Pancreatic cancer biomarker and use thereof

Abstract

The application discloses a pancreatic cancer diagnosis marker, and the marker is FAM83A. The application also discloses a kit for detecting FAM83A protein, which comprises effective FAM83A protein, two effective anti-FAM83A protein monoclonal antibodies and matched detection reagents, the two anti-FAM83A protein monoclonal antibodies are monoclonal antibody 1 and monoclonal antibody 2, the heavy chain variable region and light chain variable region sequences of the monoclonal antibody 1 are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the heavy chain variable region and light chain variable region sequences of the monoclonal antibody 2 are shown as SEQ ID NO. 3 and SEQ ID NO. 4. The kit has the advantages of wide linear range and fast detection speed.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a pancreatic cancer biomarker and its application. Background Technology

[0002] Pancreatic cancer has the highest mortality rate among all cancer types, with an average 5-year survival rate of approximately 10% in the United States. The lack of obvious early symptoms makes diagnosis difficult, leading to poor treatment outcomes and prognosis, thus increasing mortality. Radical surgery is the only curative treatment, while adjuvant chemoradiotherapy is currently the main treatment for advanced pancreatic cancer. With the continuous development of various comprehensive treatment methods, the overall survival (OS) of pancreatic cancer patients has gradually improved; however, most patients lose the opportunity for surgical treatment due to distant metastasis or local invasion. Immunotherapy has recently revolutionized anti-tumor treatment because it can mobilize the patient's immune system to enhance its anti-tumor capabilities. A new human cancer immunophenotyping classification has recently been proposed internationally: immune sensitivity, immune exemption, and immune desert, emphasizing that different immune subtypes can influence anti-tumor responses. Therefore, exploring biomarkers involved in immune subtype transitions is crucial for a better understanding of their immune mechanisms and for adjuvant immunotherapy in pancreatic cancer patients.

[0003] Our previous research has shown that extensive bioinformatics analysis has revealed several key genes playing important roles in immune subtype transitions. Therefore, bioinformatics methods can be used to screen for suitable biomarkers for the diagnosis or treatment of pancreatic cancer. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, one objective of this invention is to provide a diagnostic biomarker for pancreatic cancer, a monoclonal antibody for the biomarker, and a method for its preparation. Another objective of this invention is to provide a kit for the detection of FAM83A protein prepared using the monoclonal antibody prepared by this invention and the FAM83A protein.

[0005] Therefore, the present invention provides an application of a pancreatic cancer diagnostic biomarker, wherein the biomarker is FAM83A, and the application of FAM83A in the preparation of pancreatic cancer diagnostic reagents.

[0006] Furthermore, the present invention also provides a kit for detecting FAM83A protein, the kit comprising an effective amount of FAM83A protein, two effective amounts of monoclonal antibodies against FAM83A protein, and matching detection reagents. The two monoclonal antibodies against FAM83A protein are monoclonal antibody 1 and monoclonal antibody 2. The heavy chain variable region and light chain variable region sequences of monoclonal antibody 1 are shown in SEQ ID NO.1 and SEQ ID NO.2, and the heavy chain variable region and light chain variable region sequences of monoclonal antibody 2 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0007] Preferably, the effective amount of FAM83A protein described in this invention is a standard prepared using FAM83A protein, and the concentrations of the standard are 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, and 0 μg / ml.

[0008] Preferably, the monoclonal antibody 2 coated ELISA plate of the present invention has a coating concentration of 1 μg / ml.

[0009] Preferably, the monoclonal antibody 1 described in this invention is labeled with HRP, and the HRP-labeled monoclonal antibody 1 is diluted 10,000 times when used.

[0010] In another aspect, the present invention also provides the application of the monoclonal antibody 1 against FAM83A protein and the monoclonal antibody 2 against FAM83A protein described in 1 in the preparation of FAM83A protein detection reagent.

[0011] This invention utilizes bioinformatics techniques to identify a biomarker, FAM83A, from numerous pancreatic cancer prognostic-related genes. Based on this, a monoclonal antibody for detecting the FAM83A protein and diagnostic reagents or kits targeting the FAM83A protein were prepared.

[0012] The FAM83A protein biomarker screened in this invention has good indicative effect in pancreatic cancer and can be used as a target for the diagnosis and treatment of pancreatic cancer.

[0013] This invention screened two specific monoclonal antibodies based on the FAM83A protein, providing a good tool for the detection of FAM83A protein. Monoclonal antibody 1 and monoclonal antibody 2 both exhibit good specificity and sensitivity, making them suitable for the detection of FAM83A protein.

[0014] Based on research on the FAM83A protein and monoclonal antibodies, this invention develops a kit for the detection of the FAM83A protein. The kit possesses advantages such as high sensitivity, a wide linear range, and rapid detection speed. Therefore, this kit has significant reference value for the clinical auxiliary diagnosis of pancreatic cancer. Attached Figure Description

[0015] Figure 1 Expression analysis of FAM83A in various cancers. (A) Database-based expression of FAM83A in human cancers. (BH) Differential expression of FAM83A in tumor and normal tissues in TCGA and GTEx databases: (B) BLCA, (C) CESC, (D) HNSC, (E) LUAD, (F) LUSC, (G) PAAD, (H) ESCA. *p < 0.05; **p < 0.01; ***p < 0.001.

[0016] Figure 2 Overall survival and univariate Cox regression analysis of FAM83A in six human cancers based on TCGA. (AC), OS plots of FAM83A in LUAD (A), PAAD (B), and LUSC (C); forest plots of univariate Cox regression analysis: (DG), (D)OS, (E)DSS, (F)DFI, (G)PFI.

[0017] Figure 3 Laboratory Experiments and Clinical Significance of FAM83A in Pancreatic Cancer. (AD) Laboratory Experiments: (A) qRT-PCR results of FAM83A in tumor and normal tissues. (B) Immunohistochemical (IHC) staining of pancreatic cancer. (C) IHC staining of adjacent normal tissue in pancreatic cancer. (D) IHC staining of normal pancreatic tissue. (EJ) Clinical Significance of FAM83A: (E) Correlation analysis between FAM83A and AJCC staging. (F) Univariate Cox regression analysis. (G) Multivariate Cox regression analysis. (H) Nonograph based on FAM83A expression and N stage.

[0018] Figure 4 FAM83A Protein Content Assay Kit Standard Curve. Detailed Implementation

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0020] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0021] Example 1: Pan-cancer analysis of differential expression of FAM83A The results of the bioinformatics analysis are shown below: FAM83A (family with sequence similarity 83 member A) showed significant differential expression in 15 / 33 human cancers analyzed in the TGCA database. It was significantly overexpressed in BLCA (bladder urothelial carcinoma), BRCA (invasive breast cancer), CESC (cervical squamous cell carcinoma and cervical endometrial adenocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal cancer), GBM (glioblastoma multiforme), HNSC (head and neck squamous cell carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), PAAD (pancreatic cancer), READ (rectal adenocarcinoma), STAD (gastric adenocarcinoma), and UCEC (endometrial cancer), while significantly underexpressed in PRAD (prostate adenocarcinoma) and KICH (renal chromophobe carcinoma) compared to normal tissues. Figure 1 A).

[0022] The expression level of FAM83A in these 15 cancer types was reanalyzed using the GEPIA database. Significantly elevated expression was observed in BLCA, CESC, HNSC, LUAD, LUSC, and PAAD tissues, while expression was lower in ESCA. Figure 1 BH).

[0023] No significant differential expression was observed in the other 8 / 15 types of human cancer (Figure S1). Because the two methods demonstrated opposite expression trends for FAM83A in ESCA, ESCA was omitted from further analysis. These results suggest that upregulation of the FAM83A gene may play a crucial role in tumorigenesis.

[0024] Example 2: Prognostic Value of FAM83A Kaplan-Meier survival analysis of FAM83A in BLCA, CESC, HNSC, LUAD, LUSC, and PAAD showed that FAM83A was a positive risk factor for LUAD, LUSC, and PAAD. Figure 2 AC). Univariate Cox regression analysis showed that FAM83A was a risk factor for LUAD, LUSC, and PAAD in human cancer types. Figure 2 D), while DSS, DFI, and PFI analyses showed that FAM83A was a risk factor for LUAD and PAAD patients (D), Figure 2 EG).

[0025] Example 3: qRT-PCR and immunohistochemistry Real-time quantitative PCR: Tumor tissue and normal tissue pathologically diagnosed as pancreatic cancer were collected. Total RNA was extracted using TRIzol reagent; RNA content and purity were detected, and RNA purity was assessed based on the ratio of OD values ​​of 260 / 280 and 260 / 230; qualified RNA was reverse transcribed into single-stranded complementary DNA according to the reverse transcription kit instructions; real-time quantitative PCR was performed using the StepOnePlus real-time PCR system; 18S rDNA was used as an internal control, the threshold (Ct) was recorded, and the relative expression level was calculated. Primer sequences are shown below: FAM83A-R: 5'-tcctcgtcaaacagctccac-3'; FAM83A-F: 5'-ggtgttcgtttgtgtgctcc-3'; Immunohistochemical staining: IHC was performed on pancreatic cancer, adjacent normal tissue, and normal tissue fixed in 10% neutral formalin. According to the instructions, the collected tissues were stained with IHC, using monoclonal antibody 1 prepared in Example 5 of this invention at a dilution ratio of 1:1000.

[0026] The results showed that FAM83A was significantly highly expressed in pancreatic cancer tissues compared with the normal control group. Figure 3 A). IHC results showed that FAM83A was located in the cytoplasm, and FAM83A was also positively expressed in pancreatic cancer. Figure 3 BD).

[0027] Example 4: Independent Prognostic Analysis After conducting a clinical correlation analysis of different AJCC stages for these six cancer types, increased expression of FAM83A was found in advanced PAAD. Figure 3E). This suggests that FAM83A may be considered a harmful prognostic biomarker for pancreatic cancer patients. Including clinicopathological features in prognostic analyses showed that FAM83A and N stage were significantly associated with overall survival (OS). Univariate and multivariate regression analyses showed that FAM83A was an independent prognostic factor (E). Figure 3 F, G: P < 0.001, P = 0.029). Subsequently, we integrated FAM83A and N staging into a risk model predicting 1-, 2-, and 3-year survival rates. The area under the curve (AUC) of the multivariate ROC was 0.696 (…). Figure 3 H, I) confirmed that FAM83A is the strongest prognostic indicator. Calibration curves confirmed that the nomogram can accurately predict the 1-year, 2-year, and 3-year overall survival (OS) of pancreatic cancer patients. Figure 3 (J), indicating that FAM83A is a good predictive indicator for pancreatic cancer patients.

[0028] Example 5: Preparation and testing of monoclonal antibodies against FAM83A protein Using FAM83A protein (purchased from Wuhan Huamei Biotechnology Co., Ltd.) as the antigen, monoclonal antibodies were prepared using conventional hybridoma cell technology. The specific method is briefly described below: 1. Animal Immunization FAM83A protein was emulsified with Freund's complete adjuvant (200 μg / ml) and administered subcutaneously at multiple sites, 0.5 ml per animal. Two weeks later, the same dose and method of immunization were administered, but the adjuvant was changed to Freund's incomplete adjuvant. One week later, a third immunization was performed with the same dose, administered intraperitoneally without adjuvant. Blood samples were collected 7 days later to measure ELISA antibody titers. Animals with higher ELISA antibody titers (≥10) were selected. 5 Cell fusion was performed on the spleen cells of mice.

[0029] ELISA antibody titer assay: FAM83A protein was used to coat the microplate at 1 μg / ml, and the coating was incubated overnight at 2-8℃. After blocking, mouse serum was serially diluted 10-fold and added to the microplate at 0.1 ml / well, and incubated at 37℃ for 1 h. After washing, 5000-fold diluted HRP-labeled goat anti-mouse IgG secondary antibody was added, and the plate was incubated at 37℃ for 30 min. 0.1 ml of chromogenic reagent was added to each well, and the plate was incubated at 37℃ for 10 min. 2M sulfuric acid was added to stop the incubation, and 0.1 ml / well was added to each well. The OD450nm value was immediately measured. A positive result was defined as an OD450nm value greater than 0.1 and a P / N ratio (sample / blank control) > 2.1; otherwise, a negative result was defined as negative.

[0030] 2. Cell fusion 1×10 8 spleen cells and 1×10 7Myeloma cells SP2 / 0 were mixed in a 50ml centrifuge tube, and incomplete culture medium was added to a final volume of 30ml. The mixture was thoroughly mixed. Centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. Immediately, 1ml of preheated 50% PEG was added via a 1ml pipette over 30 seconds, while gently stirring. Preheated incomplete culture medium was added to terminate the PEG reaction. The mixture was centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. 5ml of complete culture medium was added to resuspend the cells and mix well, then the final volume of complete culture medium was added to a final volume of 50ml. The cells were aliquoted into 96-well cell culture plates (0.1ml per well) and incubated at 37°C in a 5% CO2 incubator. After 6 hours, 50μl of selection medium was added to each well. After 3 days, the medium was replaced halfway with selection medium. The growth of hybridoma cells was observed. When the cells reached more than 1 / 10 of the bottom area of ​​the well, the supernatant was aspirated, and the ELISA antibody titer was measured. Cells with higher ELISA titers (≥10) were selected. 6 Hybridoma cells were subcloned and screened using a limiting dilution method.

[0031] The selected hybridoma cells with higher performance were diluted with HT medium containing 20% ​​serum to three different dilutions: 5, 10, and 20 cells per milliliter. Each type of hybridoma cell was aliquoted into one 96-well plate, 0.1 ml / well. The cells were incubated at 37°C and 5% CO2 for 6 days. Antibody detection was initiated when visible clones appeared. Wells with only a single clone were marked under an inverted microscope, and the supernatant was used for antibody detection. Cells from antibody-positive wells were expanded and cryopreserved. Two superior hybridoma cell lines were selected and named Hybridoma Cell 1 and 2, respectively, and were cryopreserved in liquid nitrogen for future use.

[0032] 3. Production and purification of monoclonal antibodies The two obtained hybridoma cell lines were expanded and cultured, then suspended and counted for later use. 6-8 week old BALB / c mice were intraperitoneally inoculated with liquid paraffin, 0.5 ml per mouse. 7-10 days later, hybridoma cells 1 and 2 diluted with PBS were intraperitoneally inoculated, 5 × 10⁶ cells per mouse, respectively. 5 / 0.2 ml. After 5-7 days, observe the ascites production of mice daily. If the abdomen is significantly distended and the skin feels tense when touched, ascites can be collected. Usually, 3 ml of ascites can be collected from each mouse. Centrifuge the ascites (2000 rpm for 5 minutes) to remove cellular components and other precipitates, collect the supernatant, determine the antibody titer, aliquot, and store at -70℃ for later use.

[0033] The preserved ascites fluid was centrifuged at 12,000 rpm for 15 min at 4 °C to remove impurities. One portion of ascites fluid was mixed with two portions of 0.06 mol / L PPH 5.0 acetate buffer. Caprylic acid was added dropwise at a ratio of 33 μl per mL of diluted ascites fluid, with stirring at room temperature. The mixture was stirred at room temperature for 30 min, incubated at 4 °C for 2 h, and then centrifuged at 12,000 rpm for 30 min. The precipitate was discarded. The supernatant was filtered through a nylon sieve and dialyzed against 50 times its volume of PBS at 4 °C for 6 h. An equal volume of saturated ammonium sulfate solution was added to the supernatant after dialyzing. The mixture was incubated at 4 °C for at least 1 h, centrifuged at 10,000 g for 30 min, and the supernatant was discarded. The precipitate was dissolved in an appropriate amount of PBS and dialyzed against 50-100 times its volume of PBS overnight. A small amount of the dialyzed sample was appropriately diluted, aliquoted, and stored at -70 °C for later use. Two monoclonal antibodies were obtained (named Monoclonal Antibody 1 and 2, corresponding to hybridoma cells 1 and 2).

[0034] 4. Detection of monoclonal antibodies 4.1 Purity test: 20 μl of each of the two prepared monoclonal antibodies was analyzed by SDS-PAGE. The results showed that the SDS-PAGE purity of both monoclonal antibodies was greater than 90%.

[0035] 4.2 Concentration Testing: 20 μl of each of the two prepared monoclonal antibodies was taken and their concentrations were tested according to the BCA kit instructions. The concentrations of monoclonal antibodies 1 and 2 were 1.58 mg / ml and 1.89 mg / ml, respectively.

[0036] 4.3 Monoclonal Antibody Sequence Analysis The heavy chain and light chain variable regions of prepared monoclonal antibodies 1 and 2 were analyzed and determined using a sequencing method based on pancreatic cancer R amplification (see reference: "Chen Sumin, Yang Ping. Cloning and sequencing of the heavy chain variable region gene of anti-human liver cancer monoclonal antibody. Chinese Journal of Immunology, 1995, 011(001)-10~12"). The results showed that, after detection and analysis, the heavy chain and light chain variable region sequences of monoclonal antibody 1 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the heavy chain and light chain variable region sequences of monoclonal antibody 2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0037] SEQ ID NO.1: MYQSEVKLEEETVTLVQPASSTKLSCVVPRDTISNIVR QSSVFDWVAEIRLFKCRYVTHYANVQKGRFTVYTIPKNSVAKDKVNLRPEDTITDFTPIYSTFRSYWGQGSSVFISAAKT; SEQ ID NO.2: MKLDVLMTNSYSLPHKTSQASISCINVKWSIVHNSWTDYL ETYSMSSPGQSPKLLIYGASVNRFSGVPDRGAGTTLELKSGYPLKINRVEAEDGSEYYCLQGSHVPLTFGAGTTLELKR; SEQ ID NO.3: MFEVHLVESAKTLVKPETVTLSCAAPASSFSHPREEVRQT PDTFRSVVATIGSRGTMHQDWDSVTKGRPTISRYTIPALYLQVEVHKSEDTAMPREESEFYSVSEYYSKCRVNWGQGASV; SEQ ID NO.4: MLGDIVMSGSGKFMLTSGGGDRVSPKDINASQDVSTAER QNQKPGQSPDQDSYSASYRSMSSTDRFTGSLTKDDFTSPIVSVQAEDNECCQQHYSSPLGGASVELKRADAWTDIFPPSSE.

[0038] 5. Applications of Monoclonal Antibodies 5.1 Immunohistochemistry The monoclonal antibody 1 prepared using the present invention can react well with the FAM83A protein in cancer tissue, as detailed in Example 3 of the present invention.

[0039] 5.2 FAM83A Protein Content Detection Monoclonal antibody 2 was diluted to 1 μg / ml using carbonate coating buffer, 0.1 ml / well, and coated overnight at 2–8°C. Blocking was performed using PBST containing 1% BSA, 0.2 ml / well, at 37°C for 2 h. After washing, the sample (which can be serum, tissue supernatant, or body fluid, etc., generally requires dilution during testing, such as 10-fold or 20-fold dilution) and standards (6 gradients: 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, and 0 μg / ml) were added to the ELISA plate. 1 ml / well, incubate at 37℃ for 1 h; after washing, add 10000-fold diluted HRP-labeled monoclonal antibody 1 (HRP labeling was performed using a commercial kit), incubate at 37℃ for 30 min; add 0.1 ml / well of chromogenic buffer, incubate at 37℃ for 10 min, add 0.1 ml / well of 2M sulfuric acid to stop the reaction; immediately measure the OD450nm value; establish a standard curve with OD450nm as the x-axis and standard concentration as the y-axis, substitute the OD450nm value of the sample into the formula, calculate the concentration of the diluted sample, and multiply this concentration by the dilution factor to obtain the concentration of FAM83A protein in the sample. The results show ( Figure 4 This kit has advantages such as high sensitivity, wide linear range, and fast detection speed.

[0040] Application of the reagent kit: The inventors collected 20 serum samples from pancreatic cancer patients and 35 normal serum samples from a hospital in Guangxi. The prepared reagent kit was used to test all 55 serum samples. The results showed that the detection values ​​of the 20 pancreatic cancer serum samples were all greater than 10 ng / ml, and the detection values ​​of the 35 normal serum samples were all ≤0, indicating that the reagent kit has good accuracy, specificity, and sensitivity.

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A kit for detecting FAM83A protein, characterized in that, The kit includes an effective amount of FAM83A protein, two effective amounts of anti-FAM83A monoclonal antibodies, and matching detection reagents. The two anti-FAM83A monoclonal antibodies are monoclonal antibody 1 and monoclonal antibody 2. The heavy chain variable region and light chain variable region sequences of monoclonal antibody 1 are shown in SEQ ID NO.1 and SEQ ID NO.2, and the heavy chain variable region and light chain variable region sequences of monoclonal antibody 2 are shown in SEQ ID NO.3 and SEQ ID NO.

4.

2. The reagent kit according to claim 1, characterized in that, The effective amount of FAM83A protein is a standard prepared using FAM83A protein, and the concentrations of the standard are 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, and 0 μg / ml.

3. The reagent kit according to claim 1, characterized in that, The monoclonal antibody 2 was used to coat the ELISA plate at a concentration of 1 μg / ml.

4. The reagent kit according to claim 1, characterized in that, The monoclonal antibody 1 is labeled with HRP, and the HRP-labeled monoclonal antibody 1 is diluted 10,000 times when used.

5. The use of the monoclonal antibody 1 against FAM83A protein and the monoclonal antibody 2 against FAM83A protein as described in claim 1 in the preparation of a FAM83A protein detection reagent.

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