Hybridoma cell strain secreting anti-AKR1C3 monoclonal antibody, anti-AKR1C3 monoclonal antibody, application and product

Abstract

The invention belongs to the field of antibodies, and particularly relates to a hybridoma cell strain secreting an anti-AKR1C3 monoclonal antibody, the anti-AKR1C3 monoclonal antibody, application and a product. The AKR1C3 monoclonal antibody is a pair of matched antibodies which are marked as an antibody 1 and an antibody 2; the antibody 1 is secreted by a hybridoma cell strain of which the preservation number is GDMCC (China General Microbiological Culture Collection Center) No.65671, and the antibody 2 is secreted by a hybridoma cell strain of which the preservation number is GDMCC No.65670. The matched antibody provided by the invention has the characteristics of strong specificity, high sensitivity and strong stability in the aspect of detecting AKR1C3, and has better inhibitory activity on AKR1C3, thereby inhibiting the growth and metastasis of tumor cells. The antibody has important significance in multiple fields of cancer treatment, diagnosis, prognosis evaluation, biological and medical research and the like.

Description

Technical Field

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

[0002] AKR1C3 is located in the third position of the first group C subfamily of the aldehyde-keto reductase family. It is a monoplasmic cytoplasmic protein with about 320 amino acid residues and a size of about 35KDa. It is ubiquitous in the biological world and can oxidize and reduce most substrates with carboxyl as functional groups and is an intermediate product of aldehydes or ketones, which are very strong mutagens in the body. AKR1C3 can encode 17β-hydroxysteroid dehydrogenase. The aldehyde-keto reductase AKR1C3 depends on the coenzyme NADP (H) to perform oxidation and reduction functions. In addition, the aldehyde-keto reductase AKR1C3 also has multiple functions in the human body. For example, it can regulate the content of substances such as ketosteroids and lipid aldehydes in human cells and reduce their content. AKR1C3 can not only reduce the content of sterols but also participate in the metabolism of sterol enzymes. It can convert 4-steroid-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 the metabolism of steroid hormones and prostaglandins. AKR1C3 expression is significantly upregulated in liver cancer tissues, and patients with upregulated AKR1C3 liver cancer have a poor prognosis. In vivo and in vitro experiments have confirmed that AKR1C3 can significantly promote the proliferation and metastasis of liver cancer cells and can be used as a marker for liver cancer prognosis detection. 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 human prostate is much higher than that in other tissues. AKR1C3 is expressed in some sex hormone-dependent and non-hormone-dependent malignant tumors, such as glial tumors and meningiomas. It is expressed in the human adrenal glands, but the expression levels in different zona fasciculata are inconsistent. Its expression level in the zona reticularis is higher than that in the zona fasciculata.

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

[0005] In chronic myeloid leukemia (CML), AKR1C3 is associated with resistance to imatinib, a tyrosine kinase inhibitor used as a first-line targeted drug. High expression of AKR1C3 increases resistance to imatinib in CML cells and mouse models. Combination of imatinib with indomethacin, a chemical inhibitor of AKR1C3, significantly prolonged mouse survival and reduced splenomegaly, suggesting that AKR1C3 inhibition has the potential to enhance imatinib treatment. In addition, downregulated miR-379-5p in the bone marrow microenvironment was found to inhibit AKR1C3, suggesting a novel miR-379-5p / AKR1C3 / ERK signaling axis in imatinib resistance in CML. In HCC, AKR1C3 is associated with resistance to sorafenib, a targeted therapy used to treat HCC. Studies have found that upregulation of AKR1C3 enhances cell survival in response to sorafenib, while removal of AKR1C3 restores sensitivity to sorafenib. AKR1C3 is associated with a metabolic shift from fatty acid oxidation (FAO) to glycolysis, promoting sorafenib resistance in HCC cells. High expression of AKR1C3 in sorafenib-resistant patients is associated with poor prognosis. The combination of AKR1C3 inhibition and sorafenib is thought to have a more significant effect on HCC treatment and may overcome resistance.

[0006] In summary, elevated AKR1C3 expression is significantly associated with poor prognosis and anticancer treatment resistance, which suggests that AKR1C3 inhibitors have potential in cancer treatment and overcoming drug resistance. Therefore, it is urgent to develop more AKR1C3 inhibitors to provide new ideas and methods for the treatment of related diseases. Summary of the invention

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

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

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

[0010] In yet another aspect, the present invention provides a hybridoma cell line, the accession number of which is GDMCC No.65671 or GDMCC No.65670.

[0011] In yet another aspect, the present invention provides an anti-AKR1C3 monoclonal antibody, wherein the anti-AKR1C3 monoclonal antibody is secreted by the aforementioned hybridoma cell line.

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

[0013] Further specifically, the antibody 1 is typed as IgG1 and Ig-κ;

[0014] The antibody 2 was typed as IgG2a and Ig-κ.

[0015] In another aspect, the present invention provides a method for preparing the aforementioned anti-AKR1C3 monoclonal antibody, comprising culturing the aforementioned hybridoma cell line.

[0016] Specifically, the steps include:

[0017] (1) injecting the aforementioned hybridoma cell line into the peritoneal cavity of an animal, culturing for 7-10 days, and recovering ascites;

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

[0019] (2) precipitating the monoclonal antibody ascites and dissolving it with a buffer to obtain A;

[0020] (3) dialyze A against 4°C ambient buffer;

[0021] (4) Recovering the dialyzed product to obtain the anti-AKR1C3 monoclonal antibody.

[0022] In yet another aspect, the present invention provides an antibody preparation comprising the aforementioned anti-AKR1C3 monoclonal antibody.

[0023] In yet another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned anti-AKR1C3 monoclonal antibody or antibody preparation.

[0024] Specifically, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0025] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

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

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

[0028] In yet another aspect, the present invention provides 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 yet another aspect, the present invention provides 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 also includes pharmaceutically acceptable excipients.

[0032] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

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

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

[0035] (1) Strong specificity.

[0036] (2) High sensitivity: the sensitivity of paired antibodies is 0.4173 ng / mL.

[0037] (3) Good stability. After three freeze-thaw cycles, the antibody activity showed no change. After accelerated storage at 37°C for 7 days, the antibody activity decreased by less than 5%.

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

[0039] Storage Instructions 1:

[0040] Biological materials: anti-AKR1C3 monoclonal antibody 2 cell line SP2 / 0 cells;

[0041] Deposit number: GDMCC No.65670;

[0042] Classification and nomenclature: mouse hybridoma cells;

[0043] Deposit date: December 25, 2024;

[0044] Depository: Guangdong Microbiological Culture Collection Center;

[0045] Abbreviation of depository unit: GDMCC;

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

[0047] Preservation Instructions 2:

[0048] Biological materials: anti-AKR1C3 monoclonal antibody 1 cell line SP2 / 0 cells;

[0049] Deposit number: GDMCC No.65671;

[0050] Classification and nomenclature: mouse hybridoma cells;

[0051] Deposit date: December 25, 2024;

[0052] Depository: Guangdong Microbiological Culture Collection Center;

[0053] Abbreviation of depository unit: GDMCC;

[0054] Address of the preservation unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The figure shows the antibody specificity results.

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

[0057] Figure 3 Fit the curve between two points AB. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0059] Example 1

[0060] 1.1 Animal immunization

[0061] Mice were routinely immunized with human AKR1C3 recombinant antigen, i.e., AKR1C3 recombinant protein was mixed with Freund's complete adjuvant, fully emulsified, and then injected subcutaneously at multiple points in the dorsal abdomen. After that, every 2 weeks, the antigen was mixed with Freund's incomplete adjuvant and fully emulsified, and then injected intraperitoneally. One week after the fourth immunization, the serum titer was tested, and mice with high titer were taken for spleen booster immunization. After 3 days, spleen cells were taken for cell fusion.

[0062] 1.2 Preparation of feeder cells

[0063] Mouse peritoneal macrophages and thymocytes were used as feeder cells.

[0064] 1.3 Cell fusion

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

[0066] 1.4 Screening and cloning of hybridoma cells

[0067] After culturing in a cell culture incubator for 5 days, when the fused cells covered 10-30% of the bottom of the well, the positive wells for secreting antibodies were screened by conventional indirect ELISA method, that is, AKR1C3 recombinant protein was used as antigen, 100 μL / well was coated on the ELISA plate, coated overnight at 4°C, patted dry, 200 μL of blocking solution was added to each well, blocked for 2 hours, patted dry for later use; 50 μL / well of the culture supernatant of the cells to be tested was added to the coated plate, reacted at 37°C for 30 minutes, washed and patted dry, 50 μL / well of HRP-labeled goat anti-mouse IgG was added, incubated at 37°C for 30 minutes, washed and patted dry, 50 μL / well of color development solution was added, color was developed at 37°C in the dark for 10 minutes, 100 μL / well of stop solution was added to terminate the reaction, and the OD value was read with an enzyme marker. The cell wells with strong positive reactions were selected for limiting dilution cloning. After three to four rounds of cloning screening, the monoclonal cell line with a positive rate of 100% was determined to be a stable cell line. After expanded culture, it was used for ascites preparation and liquid nitrogen storage.

[0068] 1.5 Preparation and purification of monoclonal antibody ascites

[0069] Take mice around 8 weeks old, inject paraffin into the peritoneum 7-10 days later, inject hybridoma cells, collect ascites one week later, centrifuge at 1200rpm / min, 4℃ for 30min, and collect the supernatant, which is the monoclonal antibody ascites. Take the mouse ascites and dilute it with 0.06M pH 4.8 acetate buffer, centrifuge and collect the supernatant, then precipitate immunoglobulin with 50% (m / v) ammonium sulfate, place at 4℃ for 2h, centrifuge at 1200rpm / min, 4℃ for 20min, dissolve the precipitate with 0.01M pH7.4 PBS buffer, and obtain the purified ascites antibody after flow dialysis at 4℃ for 24h, which is stored at -20℃.

[0070] 1.6 Antibody Screening

[0071] (1) HRP labeling of antibodies

[0072] The AKR1C3 monoclonal antibody was labeled using the HRP rapid coupling kit (Cat. No. K011-1-S) of Inplus (Shanghai) Biopharmaceutical Technology Co., Ltd. according to the instructions. After labeling, the antibody was stored at -20°C for future use.

[0073] (2) Antibody-coated ELISA plate

[0074] AKR1C3 antibody was added to each well of the ELISA plate at 100 μL, coated overnight at 4°C, patted dry, washed 3 times, 200 μL of blocking solution was added to each well, blocked at 37°C for 2 h and patted dry to obtain the AKR1C3-coated ELISA plate.

[0075] (3) ELISA experiment

[0076] Take the antibody-coated ELISA plate, add 50 μL of AKR1C3 protein standard to each well, incubate at 37°C for 30 minutes, wash the plate 3 times, spin 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 color development solution to each well, incubate at 37°C for 10 minutes, add 100 μL of stop solution to each well, and read the OD value on the ELISA instrument. The results of the ELISA experiment are shown in Table 1.

[0077] Table 1 ELISA experimental 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 essential for patient selection and a better understanding of the function of AKR1C3 in normal physiology and disease. 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. In order to develop a pair of more specific anti-AKR1C3 antibodies, two mouse monoclonal antibodies were developed and identified in this study. Studies have shown that the two antibodies form a double antibody immune sandwich method, which can effectively identify the concentration level of AKR1C3 in the sample to be tested.

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

[0084] Table 2 Specificity test results

[0085]

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

[0087] (2) Paired antibody sensitivity test

[0088] Sensitivity is determined based on the minimum detection limit (LOB). The minimum detection limit is detected using zero concentration as a sample, and the measurement is repeated 20 times to obtain the results of 20 measurements. The mean (M) and standard deviation (SD) are calculated to obtain M+2SD. A linear equation is obtained by performing a two-point regression fit based on the measured values ​​between the zero concentration calibrator and the adjacent calibrator. The measured value of M+2SD is substituted into the above equation to obtain the corresponding concentration value, i.e., the minimum detection limit (LOB). The sensitivity results are shown in Table 3.

[0089] Table 3 Sensitivity results

[0090]

[0091] The sensitivity of paired antibodies was 0.4173 ng / mL.

[0092] (3) Stability of paired antibodies

[0093] To verify the stability of the antibody, the freeze-thaw and thermal stability of the antibody were investigated. In the freeze-thaw experiment, the anti-AKR1C3 antibody was packaged and frozen at -20℃ overnight. The antibody was taken out the next day and thawed at room temperature. After thawing, it was put into the -20℃ freezer overnight again after returning to room temperature. This freeze-thaw cycle was repeated three times, and the antibodies after three freeze-thaw cycles were compared by Elisa test to evaluate the effect of repeated freeze-thaw on antibody activity.

[0094] In the thermal stability experiment of antibodies, the labeled antibody and the coated antibody were placed in a 37°C incubator respectively, and the antibodies were taken out at 1 day, 3 days and 7 days respectively, and compared with 4°C to evaluate the effect of 37°C conditions on antibody activity. The final antibody stability results are shown in Table 4. The freeze-thaw stability and stability of the AKR1C3 antibody are very stable. There is no change in the antibody activity after three freeze-thaw cycles, and the antibody activity decreases by less than 5% after 7 days of accelerated storage at 37°C. These indicators show that the AKR1C3 paired antibodies are a pair of antibodies with good stability.

[0095] Table 4 Antibody stability experimental data

[0096]

[0097]

[0098] (4) Antibody typing and identification

[0099] The mouse monoclonal antibody Ig class / subclass identification ELISA kit (Cat. No.: BF06001) of Suzhou Biolong Technology Co., Ltd. was used for detection according to the instruction manual. The results are shown in Tables 5 and 6.

[0100] Table 5 Antibody typing test 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 test results, the typing of antibody 1 was IgG1, Ig-κ, and the typing of antibody 2 was IgG2a, Ig-κ.

[0105] Application Example 1 Immunohistochemical staining to detect AKR1C3

[0106] Immunohistochemical staining was used to detect the expression level of AKR1C3 in ex vivo paraffin-embedded human tissue sections, wherein the human tissue sections were liver cancer cell tissue sections.

[0107] (1) Antigen retrieval: When performing immunohistochemical staining, the sections were baked at 60°C for 2 h, then dewaxed and antigen retrieval was performed, and the sections were incubated at room temperature for 30 min to block nonspecific binding;

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

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

[0110] (4) Staining and sealing: Rinse the sections with water, re-stain, and then mount a coverslip for observation.

[0111] Application Example 2 Enzyme-linked immunosorbent assay (ELISA) analysis and detection of AKR1C3

[0112] The specific operations are as follows:

[0113] (1) Preparation of AKR1C3 standard

[0114] The AKR1C3 recombinant protein was prepared 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, filtered and packaged in 0.5 ml per tube, and stored at 4°C.

[0115] (2) Preparation of antibody-coated plates

[0116] AKR1C3 antibody 1 was added to each well of the ELISA plate at 100 μL, coated at 4°C overnight, the ELISA plate was patted dry, washed 3 times, 200 μL of blocking solution was added to each well, blocked at 37°C for 2 h and patted dry to obtain the AKR1C3 antibody-coated ELISA plate, which was packaged in an aluminum foil bag and vacuum sealed.

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

[0118] The AKR1C3 antibody 2 was labeled using the HRP rapid coupling kit (Cat. No.: K011-1-S) of Inplus (Shanghai) Biopharmaceutical Technology Co., Ltd. according to the instructions. After labeling, it was stored at -20°C for future use.

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

[0120] (5) Color development reaction: Add 100 μL of color development solution to each well, incubate at 37°C for 10 min, and then add 100 μL of reaction stop solution to terminate the reaction.

[0121] (6) Measure the OD value using an enzyme-labeled instrument.

[0122] (7) Calculation of results: With the sample concentration as the horizontal axis and the OD value as the vertical axis, a standard curve for this determination was drawn. The AKR1C3 concentration was calculated from the standard curve according to the OD value of the sample to be tested.

[0123] Application Example 3 Detection of AKR1C3 by Magnetic Microparticle Chemiluminescence

[0124] Detection of AKR1C3 by magnetic microparticle chemiluminescence, the reagents of which include calibrators, magnetic separation reagents and alkaline phosphatase labeling reagents

[0125] (1) Preparation of AKR1C3 Calibrator

[0126] The AKR1C3 recombinant protein was prepared 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, filtered and packaged in 0.5 ml per tube, and stored at 4°C.

[0127] (2) Preparation of magnetic separation reagent

[0128] The magnetic particles were washed and resuspended, and EDC aqueous solution was added and suspended at room temperature for 30 minutes; AKR1C3 antibody 1 was added and suspended at room temperature for 2 hours to prepare antibody-magnetic particle conjugates.

[0129] (3) Coupling of alkaline phosphatase (ALP) and labeled antibodies

[0130] The AKR1C3 antibody 2 was labeled using the alkaline phosphatase (ALP) rapid coupling kit (Cat. No. K012-1-S) of Inplus (Shanghai) Biopharmaceutical Technology Co., Ltd. according to the instructions to prepare an alkaline phosphatase labeling reagent.

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

[0132] (1) Add 50 μL of magnetic separation reagent, 50 μL of sample, and 50 μL of alkaline phosphatase labeling reagent into the detection tube in sequence, mix well, and incubate at 37°C for 30 minutes;

[0133] (2) Place the test tube on a magnetic separator, let it stand for 2 minutes, and spin dry the supernatant;

[0134] (3) Add 200 μL of cleaning solution, shake to mix, pat dry, and wash three times;

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

[0136] (5) Detecting the luminescence value using a semi-automatic luminescence detector;

[0137] (6) Result calculation: With the sample concentration as the horizontal axis and the luminescence value as the vertical axis, a standard curve is drawn. The regression equation of the sample dose-response curve is obtained by four-parameter logical fitting. Then, the concentration of the test sample can be calculated from the regression curve based on the relative luminescence intensity of the test sample. See Appendix. Figure 2 , concentration-luminescence value curve of AKR1C3.

[0138] Effect Example 1: Performance Verification of the Kit

[0139] 1.1 Kit precision

[0140] (1) Repeatability

[0141] The kit prepared in Example 3 was used to measure two samples of different concentrations, and 10 wells were measured in parallel to obtain the intra-batch coefficient of variation CV. The repeatability results are shown in Table 7.

[0142] Table 7 Repeatability

[0143]

[0144]

[0145] (2) Batch-to-batch precision

[0146] Three batches of the kit prepared in Application Example 3 were taken, and each batch of the kit was used to measure two samples with different concentrations, and the 10 wells were measured in parallel. 30 concentration values ​​were obtained for each sample, and the coefficient of variation CV was statistically analyzed.

[0147] Table 8 Inter-batch precision

[0148]

[0149] 1.2 Kit Accuracy Determination

[0150] Accuracy refers to how close a measurement is to its true value and is usually expressed as recovery.

[0151] The AKR1C3 sample (A) with a concentration of about 1000.00 ng / mL (with a concentration deviation of ±20%) was added to the low-value sample (B), and the volume ratio of A to B was 1:9. Each sample was tested three times and the average was calculated. The recovery rate R was calculated according to formula (1), and the result should meet the requirement of 85%-115%. The results are shown in Table 9.

[0152]

[0153] Where:

[0154] R─recovery rate;

[0155] V─the volume of sample A solution added;

[0156] V0─volume of sample B solution;

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

[0158] C0─Concentration of sample B solution;

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

[0160] Table 9 Accuracy and recovery

[0161]

[0162] 1.3 Linear range

[0163] The high-value sample close to the upper limit of the linear range was diluted to at least 5 concentrations according to a certain ratio, among which the low-concentration sample was close to the lower limit of the linear range. Each sample was tested according to the operating method of Application Example 3, and each dilution concentration was tested 3 times to obtain the mean value of the measurement results of each dilution concentration. The mean value of the result and the dilution concentration were fitted with a linear method using the least squares method, and the linear correlation coefficient R was calculated.2 , the correlation coefficient R of the kit 2 Should be ≥0.9900.

[0164] Table 10 Linear range test results

[0165]

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

[0167] 1.4 Minimum detection limit:

[0168] The lowest detection line is the dose that can be distinguished from the zero dose at a given significance level. The zero-concentration calibrator is used as a sample for detection, and the measurement is repeated 20 times to obtain the relative luminescence intensity (RLU) value of the 20 measurement results, and the mean (M) and standard deviation (SD) are calculated to obtain M+2SD. A linear equation is obtained by performing a two-point regression fitting based on the concentration-RLU between the zero-concentration calibrator and the adjacent calibrator. The RLU value of M+2SD is substituted into the above equation to obtain the corresponding concentration value, which is the lowest detection limit.

[0169] (1) Luminescence value (RLU) of point A is shown in Table 11:

[0170] Table 11

[0171]

[0172] (2) Luminescence value (RLU) of point B is shown in Table 12:

[0173] Table 12

[0174]

[0175] (3) The fitting curve of two points AB is shown in the attached figure. Figure 3 .

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

[0177] The paired antibodies of the present invention, combined with current detection technology, can effectively shorten the detection time of AKR1C3, and the detection has high 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 immune sandwich method, in which the double antibodies include antibody 1 and / or antibody 2; the antibody 1 is secreted by a hybridoma cell line with a preservation number of GDMCC No. 65671, and the antibody 2 is secreted by a hybridoma cell line with a preservation number of GDMCC No. 65670.

2. A hybridoma cell line, characterized in that The deposit number of the hybridoma cell line is GDMCC No.65671 and / or GDMCC No.65670.

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

4. The anti-AKR1C3 monoclonal antibody according to claim 2, characterized in that The hybridoma cell line with the deposit number of GDMCC No.65671 secretes antibody 1; the hybridoma cell line with the deposit number of GDMCC No.65670 secretes antibody 2.

5. The anti-AKR1C3 monoclonal antibody according to claim 4, characterized in that The antibody 1 is classified into IgG1 and Ig-κ; the antibody 2 is classified into IgG2a and Ig-κ.

6. The method for preparing the anti-AKR1C3 monoclonal antibody according to any one of claims 3 to 5, characterized in that: The method comprises culturing the hybridoma cell line according to claim 2.

7. The method according to claim 6, characterized in that The following steps are involved: (1) injecting the hybridoma cell line described in claim 2 into the peritoneal cavity of an animal, culturing for 7-10 days, and recovering ascites; (2) Centrifuge the ascites and collect the supernatant, which is the monoclonal antibody ascites; (2) precipitating the monoclonal antibody ascites and dissolving it with a buffer to obtain A; (3) dialyze A against 4°C ambient buffer; (4) Recovering the dialyzed product to obtain the anti-AKR1C3 monoclonal antibody.

8. An antibody preparation, characterized in that The invention comprises the anti-AKR1C3 monoclonal antibody according to any one of claims 3 to 5.

9. A kit, characterized in that: The invention comprises the anti-AKR1C3 monoclonal antibody according to any one of claims 3 to 5 or the antibody preparation according to claim 8.

10. Use of the anti-AKR1C3 monoclonal antibody according to any one of claims 3 to 5 or the antibody preparation according to claim 8, characterized in that: The uses include 1 and / or (2): (1) use in preparing a reagent or kit for detecting AKR1C3; (2) use in preparing a drug for diagnosing, preventing and / or treating cancer.

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