Hybridoma cell line secreting anti-AKR1C3 monoclonal antibody, anti-AKR1C3 monoclonal antibody and its applications and products

Abstract

The present invention belongs to the field of antibodies, and specifically relates to a hybridoma cell line secreting anti-AKR1C3 monoclonal antibodies, an anti-AKR1C3 monoclonal antibody and its applications and products. The anti-AKR1C3 monoclonal antibody is a pair of matching antibodies, denoted as antibody 1 and antibody 2; antibody 1 is secreted by the hybridoma cell line with the preservation number GDMCC No. 65671, and antibody 2 is secreted by the hybridoma cell line with the preservation number GDMCC No. 65670. The matching antibodies provided by the present invention have the characteristics of strong specificity, high sensitivity and strong stability in detecting AKR1C3, and have better inhibitory activity against AKR1C3, thereby inhibiting the growth and metastasis of tumor cells. The antibody is of great significance in many fields such as cancer treatment, diagnosis, prognosis evaluation, and biological and medical research.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies, and particularly relates to a hybridoma cell line secreting anti-AKR1C3 monoclonal antibody, an anti-AKR1C3 monoclonal antibody, and applications and products thereof. Background Art

[0002] AKR1C3 is located at the third position of the first family C subfamily of the aldo-keto reductase family, and belongs to a monomeric cytosolic protein, containing approximately more than 320 amino acid residues, with a size of about 35 KDa. It is ubiquitously present in the biological world and can oxidize and reduce most substrates with carboxyl groups as functional groups and is an intermediate product of strong mutagens such as aldehydes or ketones in the body. AKR1C3 can encode 17β-hydroxysteroid dehydrogenase, and the aldo-keto reductase AKR1C3 exerts its redox function depending on the coenzyme NADP(H). Moreover, the aldo-keto reductase AKR1C3 also has various functions in the human body. For example, it can regulate the content of substances such as ketosteroids and lipid aldehydes in human cells and thereby reduce their content. AKR1C3 can not only reduce the content of steroids but also participate in the metabolism of steroid enzymes. It can convert 4-sterene-5,17-dione into testosterone and can also convert estrogen into 17β-estradiol.

[0003] AKR1C3 is a multifunctional enzyme widely distributed in liver, prostate, and breast tissues and plays a key role in steroid hormone and prostaglandin metabolism. AKR1C3 is significantly upregulated in liver cancer tissues, and liver cancer patients with upregulated AKR1C3 have a poor prognosis. In vitro and in vivo experiments have confirmed that AKR1C3 can significantly promote the proliferation and metastasis of liver cancer cells and can be used as a prognostic detection marker for liver cancer. The research on AKR1C3 and prostate cancer has been relatively mature. A large number of experimental studies have confirmed that the expression level of AKR1C3 in the human prostate is much higher than that in other tissues, and AKR1C3 is expressed in some sex hormone-dependent and non-hormone-dependent malignant tumors, such as its expression in glioma and meningioma. It is expressed in the human adrenal gland, but the expression levels in different zonae are inconsistent, and its expression level in the reticular zone is higher than that in the fascicular zone.

[0004] In addition, among multiple public datasets, the expression of AKR1C3 was significantly elevated in esophageal adenocarcinoma (EAC) compared with normal esophageal tissues. Notably, AKR1C3 was associated with promoting the proliferation, colony formation, and migration of EAC cell lines, highlighting its crucial role in EAC development. The study also found that AKR1C3 exhibited the highest transcriptional levels in human papillomavirus (HPV)-negative oropharyngeal squamous cell carcinoma (OPSCC) samples, and the inhibition of AKR1C3 has shown the potential to enhance cisplatin efficacy, positioning AKR1C3 as a promising prognostic biomarker and a viable drug target in OPSCC.

[0005] In chronic myeloid leukemia (CML), AKR1C3 was associated with imatinib resistance. Imatinib is a tyrosine kinase inhibitor used as a first-line targeted drug. The high expression of AKR1C3 increased imatinib resistance in CML cells and mouse models. The combination of imatinib and indomethacin, a chemical inhibitor of AKR1C3, significantly prolonged mouse survival and reduced splenomegaly, indicating that AKR1C3 inhibition has the potential to enhance imatinib therapy. In addition, miR-379-5p, which was found to be downregulated in the bone marrow microenvironment, inhibited AKR1C3, suggesting the existence of a novel miR-379-5p / AKR1C3 / ERK signaling axis in imatinib resistance in CML. In hepatocellular carcinoma (HCC), AKR1C3 was associated with sorafenib resistance. Sorafenib is a targeted therapy used to treat HCC. The study found that the upregulation of AKR1C3 enhanced cell survival in response to sorafenib, while the removal of AKR1C3 restored sensitivity to sorafenib. AKR1C3 was associated with the metabolic shift from fatty acid oxidation (FAO) to glycolysis, promoting sorafenib resistance in HCC cells. The high expression of AKR1C3 in sorafenib-resistant patients was associated with poor prognosis. The combination of AKR1C3 inhibition and sorafenib was considered to have a more significant impact on HCC treatment and may overcome drug resistance.

[0006] In summary, the elevated expression of AKR1C3 was significantly associated with poor prognosis and resistance to anticancer therapy, indicating the potential of AKR1C3 inhibitors in cancer treatment and overcoming drug resistance. Therefore, there is an urgent need to develop more AKR1C3 inhibitors to provide new ideas and methods for the treatment of related diseases. Summary of the Invention

[0007] To solve the above problems, the present invention provides two hybridoma cell lines that can secrete a new pair of anti-AKR1C3 antibodies, which have strong specificity and good stability and are of great significance in disease diagnosis, drug research and development, scientific research, and clinical applications.

[0008] On the one hand, the present invention provides a method for in vitro detecting AKR1C3 in a sample for non-diagnostic purposes.

[0009] Specifically, the method is a double-antibody sandwich immunoassay method, and the double antibodies include antibody 1 and / or antibody 2; antibody 1 is secreted by the hybridoma cell line with the preservation number of GDMCC No. 65671, and antibody 2 is secreted by the hybridoma cell line with the preservation number of GDMCC No. 65670.

[0010] On the other hand, the present invention provides a hybridoma cell line with the preservation number of GDMCC No. 65671 or GDMCC No. 65670.

[0011] On the other hand, the present invention provides a monoclonal antibody against AKR1C3, and the monoclonal antibody against AKR1C3 is secreted by the aforementioned hybridoma cell line.

[0012] Specifically, the hybridoma cell line with the preservation number of GDMCC No. 65671 secretes antibody 1; the hybridoma cell line with the preservation number of GDMCC No. 65670 secretes antibody 2.

[0013] More specifically, the classification of antibody 1 is IgG1 and Ig-κ;

[0014] The classification of antibody 2 is IgG2a and Ig-κ.

[0015] On the other hand, the present invention provides a preparation method of the aforementioned monoclonal antibody against AKR1C3, including culturing the aforementioned hybridoma cell line.

[0016] Specifically, it includes the following steps:

[0017] (1) Inject the aforementioned hybridoma cell line into the abdominal cavity of an animal, culture for 7 - 10 days, and collect ascites;

[0018] (2) Centrifuge the ascites, and collect the supernatant as the monoclonal antibody ascites;

[0019] (2) Precipitate the above-mentioned monoclonal antibody ascites, and dissolve it with a buffer solution to obtain A;

[0020] (3) Dialyze A in a buffer solution at 4°C;

[0021] (4) Recover the dialysis product to obtain the monoclonal antibody against AKR1C3.

[0022] On the other hand, the present invention provides an antibody preparation including the aforementioned monoclonal antibody against AKR1C3.

[0023] On the other hand, the present invention provides a pharmaceutical composition including the aforementioned monoclonal antibody against AKR1C3 or the antibody preparation.

[0024] Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0025] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a wetting agent, an emulsifier, a preservative, an antioxidant, a buffer, an excipient, a diluent, a lubricant, a bacteriostatic agent, a suspending agent, a suspending aid, a solubilizer, a thickening agent, a stabilizer, a sweetening agent, and a fragrance.

[0026] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0027] In another aspect, the present invention provides a kit comprising the aforementioned anti-AKR1C3 monoclonal antibody or antibody preparation or pharmaceutical composition.

[0028] In another aspect, the present invention provides the use of the aforementioned anti-AKR1C3 monoclonal antibody or antibody preparation or pharmaceutical composition in the preparation of a reagent or kit for detecting AKR1C3.

[0029] In another aspect, the present invention provides the use of the aforementioned anti-AKR1C3 monoclonal antibody or antibody preparation in the preparation of a drug for diagnosing, preventing, and / or treating cancer.

[0030] Specifically, the cancer includes but is not limited to: liver cancer, prostate cancer, breast cancer, ovarian cancer, gastric cancer, bladder cancer, renal cell carcinoma, pancreatic cancer, colon cancer, non-small cell lung cancer, rectal cancer, esophageal cancer, endometrial cancer, brain cancer, or leukemia.

[0031] Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0032] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a wetting agent, an emulsifier, a preservative, an antioxidant, a buffer, an excipient, a diluent, a lubricant, a bacteriostatic agent, a suspending agent, a suspending aid, a solubilizer, a thickening agent, a stabilizer, a sweetening agent, and a fragrance.

[0033] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0034] The anti-AKR1C3 monoclonal antibody provided by the present invention has the following advantages:

[0035] (1) High specificity.

[0036] (2) High sensitivity, with the sensitivity of the paired antibody being 0.4173 ng / mL.

[0037] (3) Good stability. After three cycles of freezing and thawing, the antibody activity remains unchanged. After 7 days of acceleration at 37°C, the decrease in antibody activity is <5%.

[0038] (4) The kit prepared with the antibody of the present invention has the advantages of good repeatability, high precision, low detection limit, and good linearity.

[0039] Depositing Instructions 1:

[0040] Biological material: SP2 / 0 cells of the monoclonal antibody 2 cell line against AKR1C3;

[0041] Deposit number: GDMCC No.65670;

[0042] Classification and naming: Mouse hybridoma cells;

[0043] Deposit date: December 25, 2024;

[0044] Depositary institution: Guangdong Provincial Microbial Culture Collection Center;

[0045] Abbreviation of the depositary institution: GDMCC;

[0046] Address of the depositary institution: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0047] Depositing Instructions 2:

[0048] Biological material: SP2 / 0 cells of the monoclonal antibody 1 cell line against AKR1C3;

[0049] Deposit number: GDMCC No.65671;

[0050] Classification and naming: Mouse hybridoma cells;

[0051] Deposit date: December 25, 2024;

[0052] Depositary institution: Guangdong Provincial Microbial Culture Collection Center;

[0053] Abbreviation of the depositary institution: GDMCC;

[0054] Address of the depositary institution: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou. Description of the Drawings

[0055] Figure 1 It is a figure showing the antibody specificity result.

[0056] Figure 2It is the concentration-luminescence value curve of AKR1C3.

[0057] Figure 3 It is the fitting curve of two points A and B. Specific implementation mode

[0058] The present invention will be further elaborated in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are conventional conditions if not otherwise specified. For the experimental methods without specific conditions indicated in the embodiments, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels if not otherwise specified.

[0059] Example 1

[0060] 1.1 Animal immunization

[0061] Immunize mice with human AKR1C3 recombinant antigen according to the routine, that is, mix the AKR1C3 recombinant protein with Freund's complete adjuvant, fully emulsify it, and then inject it subcutaneously at multiple points on the back and abdomen. Then, every two weeks, take the antigen and Freund's incomplete adjuvant, fully emulsify them, and inject them intraperitoneally. One week after the fourth immunization, detect the serum titer, and select the mice with high titer for spleen booster immunization. Take the spleen cells 3 days later for cell fusion.

[0062] 1.2 Preparation of feeder cells

[0063] Take mouse peritoneal macrophages and thymocytes as feeder cells.

[0064] 1.3 Cell fusion

[0065] Take the spleen cells of immunized mice and mouse myeloma cells, mix and centrifuge them in the culture medium, remove the culture medium, add feeder cells, and dispense them into a 96-well cell plate containing feeder cells, and culture them in a 37°C cell incubator.

[0066] 1.4 Screening and cloning of hybridoma cells

[0067] After culturing in the cell incubator for 5 days, when the fused cells cover 10 - 30% of the bottom of the well, positive wells secreting antibodies are screened by the conventional indirect ELISA method. That is, the enzyme-linked immunosorbent assay (ELISA) plate is coated with the recombinant AKR1C3 protein as the antigen, 100 μL per well, and coated overnight at 4°C. After patting dry, 200 μL of blocking solution is added to each well and blocked for 2 hours, then patted dry for standby. 50 μL of the cell culture supernatant to be tested is added to each well of the coated plate, reacted at 37°C for 30 min, then washed and patted dry. 50 μL of HRP-labeled goat anti-mouse IgG is added to each well, incubated at 37°C for 30 min, then washed and patted dry. 50 μL of chromogenic solution is added to each well, and the color is developed at 37°C in the dark for 10 min. 100 μL of stop solution is added to each well to terminate the reaction, and the OD value is read with an enzyme-linked immunosorbent assay (ELISA) reader. The cell wells showing a strong positive reaction are selected for cloning by the limiting dilution method. After three to four rounds of cloning and screening, a monoclonal cell line with a 100% positive rate is determined as a stable cell line. After expanded culture, it is used for ascites preparation and cryopreservation in liquid nitrogen.

[0068] 1.5 Preparation and purification of monoclonal antibody ascites

[0069] Mice about 8 weeks old are taken. After injecting paraffin intraperitoneally for 7 - 10 days, hybridoma cells are injected. After one week, ascites is collected. Centrifuge at 1200 rpm / min at 4°C for 30 min, and the supernatant is collected, which is the monoclonal antibody ascites. Dilute the mouse ascites with 0.06 M acetate buffer at pH 4.8, centrifuge and collect the supernatant. Then precipitate the immunoglobulin with 50% (m / v) ammonium sulfate, place it at 4°C for 2 h, centrifuge at 1200 rpm / min at 4°C for 20 min. Dissolve the precipitate with 0.01 M PBS buffer at pH 7.4, and perform dialysis in a flowing state at 4°C for 24 h to obtain the purified ascites antibody, which is stored at -20°C.

[0070] 1.6 Screening of antibodies

[0071] (1) HRP labeling of antibodies

[0072] Use the HRP rapid coupling kit (product number: K011 - 1 - S) from Yingjia (Shanghai) Biomedical Technology Co., Ltd., and operate according to the instructions to label the monoclonal AKR1C3 antibody. After labeling, store it at -20°C for standby.

[0073] (2) Coating the ELISA plate with antibodies

[0074] Add the AKR1C3 antibody to each well of the ELISA plate at 100 μL per well, coat overnight at 4°C, pat dry, wash the plate 3 times, add 200 μL of blocking solution to each well, block at 37°C for 2 h and pat dry to obtain the ELISA plate coated with AKR1C3.

[0075] (3) ELISA experiment

[0076] Take the antibody-coated enzyme-linked immunosorbent assay (ELISA) plate, add 50 μL of AKR1C3 protein standard to each well, incubate at 37 °C for 30 minutes, wash the plate 3 times, shake dry, add 50 μL of enzyme-labeled antibody to each well, incubate at 37 °C for 30 minutes, then wash the plate 3 times and pat dry; add 100 μL of chromogenic solution to each well, incubate at 37 °C for 10 min, add 100 μL of stop solution to each well, and read the OD value on the microplate reader. The results of the ELISA experiment are shown in Table 1.

[0077] Table 1 ELISA experiment results

[0078]

[0079]

[0080] 1.7 Performance evaluation of paired antibodies

[0081] (1) Specificity detection of paired antibodies

[0082] A highly specific and sensitive antibody against AKR1C3 is crucial for patient selection and a better understanding of the function of AKR1C3 in normal physiology and diseases. The human AKR1s family includes AKR1A1, AKR1B1, AKR1B10, AKR1D1, and four human AKR1C isoforms (AKR1C1-4). The sequence homology between different AKR1s and AKR1C3 is greater than 84%, which makes the development of specific antibodies against AKR1C3 very challenging. To develop a pair of more specific anti-AKR1C3 antibodies, 2 mouse monoclonal antibodies were developed and identified in this study. Studies have shown that the 2 antibodies form a sandwich ELISA method and can effectively recognize the concentration level of AKR1C3 in the sample to be detected.

[0083] Purified AKR1 family proteins AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, and PBS buffer were separately coated on ELISA microplates, and then different AKR1 family proteins were detected. The binding rate was expressed as the average value of triplicate wells with standard deviation. The results of the specificity detection are shown in Table 2. Figure 1 .

[0084] Table 2 Specificity detection results

[0085]

[0086] The detection results showed that antibody 1 and antibody 2 could recognize AKR1C3 and had no cross-reaction with AKR1A1, AKR1B1, AKR1B10, AKR1C1, AKR1C2, AKR1C4, AKR1D1, and PBS.

[0087] (2) Detection of the sensitivity of the paired antibody

[0088] The sensitivity is determined according to the limit of blank (LOB). The limit of blank is detected with zero concentration as the sample. It is measured 20 times repeatedly, and the results of the 20 measurements are obtained. Calculate its average value (M) and standard deviation (SD), and obtain M + 2SD. A linear equation is obtained by two-point regression fitting based on the measured values between the zero concentration calibrator and the adjacent calibrator. Substitute the measured value of M + 2SD into the above equation to find the corresponding concentration value, that is, the limit of blank (LOB). The sensitivity results are shown in Table 3.

[0089] Table 3 Sensitivity results

[0090]

[0091] The sensitivity of the paired antibody is 0.4173 ng / mL.

[0092] (3) Stability of the paired antibody

[0093] To verify the stability of the antibody, the freeze-thaw and thermal stabilities of the antibody were investigated. For the freeze-thaw experiment of the antibody, the aliquoted anti-AKR1C3 antibody was placed at -20 °C and frozen overnight. The next day, the antibody was taken out and thawed at room temperature. After thawing and returning to room temperature, it was put back at -20 °C and frozen overnight again. This was repeated three times, and the antibody after three freeze-thaw cycles was detected by ELISA for comparison to evaluate the effect of repeated freeze-thaw on the antibody activity.

[0094] For the thermal stability experiment of the antibody, the labeled antibody and the coated antibody were respectively placed in an incubator at 37 °C. The antibodies were taken out and compared with those at 4 °C on the 1st, 3rd, and 7th days respectively to evaluate the effect on the antibody activity under the condition of 37 °C. Finally, the antibody stability results are shown in Table 4. The freeze-thaw stability and stability of the AKR1C3 antibody are both very stable. After three freeze-thaw cycles, the antibody activity has no change, and after 7 days of acceleration at 37 °C, the antibody activity decline is <5%. These indicators all show that the AKR1C3 paired antibody is a pair of antibodies with good stability.

[0095] Table 4 Antibody stability experiment data

[0096]

[0097]

[0098] (4) Typing and identification of the antibody

[0099] Use the mouse monoclonal antibody Ig class / subclass identification ELISA kit (product number: BF06001) of Suzhou Bio-long Biotechnology Co., Ltd. and detect according to the method described in the instruction manual. The results are shown in Table 5 and Table 6.

[0100] Table 5 Antibody Typing Detection Results

[0101]

[0102] Table 6 Antibody Typing

[0103] Antibody 1 Antibody 2 Subclass IgG1 IgG2a Subtype Ig-κ Ig-κ

[0104] According to the antibody typing detection results, the typing of antibody 1 is IgG1, Ig-κ, and the typing of antibody 2 is IgG2a, Ig-κ.

[0105] Detect AKR1C3 by immunohistochemical staining method in Application Example 1

[0106] Detect the expression level of AKR1C3 in paraffin-embedded human tissue sections in vitro by immunohistochemical staining method, where the human tissue sections are liver cancer cell tissue sections.

[0107] (1) Antigen retrieval: When performing immunohistochemical staining, bake the sections at 60 °C for 2 hours, then dewax and perform antigen retrieval. Incubate the sections at room temperature for 30 minutes to block non-specific binding;

[0108] (2) Primary antibody incubation: Mix the sections after antigen retrieval with the working solution prepared from AKR1C3 antibody 1 or antibody 2, incubate at 37 °C for 1 hour, rinse the sections, and then incubate the tissue sections for 10 - 15 minutes to inhibit endogenous peroxidase;

[0109] (3) Secondary antibody incubation: After incubating with the primary antibody, mix the sections with the working solution prepared from goat anti-mouse secondary antibody and incubate at 37 °C for 1 hour.

[0110] (4) Staining and mounting: Rinse the sections with water, perform counterstaining, and then mount the cover glass for observation.

[0111] Detect AKR1C3 by enzyme-linked immunosorbent assay (ELISA) analysis in Application Example 2

[0112] The specific operations are as follows:

[0113] (1) Prepare AKR1C3 standard products

[0114] Prepare concentration points of 0 ng / mL, 4.12 ng / mL, 12.35 ng / mL, 37.04 ng / mL, 111.11 ng / mL, 333.33 ng / mL, 1000 ng / mL, and 3000 ng / mL by formulating AKR1C3 recombinant protein, filter and aliquot, 0.5 ml per tube, and store at 4 °C.

[0115] (2) Make antibody-coated plates

[0116] Add 100 μL of AKR1C3 antibody 1 to each well of the ELISA plate, coat overnight at 4 °C, pat dry the ELISA plate, wash the plate 3 times, add 200 μL of blocking solution to each well, block at 37 °C for 2 h and pat dry, then the ELISA plate coated with AKR1C3 antibody is obtained and vacuum-sealed with an aluminum foil bag.

[0117] (3) Preparation of horseradish peroxidase (HRP)-labeled antibody

[0118] Use the HRP rapid conjugation kit (product number: K011-1-S) from Yijia (Shanghai) Biomedical Technology Co., Ltd., operate according to the instruction manual to label AKR1C3 antibody 2, and store it at -20 °C for later use after labeling.

[0119] (4) Antigen-antibody reaction: Take the antibody-coated plate, add 50 μL of the sample to each well, incubate at 37 °C for 30 minutes, repeat washing the plate 3 times, add 100 μL of the HRP-labeled antibody to each well, incubate at 37 °C for 30 minutes, and repeat washing the plate 3 times.

[0120] (5) Color reaction: Add 100 μL of the chromogenic solution to each well in sequence, incubate at 37 °C for 10 minutes, and then add 100 μL of the reaction termination solution to end the reaction.

[0121] (6) Measure the OD value with an ELISA reader.

[0122] (7) Result calculation: Use the sample concentration as the abscissa and the OD value as the ordinate to plot the standard curve of this measurement, and calculate the AKR1C3 concentration from the OD value of the sample to be measured according to the standard curve.

[0123] Application Example 3 Detection of AKR1C3 by magnetic particle chemiluminescence method

[0124] The reagents for detecting AKR1C3 by magnetic particle chemiluminescence method include calibrators, magnetic separation reagents, and alkaline phosphatase-labeled reagents

[0125] (1) Preparation of AKR1C3 calibrators

[0126] Prepare AKR1C3 recombinant protein into concentration points of 0 ng / mL, 4.12 ng / mL, 12.35 ng / mL, 37.04 ng / mL, 111.11 ng / mL, 333.33 ng / mL, 1000 ng / mL, and 3000 ng / mL, filter and aliquot, 0.5 ml per tube, and store at 4 °C.

[0127] (2) Preparation of magnetic separation reagents

[0128] Resuspend the magnetic particles by washing, add an aqueous solution of EDC, and suspend at room temperature for 30 min; add AKR1C3 antibody 1 and suspend at room temperature for 2 hours to prepare an antibody-magnetic particle conjugate.

[0129] (3) Conjugation of alkaline phosphatase (ALP) with the labeled antibody

[0130] Use the alkaline phosphatase (ALP) rapid conjugation kit (product number: K012-1-S) from Yijia (Shanghai) Biomedical Technology Co., Ltd., operate according to the instructions, label AKR1C3 antibody 2, and prepare an alkaline phosphatase-labeled reagent.

[0131] The chemiluminescence detection method for magnetic particles is as follows:

[0132] (1) Sequentially add 50 μL of magnetic separation reagent, 50 μL of sample, and 50 μL of alkaline phosphatase-labeled reagent into the detection tube, mix evenly, and incubate at 37 °C for 30 minutes;

[0133] (2) Place the detection tube on the magnetic separator, let it stand for 2 minutes, and centrifuge to discard the supernatant;

[0134] (3) Add 200 μL of washing solution, mix by oscillation, pat dry, and wash 3 times;

[0135] (4) Add 100 μL of substrate solution to the detection tube and mix evenly;

[0136] (5) Detect the luminescence value with a semi-automatic luminescence detector;

[0137] (6) Result calculation: With the sample concentration as the abscissa and the luminescence value as the ordinate, plot a standard curve, use four-parameter logistic fitting to obtain the regression equation of the sample dose-response curve, and then the concentration of the test sample can be calculated back from the regression curve according to the relative luminescence intensity of the test sample. See the attached Figure 2 , the concentration-luminescence value curve of AKR1C3.

[0138] Effect example 1 Performance verification of the kit

[0139] 1.1 Precision of the kit

[0140] (1) Repeatability

[0141] Use the kit prepared in Application Example 3 to measure two samples with different concentrations respectively, and perform 10 parallel determinations to obtain the within-batch coefficient of variation CV. The repeatability results are shown in Table 7.

[0142] Table 7 Repeatability

[0143]

[0144]

[0145] (2)Inter - batch precision

[0146] Take three batches of the kits prepared in Application Example 3. For each batch of kits, measure samples at two different concentrations, with 10 - well parallel measurements. Obtain 30 concentration measurement values for each sample, and statistically analyze the coefficient of variation CV among them.

[0147] Table 8 Inter - batch precision

[0148]

[0149] 1.2 Determination of the accuracy of the kit

[0150] Accuracy refers to the degree to which the measured value approaches the true value, usually expressed by the recovery rate.

[0151] Add AKR1C3 sample (A) with a concentration of approximately 1000.00 ng / mL (allowing a concentration deviation of ±20%) to the low - value sample (B). The volume ratio of added A to B is 1:9. Each sample is detected 3 times repeatedly, and the mean value is calculated. Calculate the recovery rate R according to formula (1), and the result should meet the requirement of 85% - 115%. The results are shown in Table 9.

[0152]

[0153] In the formula:

[0154] R ─ Recovery rate;

[0155] V ─ Volume of added sample A solution;

[0156] V0 ─ Volume of sample B solution;

[0157] C ─ Detection concentration after adding sample A solution to sample B solution;

[0158] C0 ─ Concentration of sample B solution;

[0159] C s ─ Concentration of sample A solution.

[0160] Table 9 Accuracy and recovery rate

[0161]

[0162] 1.3 Linear range

[0163] Dilute the high - value sample close to the upper limit of the linear range to at least 5 concentrations in a certain proportion, where the low - concentration sample is close to the lower limit of the linear interval. Test each sample according to the operation method in Application Example 3, repeat the test 3 times for each dilution concentration, and find the mean value of the measurement results for each dilution concentration. Fit the mean value of the results and the dilution concentration by the least - squares method to calculate the linear correlation coefficient R2 , the correlation coefficient R of the kit 2 should be ≥ 0.9900.

[0164] Table 10 Detection results of linear range

[0165]

[0166] Experimental conclusion: R 2 = 0.9999, meeting the requirements.

[0167] 1.4 Minimum detection limit:

[0168] The minimum detection limit is the dose that can be distinguished from the zero dose at a given significance level. Using the zero-concentration calibrator as a sample for detection, repeat the measurement 20 times, obtain the relative luminescence intensity (RLU) values of the 20 measurement results, calculate their average value (M) and standard deviation (SD), obtain M + 2SD, and perform two-point regression fitting based on the concentration-RLU between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation. Substitute the RLU value of M + 2SD into the above equation to find the corresponding concentration value, which is the minimum detection limit.

[0169] (1) The luminescence value (RLU) at point A is shown in Table 11:

[0170] Table 11

[0171]

[0172] (2) The luminescence value (RLU) at point B is shown in Table 12:

[0173] Table 12

[0174]

[0175] (3) The fitting curve of points A and B is shown in the appendix Figure 3 .

[0176] (4) Calculated according to the formula, the minimum detection limit = 0.065 ng / mL.

[0177] The paired antibodies of the present invention, combined with the current detection technology, can effectively shorten the detection time of AKR1C3, and have high detection sensitivity and good specificity, indicating that the detection of AKR1C3 can play an effective diagnostic role in the detection of early liver cancer.

Claims

1. A method for detecting AKR1C3 in a sample, characterized in that, The method is a double-antibody sandwich immunoassay. The double antibodies in the double-antibody sandwich immunoassay include antibody 1 and / or antibody 2. Antibody 1 is secreted by the hybridoma cell line with the deposit number GDMCC No. 65671, and antibody 2 is secreted by the hybridoma cell line with the deposit number GDMCC No. 65670.

2. A hybridoma cell line, characterized in that, The deposit numbers of the hybridoma cell lines are GDMCC No. 65671 or GDMCC No. 65670.

3. Anti-AKR1C3 monoclonal antibody, characterized in that, The anti-AKR1C3 monoclonal antibody is secreted by the hybridoma cell line described in claim 2.

4. The anti-AKR1C3 monoclonal antibody according to claim 3, characterized in that, The antibody secreted by the hybridoma cell line with the deposit number GDMCC No. 65671 is classified as IgG1 and Ig-κ. The antibody secreted by the hybridoma cell line with the deposit number GDMCC No. 65670 is classified as IgG2a and Ig-κ.

5. The preparation method of the anti-AKR1C3 monoclonal antibody according to any one of claims 3-4, characterized in that, It includes culturing the hybridoma cell line described in claim 2.

6. The preparation method according to claim 5, characterized in that, It includes the following steps: (1) Culturing the hybridoma cell line described in claim 2 that has been injected into the abdominal cavity of an animal for 7 - 10 days, and recovering the ascites. (2) Centrifuging the ascites and collecting the supernatant, which is the monoclonal antibody ascites. (2) Precipitating the above monoclonal antibody ascites and dissolving it with a buffer to obtain A. (3) Dialyzing A in a buffer at 4°C. (4) Recovering the dialyzed product to obtain the anti-AKR1C3 monoclonal antibody.

7. An antibody preparation, characterized in that, It includes the anti-AKR1C3 monoclonal antibody described in any one of claims 3 - 4.

8. A kit, characterized in that, It includes the anti-AKR1C3 monoclonal antibody described in any one of claims 3 - 4 or the antibody preparation described in claim 7.

9. Use of the anti-AKR1C3 monoclonal antibody described in any one of claims 3 - 4 in the preparation of a reagent or kit for detecting AKR1C3.

10. Use of the antibody preparation described in claim 7 in the preparation of a reagent or kit for detecting AKR1C3.

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