coated in the kit). This method is simple to use, highly sensitive (minimum detection limit 2.6 ng / mL), and applicable to a variety of biological samples. This method can be used to develop in vitro diagnostic kits, and the technology can be expanded to detect body fluids for cancer (such as leukemia and lung cancer) or inflammatory diseases, demonstrating broad clinical translational potential and commercial prospects.

[0120] The present invention provides a novel monoclonal antibody that specifically targets the N-terminal domain of ANXA1 and is used to detect ANXA1 expression in blood and tissue samples. This antibody is intended to:

[0121] 1. Improve diagnostic specificity: By targeting the N-terminal domain and leveraging its key role in membrane interactions, the ability to identify cancer-specific expressions is enhanced, especially for diseases such as hairy cell leukemia, breast cancer, and bile duct cancer.

[0122] 2. Broaden the scope of application: Fill the gaps in existing technologies in the detection of non-EMTCTC and leukemia cells, and achieve diagnostic coverage for a wider range of cancer types.

[0123] 3. Dual functionality: It can be used not only for tissue immunoassays, but also for detecting serum anti-ANXA1 antibody levels, providing a comprehensive tool for early diagnosis and prognostic assessment.

[0124] 4. Mechanistic Support: ANXA1 affects tumor cell proliferation, migration, and metastasis through signaling pathways such as PI3K / AKT, mTOR-S6, EMT, and FPR, providing a solid biological basis for the diagnostic target of this antibody.

[0125] In summary, the present invention, by developing a novel ANXA1 monoclonal antibody and related detection methods, meets the existing demand for highly specific and widely used diagnostic tools, providing an innovative solution for the early diagnosis and prognosis assessment of various cancers, such as breast cancer, bile duct cancer, and lung cancer.

[0126] The embodiments of the present invention are only used to illustrate the present invention and do not limit the scope of the claims. Other substantially equivalent alternatives that can be thought of by those skilled in the art are all within the scope of protection of the present invention.

Claims

1. An antibody specific for Annexin A1, characterized in that Included are monoclonal antibodies that specifically bind to an epitope within the N-terminal domain of human Annexin A1 (ANXA1).

2. The annexin A1-specific antibody according to claim 1, characterized in that The epitope includes amino acid residues 1-30 of ANXA1 (MAMVSEFLKQAWFIENEEQEYVQTVKSSKG).

3. The annexin A1-specific antibody according to claim 1, characterized in that The monoclonal antibody heavy chain sequence is shown in antibody sequence No.

1.

4. The annexin A1-specific antibody according to claim 1, characterized in that The monoclonal antibody light chain sequence is shown in antibody sequence No.

2.

5. A method for using an annexin A1-specific antibody, characterized in that: A method for diagnosing hairy cell leukemia (HCL) or acute myeloid leukemia (AML) in a subject, comprising: a) contacting a blood or bone marrow sample of a subject with the antibody of claim 1; b) Detecting the binding of the antibody to cell surface ANXA1 by flow cytometry; c) Diagnosis of HCL or AML based on elevated ANXA1 expression.

6. A method for using an annexin A1-specific antibody, characterized in that: A method for detecting circulating tumor cells (CTCs) in a cancer subject is employed, comprising: a) isolating cells from a blood sample of a subject; b) contacting the isolated cells with the antibody of claim 1; c) detecting ANXA1 on the surface of CTCs by flow cytometry, wherein the cancer is selected from pancreatic cancer, breast cancer, or lung cancer.

7. A method for using an annexin A1-specific antibody, characterized in that: A method for diagnosing or prognosing cancer in a subject, comprising: a) contacting a tissue sample of a subject with the antibody of claim 1; b) Detecting the binding of the antibody to ANXA1 by immunohistochemistry; c) Determining the presence or progression of cancer based on the ANXA1 expression pattern.

8. A diagnostic kit for annexin A1-specific antibodies, characterized in that: The antibody of claim 1, a fluorophore-conjugated secondary antibody, and reagents for performing flow cytometry or immunohistochemistry.

Images