Monoclonal antibody against human aldo-keto reductase 1B10 protein and application thereof

Abstract

This invention provides a hybridoma cell line that produces a monoclonal antibody against human aldehyde-ketone reductase 1B10 protein and its applications. The hybridoma cell lines are named 8D7-A9-E2 and 8D7-A9-H4, and are deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession numbers CCTCC NO: C202141 and CCTCC NO: C202142, respectively, on January 28, 2021. The monoclonal antibody secreted by the hybridoma cells has a high affinity for human aldehyde-ketone reductase 1B10 protein. A human aldehyde-ketone reductase 1B10 protein detection kit constructed using the monoclonal antibody exhibits high sensitivity and specificity, and shows good performance in various detection methods, including enzyme-linked immunosorbent assay (ELISA), time-resolved immunofluorescence assay, and chemiluminescence detection.

Description

Technical Field

[0001] This invention belongs to the field of immunochemical technology, specifically relating to a monoclonal antibody against human aldehyde-ketone reductase 1B10 protein and its application. Background Technology

[0002] Liver diseases progress gradually from fatty liver (hepatitis), liver fibrosis, cirrhosis, to liver cancer. Currently, there is a lack of serum markers for early diagnosis, accurate diagnosis, and monitoring of these liver disease progressions. Liver diseases often lead to abnormal liver function, and indicators for monitoring liver function include aspartate aminotransferase (AST) and alanine aminotransferase (ALT). However, these markers are still insufficient for early diagnosis and monitoring disease progression. Liver cancer, lung cancer, and breast cancer are malignant tumors that threaten human health. Currently, there is a lack of effective methods for early detection, efficacy assessment, prognosis evaluation, or recurrence monitoring of liver cancer, lung cancer, and breast cancer. Therefore, there is an urgent need for a new target with high specificity and sensitivity that can be used for early screening, early diagnosis, efficacy assessment, prognosis evaluation, or recurrence monitoring of liver function, liver diseases, and various human cancers, as well as an easy-to-use, harmless, and clinically valuable detection method.

[0003] AKR1B10 protein is expressed at low levels in normal livers. When the liver is damaged, AKR1B10 protein is expressed, or the amount of AKR1B10 protein entering the blood increases. Therefore, AKR1B10 protein can be used for early screening, early diagnosis, efficacy assessment, prognosis evaluation, and recurrence monitoring of liver function, liver diseases (fatty liver, hepatitis, liver fibrosis, cirrhosis), and liver cancer.

[0004] AKR1B10 protein is highly expressed in lung cancer, breast cancer, and other tumors, promoting tumor growth and metastasis, and is associated with tumor drug resistance. Inhibiting or knocking down the AKR1B10 gene can suppress tumor growth and metastasis, increase tumor sensitivity to chemotherapeutic drugs, and promote tumor radiotherapy resistance. AKR1B10 protein expressed in tumor cells can be secreted into the bloodstream via the lysosomal pathway, leading to elevated AKR1B10 protein levels in the blood of cancer patients. Therefore, detecting AKR1B10 protein levels in the blood can help diagnose tumor occurrence / recurrence and assess the effectiveness of anti-tumor treatments.

[0005] Therefore, developing a series of diagnostic kits based on the AKR1B10 protein for screening, diagnosis, efficacy assessment, prognosis evaluation, or recurrence monitoring of liver function, liver diseases (fatty liver, liver fibrosis, cirrhosis, etc.), and human tumors has significant clinical application value. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention develops a novel specific monoclonal antibody targeting the human tumor-specific antigen Aldo-Keto Reductase 1B10 (AKR1B10) protein, and uses this antibody to develop a sensitive, specific, and efficient diagnostic kit for cancer screening, diagnosis, efficacy assessment, prognosis evaluation, or recurrence monitoring.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a hybridoma cell line that produces a monoclonal antibody against human aldehyde-ketone reductase 1B10 protein. The hybridoma cell lines are named 8D7A9E2 and 8D7A9H4, and are deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession numbers CCTCC NO: C202141 and CCTCC NO: C202142, respectively, and the deposit date is January 28, 2021.

[0009] This invention obtained two murine monoclonal antibody cell lines, namely 8D7A9E2 and 8D7A9H4, with accession numbers CCTCC NO: C202141 and CCTCC NO: C202142, respectively. They were deposited at the China Center for Type Culture Collection on January 28, 2021, at Wuhan University, Wuhan, China, and were found to be viable.

[0010] In a second aspect, the present invention provides a monoclonal antibody against human aldehyde-ketone reductase 1B10 protein, said monoclonal antibody being produced by the hybridoma cell line described in the first aspect that produces the monoclonal antibody against human aldehyde-ketone reductase 1B10 protein.

[0011] Thirdly, the present invention provides a method for preparing the monoclonal antibody against human aldehyde reductase 1B10 protein as described in the second aspect. The method for preparing the monoclonal antibody includes: culturing the hybridoma cell line that produces the monoclonal antibody against human aldehyde reductase 1B10 protein as described in the first aspect, performing separation and purification, and obtaining the monoclonal antibody against human aldehyde reductase 1B10 protein.

[0012] Fourthly, the present invention provides the use of the monoclonal antibody against human aldehyde-ketone reductase 1B10 protein described in the second aspect in the preparation of kits for screening, diagnosis, efficacy assessment, prognosis evaluation, or recurrence monitoring of human liver function, human liver diseases, and cancer.

[0013] Preferably, the human liver disease includes hepatitis, fatty liver, liver fibrosis, or cirrhosis.

[0014] Preferably, the cancer includes liver cancer, lung cancer, or breast cancer.

[0015] Preferably, the kit contains reagents that specifically detect the level of AKR1B10 protein in a sample.

[0016] Preferably, the kit further includes an AKR1B10 protein standard.

[0017] Preferably, the samples detected by the kit include any one or a combination of at least two of the following: serum, plasma, urine, cerebrospinal fluid, ascites, breast milk, intestinal fluid, stool, saliva, vaginal secretions, or tissue samples.

[0018] In this invention, using the kit for screening, diagnosis, efficacy assessment, prognostic evaluation, or recurrence monitoring includes:

[0019] (a) Measure the level of AKR1B10 protein in biological samples from subjects using the reagent specifically designed to detect AKR1B10 protein;

[0020] (b) Compare the levels measured in (a) with the corresponding levels in normal samples.

[0021] This invention discovers that AKR1B10 protein is a secreted human aldehyde-ketone reductase that is highly expressed in the gastrointestinal epithelial tissue of healthy individuals and lowly expressed in the liver tissue. Since the AKR1B10 protein expressed and secreted by the gastrointestinal epithelium and liver of normal individuals enters the intestine and is excreted, the concentration of AKR1B10 protein in the blood of normal individuals is very low.

[0022] Fifthly, the present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting human aldehyde-ketone reductase 1B10 protein, wherein the ELISA kit contains the anti-human aldehyde-ketone reductase 1B10 protein monoclonal antibody as described in the second aspect.

[0023] Preferably, the monoclonal antibody is an HRP-labeled anti-human aldehyde reductase 1B10 protein monoclonal antibody.

[0024] Preferably, the enzyme-linked immunosorbent assay kit further comprises any one or a combination of at least two of the following: an enzyme-linked immunosorbent assay plate coated with a specific capture antibody, a coating buffer, a sample diluent, a blocking buffer, or an elution buffer.

[0025] In this invention, the enzyme-linked immunosorbent assay kit includes:

[0026] Enzyme-linked immunosorbent assay (ELISA) plates coated with specific capture antibodies; Coating buffer: 1×PBS, pH 7.3±0.1; Sample dilution buffer: 1×PBS, 1-5% BSA, 0.05-1% NaN3, pH 7.3±0.1; Blocking buffer: 1×PBS, 3-5% BSA; Elution buffer PBST: 1×PBS, 0.05-0.06% Tween-20; HRP-labeled monoclonal antibody.

[0027] In a sixth aspect, the present invention provides a time-resolved immunofluorescence assay kit for detecting human aldehyde reductase 1B10 protein, wherein the time-resolved immunofluorescence assay kit comprises the anti-human aldehyde reductase 1B10 protein monoclonal antibody described in the second aspect.

[0028] Preferably, the monoclonal antibody is a biotin-labeled monoclonal antibody against human aldehyde reductase 1B10 protein.

[0029] Preferably, the enzyme-linked immunosorbent assay kit further comprises a time-resolved fluorescence immunoassay plate coated with specific capture antibodies, a coating buffer, a sample diluent, a blocking buffer, an elution buffer, or any one or a combination of at least two of Streptavidin-Eu.

[0030] In this invention, the time-resolved fluorescence immunoassay kit (TRFIA) comprises:

[0031] Time-resolved fluorescence immunoassay (TRFIA) plate coated with specific capture antibody; Coating buffer: 1×PBS, pH 7.3±0.1; Sample dilution buffer: 1×PBS, 1-5% BSA, 0.05-1% NaN3, pH 7.3±0.1; Blocking buffer: 1×PBS, 3-5% BSA; Elution buffer PBST: 1×PBS, 0.05-0.06% Tween-20; Biotin-labeled monoclonal antibody; Streptavidin-Eu.

[0032] In a seventh aspect, the present invention provides a chemiluminescent detection kit for detecting human aldehyde-ketone reductase 1B10 protein, the chemiluminescent detection kit comprising the anti-human aldehyde-ketone reductase 1B10 protein monoclonal antibody as described in the second aspect.

[0033] Preferably, the monoclonal antibody is an anti-human aldehyde reductase 1B10 protein monoclonal antibody containing a chemiluminescent label, wherein the chemiluminescent label includes any one of acridine ester, ruthenium tripyridine, alkaline phosphatase, or luminol.

[0034] Preferably, the chemiluminescence detection kit further comprises any one or a combination of at least two of the following: chemiluminescent magnetic microparticles coated with specific capture antibodies, sample diluent, elution buffer, or luminescent substrate.

[0035] In this invention, the chemiluminescence detection kit includes:

[0036] Chemiluminescent magnetic microparticles coated with specific capture antibodies; sample dilution buffer: 1×PBS, 1-5% BSA, 0.05-1% NaN3, pH 7.3±0.1; elution buffer PBST: 1 PBST, 0.05-0.06% Tween-20; specific monoclonal antibody containing chemiluminescent markers; the chemiluminescent markers include acridinium ester, ruthenium tripyridine, alkaline phosphatase, and luminol; luminescent substrate reagent.

[0037] Eighthly, the present invention provides an immunoturbidimetric assay kit for detecting human aldehyde-ketone reductase 1B10 protein, the immunoturbidimetric assay kit comprising the anti-human aldehyde-ketone reductase 1B10 protein monoclonal antibody as described in the second aspect.

[0038] Preferably, the immunoturbidimetric assay kit further comprises any one or a combination of at least two of the following: reagent R1, reagent R2, calibration solution, or detection antibody. The reagent R1 is composed of phosphate buffer, polyethylene glycol, and sodium azide. The calibration solution is a human AKR1B10 antigen solution. The detection antibody is a polyclonal antibody obtained by immunizing goats or rabbits with the full-length AKR1B10 antigen.

[0039] Preferably, the R1 reagent contains 20-30 mmol / L Tris buffer, 5-15 g / L polyethylene glycol 8000, and 0.5-1% sodium azide, with a pH of 8.2.

[0040] Preferably, the R2 reagent contains 2-15 mg / mL of specific monoclonal antibody and 0.5-1% sodium azide, with a pH of 7.5.

[0041] Preferably, the concentration of human AKR1B10 antigen in the calibration solution is 0 ng / mL, 0.0625 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL, or 32 ng / mL.

[0042] The kits developed in this invention for detecting AKR1B10 protein using enzyme-linked immunosorbent assay (ELISA), time-resolved fluorescence immunoassay, chemiluminescence immunoassay, and immunoturbidimetric assay are applicable to liver function tests, liver diseases, and early diagnosis, population screening, efficacy assessment, recurrence monitoring, and prognostic evaluation of liver cancer, lung cancer, and breast cancer. They have the advantages of being rapid, objective, and highly accurate.

[0043] Liver function: The human AKR1B10 protein is mainly expressed in gastrointestinal epithelial cells and hepatocytes. The AKR1B10 protein expressed by gastrointestinal epithelial cells and hepatocytes enters the digestive tract directly or indirectly through the biliary system and is excreted from the body. However, when liver function is impaired, the AKR1B10 protein produced by hepatocytes enters the bloodstream, leading to an increase in serum AKR1B10 protein concentration.

[0044] Liver diseases: When hepatitis is severe, or after fatty liver (including alcoholic fatty liver and non-alcoholic fatty liver) is formed, or after it develops into liver fibrosis or cirrhosis, the expression of AKR1B10 protein in hepatocytes increases, and more of it enters the bloodstream.

[0045] Cancer diagnosis: When tumors are very small and patients have no clinical symptoms, early diagnosis of liver cancer, lung cancer, and breast cancer can be made by detecting changes in serum AKR1B10 protein.

[0046] Treatment guidance: Changes in serum AKR1B10 protein levels in patients with liver cancer, lung cancer, and breast cancer undergoing treatment can help determine the effectiveness of the treatment.

[0047] Monitoring for recurrence: Regularly monitor changes in serum AKR1B10 protein levels in treated liver cancer, lung cancer, and breast cancer patients; elevated levels indicate cancer recurrence.

[0048] Prognostic assessment: The level of AKR1B10 protein in the serum of patients with liver cancer, lung cancer, and breast cancer is related to tumor size, metastasis, recurrence rate, and survival rate, and can predict treatment effectiveness and patient prognosis.

[0049] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] (1) The present invention develops a new specific monoclonal antibody against the human tumor-specific antigen aldehyde-ketone reductase 1B10 protein. The monoclonal antibody described in the present invention has good specificity, high affinity for AKR1B10 protein, and no cross-reaction with other unrelated proteins.

[0052] (2) The present invention has developed a series of sensitive, specific and efficient diagnostic kits based on the monoclonal antibody. These kits have shown good results in various detection methods, including enzyme-linked immunosorbent assay (ELISA), time-resolved immunofluorescence assay (TRI), and chemiluminescence assay. They can be widely used in cancer screening, diagnosis, efficacy assessment, prognosis evaluation or recurrence monitoring. Attached Figure Description

[0053] Figure 1 This is an SDS-PAGE image of the AKR1B10 monoclonal antibody purified using the AKR1B10 antigen strain. Lane M: protein molecular weight marker; Lanes 1-8: number of tubes eluted during purification;

[0054] Figure 2 The results show the specificity of the purified murine AKR1B10 monoclonal antibody.

[0055] Figure 3A The results of immunohistochemical analysis of liver cancer tissue;

[0056] Figure 3B The results of immunohistochemical detection of adjacent tissues in liver cancer;

[0057] Figure 4A The results of immunohistochemical analysis of breast cancer tissue;

[0058] Figure 4B Immunohistochemical results of adjacent normal tissue in breast cancer;

[0059] Figure 5A Immunohistochemical results of human lung cancer tissue;

[0060] Figure 5B Immunohistochemical results of adjacent non-cancerous tissue in human lung cancer;

[0061] Figure 6 A standard curve for a chemiluminescence assay kit to detect AKR1B10 protein;

[0062] Figure 7 Serum AKR1B10 concentrations in normal individuals and breast cancer patients;

[0063] Figure 8 Serum AKR1B10 concentrations in normal individuals and lung cancer patients;

[0064] Figure 9 Serum AKR1B10 concentrations in normal individuals and liver cancer patients. Detailed Implementation

[0065] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0066] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Invention Details

[0068] This invention relates to a specific monoclonal antibody against the human tumor-specific antigen aldehyde-ketone reductase 1B10 protein (hereinafter referred to as AKR1B10). The AKR1B10 protein is isolated and identified from human primary tumor tissues. The AKR1B10 protein has 316 amino acids and is significantly highly expressed in human liver cancer, lung cancer, and breast cancer tissues, playing a crucial role in promoting tumor cell growth, proliferation, invasion, and metastasis, as well as in the occurrence and development of tumors.

[0069] The AKR1B10 monoclonal antibody involved in this invention was produced by hybridoma cells obtained by immunizing mice with full-length AKR1B10 to obtain lymphocytes expressing the monoclonal AKR1B10 antibody, and then hybridizing these cells with myeloma cells. To demonstrate the specificity of the AKR1B10 monoclonal antibody, breast cancer cells MCF7 / Vector (MCF7 / V) transfected with an empty vector and not expressing AKR1B10, breast cancer cells MCF7 / AKR1B10 (MCF7 / A) transfected with AKR1B10, embryonic kidney cells 293T expressing AKR1B1 but not AKR1B10, and lung cancer cells A549 highly expressing AKR1B10 were cultured. Total protein was extracted from these cells, and Western blotting was used to detect that the anti-AKR1B10 monoclonal antibody had high specificity. It was found that there was no cross-reactivity with AKR1B1 protein, but it could specifically bind to AKR1B10.

[0070] The anti-AKR1B10 protein monoclonal antibody involved in this invention can be successfully detected by immunohistochemistry in human liver cancer, lung cancer, and breast cancer tissues (see [link to relevant documentation]). Figure 3A , 3B 4A, 4B, 5A and 5B).

[0071] This invention discovers that tumor cells can secrete AKR1B10 protein into the peripheral blood circulation, and that AKR1B10 protein levels are increased in the serum of patients with liver cancer, lung cancer, and breast cancer. Therefore, AKR1B10 protein can serve as a novel tumor serum marker for screening, early diagnosis, treatment guidance, recurrence monitoring, and prognosis of liver cancer, lung cancer, and breast cancer. Furthermore, this invention also discovers that normal human intestinal epithelial cells can secrete AKR1B10 protein into intestinal fluid, and that AKR1B10 protein can be detected in intestinal fluid and stool.

[0072] The kit for detecting AKR1B10 protein provided by the present invention includes a monoclonal antibody that specifically recognizes AKR1B10 protein, wherein the monoclonal antibody is coated onto magnetic beads in the kit.

[0073] This invention is based on a newly developed monoclonal antibody against the AKR1B10 protein and utilizes a sandwich chemistry detection method to establish a kit for detecting serum AKR1B10 antigen. This kit is suitable for early diagnosis and large-scale population screening of liver cancer, lung cancer, and breast cancer, and can also be used to guide treatment, predict prognosis, and monitor patients with recurrence.

[0074] The sandwich chemiluminescence detection kit for detecting AKR1B10 antigen provided by this invention mainly comprises: immunomagnetic beads coated with a specific polyclonal antibody that captures AKR1B10 protein; and acridine-bound mouse monoclonal antibody against AKR1B10. Both the capture antibody and the detection antibody can bind to the AKR1B10 antigen. The capture antibody is an AKR1B10 polyclonal antibody obtained after immunizing goats with AKR1B10 protein.

[0075] The AKR1B10 polyclonal antibody was prepared by immunizing goats with the AKR1B10 specific polypeptide (ARL-1 specific antibodies and uses thereof, USPatent#:US13 / 017618) antigen.

[0076] The sandwich chemiluminescence assay kit for detecting AKR1B10 antigen also includes: sample diluent / antibody diluent (1×PBS, 1% BSA, 0.05% NaN3, pH 7.3±0.1); standards (different concentrations of pure AKR1B10 antigen: 0 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL and 32 ng / mL).

[0077] The capture antibody is an AKR1B10 polyclonal antibody obtained after immunizing goats with the AKR1B10 protein; the detection antibody is a murine monoclonal antibody obtained by immunizing mice with the full-length AKR1B10 antigen.

[0078] Another object of the present invention is to provide a method for preparing a sandwich chemiluminescence detection kit for detecting AKR1B10 antigen. The preparation method includes the following steps:

[0079] Immunomagnetic beads were coated with goat anti-AKR1B10 protein polyclonal antibody. These immunomagnetic beads were diluted with reagent diluent (PBS, pH 7.4, 1.0% skim milk powder, 0.05% NaN3) to prepare a magnetic bead solution (final concentration 0.2 mg / mL) for reagent kit chamber A. Acridinium ester-labeled mouse anti-AKR1B10 protein monoclonal antibody (hereinafter referred to as "mouse anti-AKR1B10 monoclonal antibody") was diluted with reagent diluent (PBS, pH 7.4, 1.0% skim milk powder, 0.05% NaN3) to prepare reagent kit chamber D. Chambers A and D were sealed with bottle caps and stored at 4°C for later use.

[0080] When using, add 50 μL of serum sample or standard (0 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL and 32 ng / mL AKR1B10 purified protein dilution) to 50 μL of rabbit anti-AKR1B10 antibody-coated magnetic beads (cavity A), incubate for 15 min, and then perform the first magnetic separation and washing. After washing, add 100 μL of acridine ester-bound mouse anti-AKR1B10 monoclonal antibody (cavity D), incubate for 15 min, and then perform the second magnetic separation and washing. After washing, add pre-excitation solution and excitation solution to the test tube and read the relative luminescence value.

[0081] Optionally, the kit may contain the following reagents: AKR1B10 protein standard, quality control, pre-activation solution, activation solution, washing solution, or instructions, etc.

[0082] The reagents used in the following specific embodiments were purchased from Sigma-Aldrich Co., LLC, Peirce, and ThermoFisher.

[0083] Example 1: Preparation of mouse anti-AKR1B10 protein monoclonal antibody hybridoma cell line

[0084] The full-length AKR1B10 protein (Gene ID: 57016) was diluted to 1 mg / mL, mixed with an equal volume of Freund's complete adjuvant, and thoroughly emulsified. Five 6-week-old Balb / c mice were subcutaneously injected at multiple sites, with an antigen dose of 50 μg per mouse.

[0085] Three weeks later, the same dose of Freund's incomplete adjuvant-treated antigen was injected. The antigen was then injected every two weeks for a total of three times. The dose for each immunization was 25 μg / animal.

[0086] Three days before cell fusion, a booster immunization was performed by intraperitoneal injection of AKR1B10 protein without adjuvant.

[0087] Three days later, blood was collected from the mouse eyeballs. The blood was placed at 37°C for 0.5 hours, then at 4°C for 1 hour, and centrifuged at 3000 rpm for 5 minutes. The separated serum was used as positive serum.

[0088] Mice were euthanized and disinfected by immersion in 75% alcohol for 5 minutes. The mice were then removed, and their limbs were fixed to a dissecting board in a laminar flow hood. Under aseptic conditions, the abdominal skin and peritoneum were sequentially cut open to fully expose the abdominal cavity. The spleen was removed from the left side of the abdominal cavity using a set of sterile scissors and forceps. It was placed on a filter screen in a sterile glass culture dish containing a small amount of serum-free DMEM medium. The fascia on the spleen was removed, and the spleen cells were ground using a glass syringe. The flushed spleen cells were washed twice with serum-free DMEM medium (centrifuged at 1500 rpm for 5 minutes), resuspended, and counted. The cell concentration was adjusted to 1 × 10⁶ cells / mL with serum-free DMEM medium. 7 The cell suspension was prepared at a density of 1 cell per mL, yielding approximately 10 mL of immune spleen cells.

[0089] Quickly remove the Sp2 / 0 myeloma cells from the liquid nitrogen tank and place them in 37°C water. Shake the cryovials continuously to thaw them as quickly as possible. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in DMEM medium containing 10% fetal bovine serum, transfer them to cell culture flasks, and incubate at 37°C in a 5% CO2 saturated humidity incubator.

[0090] Ten days later, on the day of fusion, Sp2 / 0 cells with uniform morphology, clear boundaries, and good growth were collected. The nutrient solution was discarded, and the cells were washed with serum-free DMEM nutrient solution and resuspended. Cells were counted using a cell counting chamber, and the cells were diluted to 10⁻⁶. 6 The cells are fused at 37°C in a 5% CO2 incubator at a volume of approximately 5 mL.

[0091] Prepared Sp2 / 0 myeloma cells and immune spleen cells were mixed at a ratio of 1:10 and placed in a 50 mL centrifuge tube. The mixture was washed once with serum-free DMEM medium, centrifuged at 1000 rpm for 10 min, and the cells were loosened by gently tapping the bottom of the centrifuge tube with a finger.

[0092] Place the centrifuge tube in a 37°C water bath. Using a 1mL pipette, add 1mL of PEG2000 to the centrifuge tube while gently stirring. Add the PEG2000 over an average of 1 minute, then allow it to stand gently for 1 minute. Slowly add 1mL of serum-free DMEM medium over 1 minute, then add 1 drop every 1 second until the volume is adjusted to 45mL. Centrifuge at 1200rpm for 8 minutes and discard the supernatant. Resuspend the fused cells gently to avoid dispersing the newly fused cells. Add 100mL of HAT medium (HAT + 90% DMEM + 10% fetal bovine serum) and begin seeding 10 wells of a 96-well plate. Do not change the medium for 7 days before testing.

[0093] Anti-AKR1B10 antibody was detected using an indirect ELISA method to screen hybridoma cell lines capable of secreting specific antibodies. Further limiting dilution was used to obtain hybridoma cell lines secreting monoclonal antibodies against a single epitope. In this example, two murine monoclonal antibody cell lines were obtained: 8D7A9E2 and 8D7A9H4.

[0094] Example 2: Preservation of Hybridoma Cell Lines

[0095] This embodiment preserves the hybridoma cell lines with clone numbers 8D7A9E2 and 8D7A9H4 described in Example 1. The cells are deposited at the China Center for Type Culture Collection, Wuhan, China.

[0096] Wuhan University, with accession numbers CCTCC NO: C202141 and CCTCC NO: C202142, and accession date January 28, 2021.

[0097] Example 3: Purification of anti-AKR1B10 protein monoclonal antibody

[0098] In this embodiment, hybridoma cell lines with clone numbers 8D7A9E2 and 8D7A9H4 were cultured, and monoclonal antibodies were purified using protein G affinity chromatography.

[0099] The experimental steps are as follows:

[0100] (1) Preparation of antibody culture medium

[0101] Hybridoma cells were expanded and cultured in vitro, and the culture medium was collected.

[0102] (2) Purification of monoclonal antibodies

[0103] Anti-AKR1B10 antibodies in the culture medium were purified using an AKR1B10 antigen purification column.

[0104] (3) Purity was determined by SDS-PAGE electrophoresis.

[0105] Purity was identified by SDS-PAGE electrophoresis and concentration was determined by Nanodrop. The SDS-PAGE results of the purified murine AKR1B10 monoclonal antibody are shown below. Figure 1 As shown, by Figure 1 It can be seen that the purified monoclonal antibody band size is correct and the purity is >95%. Lane M: protein molecular weight marker, loading volume 5 μL; Lanes 1-8: number of tubes eluted during purification, loading volume 5 μL.

[0106] Example 4: Specificity detection of monoclonal antibodies against anti-AKR1B10 protein

[0107] The detection steps are as follows:

[0108] (1) MCF7 / Vector (MCF7 / V) breast cancer cells transfected with empty vector and not expressing AKR1B10, MCF7 / AKR1B10 (MCF7 / A) breast cancer cells transfected with AKR1B10, 293T embryonic kidney cells expressing AKR1B1 but not expressing AKR1B10, and A549 lung cancer cells expressing high AKR1B10 were cultured.

[0109] (2) Extract total protein from these cells, determine protein concentration, take 30 μg of each sample, adjust the volume to 30 μL, add SDS, and incubate at 95℃ for 10 min.

[0110] (3) Add the sample to the SDS-PAGE sample well, transfer the film after the gel is run, stain with green spring red, and then take a picture.

[0111] (4) Cover the membrane with 5% skim milk, then add 1:1000 AKR1B10 monoclonal mouse antibody, wash three times with PBST, incubate the membrane with HRP-labeled anti-mouse secondary antibody, develop the color, and take a picture to obtain the desired result. Figure 2 .

[0112] Test results as follows Figure 2 As shown, Figure 2 The results show the specificity of the purified murine AKR1B10 monoclonal antibody. In the figure, MCF7 / V represents MCF7 / Vector (MCF7 / V), a breast cancer cell line transfected with an empty vector that does not express AKR1B10; MCF7 / A represents MCF7 / AKR1B10 (MCF7 / A), a breast cancer cell line transfected with AKR1B10; 293T represents embryonic kidney cells 293T that express AKR1B1 but not AKR1B10; and A549 represents lung cancer cells A549 that highly express AKR1B10. The results indicate that the anti-AKR1B10 monoclonal antibody has high specificity and no cross-reactivity with AKR1B1 protein.

[0113] Example 5: Detection of the binding activity of the anti-AKR1B10 protein monoclonal antibody to the AKR1B10 protein.

[0114] (1) Dissolve the full-length AKR1B10 protein in PBS (pH 7.3) at a concentration of 10 μg / mL, coat the ELISA plate, then block it with 3% skim milk powder in MPBS solution at room temperature for 2 h, and wash it 3 times with PBS.

[0115] (2) Prepare AKR1B10 antigen PBS solutions with concentrations of 0.1 nmol / L, 0.25 nmol / L, 0.5 nmol / L, 1 nmol / L, 5 nmol / L, 10 nmol / L, 50 nmol / L, 100 nmol / L, 500 nmol / L, and 1000 nmol / L in a row of test tubes. Add 1 nmol / L antibody solution to each test tube, and the total volume of each test tube is 400 μL.

[0116] (3) After incubating the test tube at room temperature for 30 min, add 100 μL of the reaction mixture to the antigen plate. Repeat the process for 3 parallel wells for each concentration and incubate for 10 min.

[0117] (4) Wash the antigen plate three times with TBST, add 1:5000 of alkaline phosphatase (AP)-conjugated mouse monoclonal antibody, and incubate for 10 min.

[0118] (5) Wash the antigen plate three times with TBST, add 10 μL of AP substrate p-nitrophenyl phosphate (p-NPP) and after 5 min, use a multi-functional microplate reader to detect the fluorescence value at a wavelength of 405 nm.

[0119] (6) The detection results show that the antibody antigen dissociation constant Kd is 5.2 nmol / L.

[0120] Example 6: Anti-AKR1B10 protein monoclonal antibody used for immunohistochemical detection

[0121] The detection steps are as follows:

[0122] (1) Collect tissue samples from liver cancer, breast cancer, and lung cancer.

[0123] (2) Fix, dehydrate, embed, and slice the tissue samples.

[0124] (3) Block the tissue sections, then incubate with 1:200 anti-AKR1B10 monoclonal mouse antibody, wash three times, add HRP-labeled mouse secondary antibody, add chromogenic agent, and observe the expression level of AKR1B10 in the tissue under a microscope.

[0125] The expression of AKR1B10 protein in human hepatocellular carcinoma tissues was detected using an anti-AKR1B10 monoclonal antibody. The results are as follows: Figure 3A and Figure 3B As shown; Figure 3A The results of immunohistochemical testing of liver cancer tissue. Figure 3B The results of immunohistochemical detection of adjacent tissues of liver cancer show that the AKR1B10 protein in liver cancer tissue can be detected by anti-AKR1B10 protein monoclonal antibody.

[0126] The expression of AKR1B10 protein in human breast cancer tissues was detected using an anti-AKR1B10 protein monoclonal antibody. The results are as follows: Figure 4A and Figure 4B As shown; Figure 4A The results of immunohistochemical testing of breast cancer tissue. Figure 4B The results of immunohistochemical detection of adjacent normal tissue in breast cancer showed that the AKR1B10 protein in breast cancer tissue could be detected by anti-AKR1B10 protein monoclonal antibody.

[0127] The expression of AKR1B10 protein in human lung cancer tissues was detected using an anti-AKR1B10 protein monoclonal antibody. The results are as follows: Figure 5A and Figure 5B As shown; Figure 5A The results of immunohistochemical analysis of human lung cancer tissue. Figure 5B The results of immunohistochemical detection of adjacent normal tissues of human lung cancer show that the AKR1B10 protein in human lung cancer tissues can be detected by anti-AKR1B10 protein monoclonal antibody.

[0128] Example 7: Preparation of an enzyme-linked immunosorbent assay kit for detecting AKR1B10 protein.

[0129] The monoclonal antibody obtained in Example 3 (i.e., the AKR1B10 monoclonal antibody extracted and purified from the preserved 8D7A9E2 and 8D7A9H4 cell lines) was used as the detection antibody, and the detection kit included:

[0130] Enzyme-linked immunosorbent assay (ELISA) plates coated with specific capture antibodies;

[0131] Coating buffer: 1×PBS, pH 7.3±0.1;

[0132] Sample dilution buffer: 1×PBS, 1% BSA, 0.05% NaN3, pH 7.3±0.1;

[0133] Blocking solution: 1×PBS, 3% BSA;

[0134] Elution buffer PBST: 1×PBS, 0.05% Tween-20;

[0135] HRP-labeled anti-AKR1B10 protein monoclonal antibody.

[0136] The AKR1B10 protein was detected using the enzyme-linked immunosorbent assay kit described above. The detection steps are as follows:

[0137] (1) Dissolve 10 μg / mL of goat anti-AKR1B10 polyclonal antibody in coating buffer and add 200 μL to each well of the ELISA plate.

[0138] (2) Block the ELISA plate with 200 μL of blocking solution;

[0139] (3) Add different concentrations of AKR1B10 protein (0 ng / mL, 0.0625 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL and 32 ng / mL) and incubate at room temperature for 1 h;

[0140] (4) Remove the protein solution, wash three times with elution buffer, add 1:2000 mouse anti-AKR1B10 monoclonal antibody, and incubate for 30 min;

[0141] (5) Wash three times with elution buffer and add 1:5000 HRP-labeled anti-mouse secondary antibody;

[0142] (6) Wash three times with elution buffer, add substrate TMB, incubate for 15 min, then add stop solution and measure fluorescence value at 450 nm.

[0143] The detection results are shown in Table 1. The limit of detection of this kit is 0.25 ng / mL, and it has a good linear relationship between 0.125-32 ng / mL.

[0144] Table 1

[0145] Antigen concentration (ng / mL) <![CDATA[OD 450 ]]> 0 1866 0.0625 1911 0.125 1932 0.25 1990 0.5 2300 1 2745 2 3458 4 5687 8 7865 16 12543 32 23456

[0146] Example 8: Preparation of a time-resolved fluorescence immunoassay kit (TRFIA) for detecting AKR1B10 protein.

[0147] The monoclonal antibody obtained in Example 3 (i.e., the AKR1B10 monoclonal antibody extracted and purified from the preserved 8D7A9E2 and 8D7A9H4 cell lines) was used as the detection antibody, and the detection kit included:

[0148] Coating buffer: 1×PBS, pH 7.3±0.1;

[0149] Sample dilution buffer: 1×PBS, 1% BSA, 0.05% NaN3, pH 7.3±0.1;

[0150] Blocking solution: 1×PBS, 3% BSA;

[0151] Elution buffer PBST: 1×PBS, 0.05% Tween-20;

[0152] Specific monoclonal antibodies containing biotin labeling;

[0153] Streptavidin-Eu, europium-labeled streptavidin.

[0154] The AKR1B10 protein was detected using the enzyme-linked immunosorbent assay kit described above. The detection steps are as follows:

[0155] (1) Dissolve 2-10 μg / mL of goat anti-AKR1B10 polyclonal antibody in coating buffer and add 100-200 μL to each well of a high-binding ELISA plate;

[0156] (2) Block the ELISA plate with 200-250 μL of blocking solution;

[0157] (3) Add different concentrations of AKR1B10 protein (0 ng / mL, 0.0625 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL and 32 ng / mL) and incubate at room temperature for 1 h;

[0158] (4) Remove the protein solution, wash 3 times with elution buffer, add 1:1000 biotin-labeled mouse anti-AKR1B10 monoclonal antibody, and incubate for 30 min;

[0159] (5) Wash three times with elution buffer, add 1:5000 Streptavidin-Eu, and incubate for 15 min;

[0160] (6) Wash three times with elution buffer and measure the fluorescence value at 613 nm under 340 nm excitation light.

[0161] The detection results are shown in Table 2. The limit of detection of this kit is 0.125 ng / mL, and it has a good linear relationship between 0.125-32 ng / mL.

[0162] Table 2

[0163] Antigen concentration (ng / mL) <![CDATA[OD 450 ]]> 0 213 0.0625 220 0.125 223 0.25 452 0.5 821 1 1632 2 3345 4 7365 8 13454 16 23455 32 32346

[0164] Example 9: Preparation of a chemiluminescent detection kit for detecting AKR1B10 protein

[0165] The monoclonal antibody obtained in Example 3 (i.e., AKR1B10 monoclonal antibody extracted and purified from the preserved 8D7A9E2 and 8D7A9H4 cell lines) was used as the detection antibody and labeled with acridinium ester; the AKR1B10 polyclonal antibody obtained by immunizing goats with wild-type full-length AKR1B10 protein was used as the capture antibody, and the capture antibody was linked to immunomagnetic beads to construct a chemiluminescent detection kit for detecting human aldehyde-ketone reductase 1B10 protein.

[0166] In this embodiment, the antibody purification kit (Sulfolink@Immobilization Kit for Peptides) was purchased from Thermo Scientific (catalog number 44999); the acrid ester labeling of monoclonal antibodies and the immunomagnetic bead conjugation of polyclonal antibodies were both handled by Shenzhen Yahuilong Biotechnology Co., Ltd.; the reagents used were prepared in-house: reagent dilution solution: PBS solution at pH 7.4, 1.0% skim milk powder, 0.05% NaN3.

[0167] Rabbit anti-AKR1B10 antibody-coated magnetic beads (cavity A of the kit): Prepare magnetic beads to a final concentration of 0.2 mg / mL using reagent diluent.

[0168] Acridine-bound mouse monoclonal antibody AKR1B10 (cavity D of the kit): Dilute the acridine-bound mouse monoclonal antibody AKR1B10 to 1:50 with reagent diluent.

[0169] Standards (AKR1B10 antigen at different concentrations: 0 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL and 32 ng / mL).

[0170] The chemiluminescent assay reagent for detecting AKR1B10 protein was loaded into the reagent window of the YHLO iFlash 3000 instrument. Standards (different concentrations of AKR1B10 antigen: 0 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL, and 32 ng / mL) were placed on the instrument's sample rack. The AKR1B10 detection item was added to the instrument's detection program, the detection program was started, the detection values ​​of the standards were obtained, and a standard curve was plotted.

[0171] The detection results of the chemiluminescence detection kit are as follows: Figure 6 As shown, Figure 6A standard curve for a chemiluminescent assay kit to detect AKR1B10 protein was prepared. The capture antibody attached to the magnetic beads was a polyclonal antibody against AKR1B10 obtained after immunizing goats with AKR1B10 protein. The detection antibody labeled with the luminescent material was a purified monoclonal antibody obtained by immunizing mice with full-length AKR1B10 antigen. Different amounts of purified AKR1B10 protein were used as test samples to determine the limit of detection.

[0172] The results show that the capture antibody and detection antibody obtained in this embodiment have high sensitivity and specificity. The chemiluminescence detection method in this embodiment has high specificity, high degree of automation, and high sensitivity, and can detect AKR1B10 protein at 0.5 ng / mL.

[0173] Example 10: Detection of AKR1B10 concentration in serum of normal individuals, breast cancer patients, and liver cancer patients using a chemiluminescence detection kit.

[0174] Serum sample detection: In this embodiment, the chemiluminescent detection reagent for detecting AKR1B10 protein prepared in Example 9 was loaded into the reagent window of the Yahuilong iFlash 3000. Serum samples and standards (different concentrations of AKR1B10 antigen: 0 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL and 32 ng / mL) were placed on the instrument's sample rack. The AKR1B10 detection item was added to the instrument's detection program, the detection program was started, and the detection values ​​of the sample and standard were obtained. According to the standard curve, the concentration of AKR1B10 in the serum sample was calculated, and the serum AKR1B10 concentration was obtained.

[0175] Analysis of test results:

[0176] Serum samples from 50 healthy individuals, 50 breast cancer patients, 30 lung cancer patients, and 30 primary liver cancer patients were analyzed. The results were analyzed using Graphpad 4.0 statistical software.

[0177] The detection results of the chemiluminescence detection kit are as follows: Figure 7 , Figure 8 and Figure 9 As shown. Figure 7 The concentrations of AKR1B10 in the serum of healthy individuals and breast cancer patients. Figure 8 The concentrations of AKR1B10 in the serum of healthy individuals and lung cancer patients. Figure 9 The concentrations of AKR1B10 in the serum of healthy individuals and patients with liver cancer are given.

[0178] The t-test results showed that the mean KR1B10 antigen levels in patients with breast cancer, lung cancer, and liver cancer were significantly higher than those in healthy individuals (p<0.001). The average serum KR1B10 antigen concentration was 150 pg / mL in healthy individuals, 812 pg / mL in breast cancer patients, 1102 pg / mL in lung cancer patients, and 1102 pg / mL in liver cancer patients.

[0179] In summary, this invention has developed a novel specific monoclonal antibody against the AKR1B10 protein. This monoclonal antibody exhibits high specificity and affinity for the AKR1B10 protein, and shows no cross-reactivity with other unrelated proteins. The AKR1B10 protein, as a tumor serum marker, possesses strong specificity for liver cancer, lung cancer, and breast cancer. The anti-AKR1B10 protein monoclonal antibody of this invention exhibits high binding activity to the AKR1B10 protein. Based on this, this invention has developed a series of experimental methods and chemiluminescent detection kits for detecting AKR1B10 protein in serum. These detection kits can be widely used in cancer screening, diagnosis, treatment efficacy assessment, prognostic evaluation, and recurrence monitoring.

[0180] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A monoclonal antibody against human aldehyde-ketone reductase 1B10 protein, characterized in that, The monoclonal antibody was produced by hybridoma cell lines that generate monoclonal antibodies against human aldehyde-ketone reductase 1B10 protein. These hybridoma cell lines are named 8D7A9E2 and 8D7A9H4, and are deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession numbers CCTCC NO: C202141 and CCTCC NO: C202142, respectively, on January 28, 2021. The affinity dissociation constant Kd between the monoclonal antibody and AKR1B10 protein is 5.2 nmol / L, and the limit of detection (LOD) of the monoclonal antibody in time-resolved immunofluorescence assay is 0.125 ng / mL.

2. The method for preparing the monoclonal antibody against human aldehyde-ketone reductase 1B10 protein according to claim 1, characterized in that, The method for preparing the monoclonal antibody includes: culturing the hybridoma cell line that produces the anti-human aldehyde reductase 1B10 protein monoclonal antibody as described in claim 1, separating and purifying it to obtain the anti-human aldehyde reductase 1B10 protein monoclonal antibody.

3. The use of the anti-human aldehyde reductase 1B10 protein monoclonal antibody as described in claim 1 in the preparation of kits for screening, diagnosis, efficacy assessment, prognostic evaluation, or recurrence monitoring of liver cancer, lung cancer, or breast cancer.

4. The application according to claim 3, characterized in that, The kit contains reagents that specifically detect the level of AKR1B10 protein in a sample.

5. The application according to claim 3, characterized in that, The kit also contains AKR1B10 protein standards.

6. The application according to claim 3, characterized in that, The kit can detect samples including any one or a combination of at least two of the following: serum, plasma, urine, cerebrospinal fluid, ascites, breast milk, intestinal fluid, stool, saliva, vaginal secretions, or tissue samples.

7. An enzyme-linked immunosorbent assay kit for detecting human aldehyde-ketone reductase 1B10 protein, characterized in that, The enzyme-linked immunosorbent assay kit contains the monoclonal antibody against human aldehyde-ketone reductase 1B10 protein as described in claim 1.

8. The enzyme-linked immunosorbent assay kit according to claim 7, characterized in that, The monoclonal antibody is an HRP-labeled anti-human aldehyde reductase 1B10 protein monoclonal antibody.

9. The enzyme-linked immunosorbent assay kit according to claim 7, characterized in that, The enzyme-linked immunosorbent assay kit further comprises any one or a combination of at least two of the following: an enzyme-linked immunosorbent assay plate coated with specific capture antibodies, a coating buffer, a sample diluent, a blocking buffer, or an elution buffer.

10. A time-resolved immunofluorescence assay kit for detecting human aldehyde-ketone reductase 1B10 protein, characterized in that, The time-resolved immunofluorescence assay kit contains the monoclonal antibody against human aldehyde reductase 1B10 protein as described in claim 1.

11. The time-resolved immunofluorescence assay kit according to claim 10, characterized in that, The monoclonal antibody is a biotin-labeled anti-human aldehyde reductase 1B10 protein monoclonal antibody.

12. The time-resolved immunofluorescence assay kit according to claim 10, characterized in that, The time-resolved immunofluorescence assay kit further comprises a time-resolved fluorescence immunoassay plate coated with a specific capture antibody, a coating buffer, a sample diluent, a blocking buffer, an elution buffer, or any one or a combination of at least two of Streptavidin-Eu.

13. A chemiluminescent assay kit for detecting human aldehyde-ketone reductase 1B10 protein, characterized in that, The chemiluminescence detection kit contains the monoclonal antibody against human aldehyde reductase 1B10 protein as described in claim 1.

14. The chemiluminescence detection kit according to claim 13, characterized in that, The monoclonal antibody is an anti-human aldehyde-ketone reductase 1B10 protein monoclonal antibody containing a chemiluminescent label, wherein the chemiluminescent label includes any one of acridine ester, ruthenium tripyridine, alkaline phosphatase, or luminol.

15. The chemiluminescence detection kit according to claim 13, characterized in that, The chemiluminescence detection kit also includes any one or a combination of at least two of the following: chemiluminescent magnetic microparticles coated with specific capture antibodies, sample diluent, elution buffer, or luminescent substrate.

16. An immunoturbidimetric assay kit for detecting human aldehyde-ketone reductase 1B10 protein, characterized in that, The immunoturbidimetric assay kit contains the monoclonal antibody against human aldehyde reductase 1B10 protein as described in claim 1.

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