An aldosterone monoclonal antibody mAb4 or an antigen-binding fragment thereof, and a preparation method and application thereof

Abstract

The application belongs to the technical field of immune detection, and discloses an aldosterone antibody mAb4 or an antigen binding fragment thereof, and a preparation method and application thereof. The aldosterone antibody mAb4 or the antigen binding fragment thereof comprises a light chain variable region VL and a heavy chain variable region VH, the light chain variable region VL comprises complementary determining regions LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the complementary determining regions LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO. 1-3; the heavy chain variable region VH comprises complementary determining regions HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the complementary determining regions HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO. 4-6. The aldosterone monoclonal antibody mAb4 provided by the application has a detection sensitivity of less than 10 pg / mL when used for detecting an aldosterone standard product; the antibody is applied to a magnetic chemiluminescence system to carry out clinical sample detection, and the detection result has excellent correlation with a clinical reference standard in a concentration range of 0-1000 pg / mL, and can be widely applied to clinical auxiliary diagnosis and curative effect evaluation of primary hypertension and various aldosterone metabolism abnormality related diseases, and has extremely high clinical application value.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, specifically to an aldosterone monoclonal antibody mAb4 or its antigen-binding fragment, its preparation method, and its application. Background Technology

[0002] Aldosterone is synthesized and released by the zona glomerulosa cells of the adrenal cortex and belongs to the mineralocorticoid steroid class. Its core physiological target is the kidney tissue, and it is a key regulatory molecule for maintaining blood volume, blood pressure homeostasis, and sodium and potassium electrolyte balance.

[0003] From a physiological regulatory perspective, aldosterone acts on the epithelial cells of the distal convoluted tubule and collecting duct of the kidney, enhancing the body's reabsorption efficiency of sodium ions while promoting the excretion of potassium and hydrogen ions, thereby maintaining the dynamic balance of electrolytes and body fluids. Aldosterone secretion is mainly regulated by the renin-angiotensin pathway: when blood pressure decreases or sodium reserves are insufficient, juxtaglomerular cells in the kidney secrete renin, which gradually generates angiotensin II, inducing the synthesis and release of aldosterone. In addition, elevated serum potassium and decreased serum sodium can directly stimulate aldosterone secretion; angiotensin-converting enzyme drugs and physiological states during pregnancy can also alter its expression levels in the body.

[0004] Aldosterone secretion disorders can induce a variety of clinical symptoms. Excessive hormone secretion is often induced by lesions such as adrenal adenomas, leading to primary aldosteronism, with typical signs including hypertension and hypokalemia. Insufficient hormone secretion is commonly seen in adrenal insufficiency, causing electrolyte disturbances such as hyperkalemia and hyponatremia. Currently, clinical screening mainly relies on two core indicators: plasma aldosterone quantification and the aldosterone / renin activity ratio.

[0005] Mass spectrometry is currently the benchmark method for aldosterone quantification, but its sample pretreatment process is cumbersome, the detection cycle is long, and the automation threshold for the supporting equipment is high, making it unsuitable for large-scale routine screening in hospitals. Immunoassay, with its advantages of ease of operation and fast detection speed, has become the mainstream clinical alternative. However, aldosterone is a small molecule steroid with a scarcity of antigen sites it can recognize. Existing immunoassay kits all use a competitive detection mode, which generally suffers from insufficient detection sensitivity, poor specificity, high false positive rate, and complex manufacturing processes.

[0006] Therefore, there is an urgent need to develop highly specific and high-affinity aldosterone monoclonal antibodies to build a dual-antibody sandwich aldosterone detection system and solve various defects in existing clinical testing technologies. Summary of the Invention

[0007] To overcome the above-mentioned technical problems, this application discloses an aldosterone monoclonal antibody mAb4 with excellent specificity and high affinity, and its antigen-binding fragment, and also provides the preparation method and application of the antibody and antigen-binding fragment.

[0008] In a first aspect, this application provides an aldosterone monoclonal antibody mAb4 or its antigen-binding fragment, comprising a light chain variable region VL and a heavy chain variable region VH. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.1 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.1. The amino acid sequence of LCDR2 is DVS (SEQ ID NO.2) or a variant having one amino acid substitution, deletion, or addition compared to DVS. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.3; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is the sequence shown in SEQ ID NO.4 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.4. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.5 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6 or a variant with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO.6; Furthermore, the antibody mAb4 or its antigen-binding fragment can specifically bind aldosterone.

[0009] Further, the light chain variable region VL comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.7, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified; the heavy chain variable region VH comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.8, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified.

[0010] Furthermore, amino acid sequences with 95%, 96%, 97%, 98%, or 99% identity.

[0011] Furthermore, the light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.8.

[0012] Furthermore, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

[0013] Secondly, this application also provides a biomaterial selected from polynucleotides, carriers, or cells. The polynucleotide encodes the aldosterone monoclonal antibody mAb4 or its antigen-binding fragment as described in the first aspect; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the vector, or is capable of expressing the monoclonal antibody mAb4 or its antigen-binding fragment as described in the first aspect.

[0014] Furthermore, the cells are eukaryotic cells, preferably mammalian cells, more preferably 293 cells or CHO cells.

[0015] Thirdly, this application also provides a method for preparing the aldosterone antibody mAb4 or its antigen-binding fragment as described in the first aspect, comprising culturing cells as described in the second aspect, wherein the cells are prepared by transforming the cells with a polynucleotide encoding an aldosterone monoclonal antibody mAb4 or its antigen-binding fragment, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprising co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0016] Fourthly, this application also provides for the application of the aldosterone antibody mAb4 described in the first aspect or its antigen-binding fragment, or the biological material described in the second aspect, wherein the application is selected from one or more of the following: 1) Detection of aldosterone for purposes other than disease diagnosis and treatment; 2) Prepare immunoassay products for detecting aldosterone; 3) Used for purifying aldosterone; 4) Prepare products for purifying aldosterone.

[0017] In this application, the application is for non-diagnostic and non-therapeutic purposes. Specifically, it can be used to detect aldosterone content in cell culture medium, animal heart, kidney, bone, inner ear, colon, and other tissues, and to study the non-disease-related physiological regulatory effects of aldosterone on osteoblast proliferation, vascular fibrosis, inner ear lymphatic balance, and intestinal electrolyte transport. It can also be used to quantify plasma and urine aldosterone in animals that have been given the drug for a long period of time, to evaluate whether the test drug interferes with the secretory function of the adrenal cortex, only as a toxicological indicator, and not for animal disease diagnosis. It can also be used to detect aldosterone levels in large-scale breeding poultry, livestock, and aquatic animals in batches, to assess the effects of breeding density, water temperature, salinity, and feed salt on animal physiological homeostasis, only to optimize breeding processes, and not to determine animal diseases, etc.

[0018] Fifthly, this application also provides an aldosterone immunoassay product, the immunoassay product comprising the aldosterone antibody mAb4 or its antigen-binding fragment as described in the first aspect or the biological material as described in the second aspect.

[0019] Furthermore, the aldosterone immunoassay product is a reagent or kit for detection using enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), immunoprecipitation, and magnetic microparticle chemiluminescence immunoassay.

[0020] Compared to existing technologies, the aldosterone monoclonal antibody mAb4 or its antigen-binding fragment of this application can bind to aldosterone with high specificity and high affinity, with an affinity constant Ka reaching 2.5 × 10⁻⁶. 9 L / mol. The aldosterone monoclonal antibody mAb4 of this application can also be used to prepare various immunoassay kits for detecting aldosterone, especially for use in immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence methods. The double-antibody sandwich chemiluminescence platform for detecting aldosterone standard antigens has a detection sensitivity of less than 10 pg / mL. Magnetochemiluminescence detection of clinical samples shows good clinical correlation within the sample range of 0-1000 pg / mL. It can be widely used in the clinical auxiliary diagnosis and efficacy evaluation of essential hypertension and various aldosterone metabolism disorders, possessing extremely high clinical application value. Attached Figure Description

[0021] Figure 1 This is an electrophoresis diagram of the full-length amplification products of the mAb4 heavy and light chains, where M is the DNA molecular weight marker.

[0022] Figure 2 The mAb4 sandwich ELISA method was used to detect cross proteins and target antigen samples. The vertical axis represents the detected OD value.

[0023] Figure 3The standard curve was used for the detection of aldosterone standard antigen by mAb4 magnetic microparticle chemiluminescence immunoassay. The x-axis represents the concentration of aldosterone standard antigen (pg / mL), and the y-axis represents the detected luminescence value. The R-value of the standard curve is... 2 =0.9994, linear detection range 0-1000 pg / mL, derive the sample concentration calculation formula: y = -2.4064x 2 + 6991.7x - 52757.

[0024] Figure 4 The standard curve was used to detect clinical samples using mAb4 magnetic microparticle chemiluminescence immunoassay. The x-axis represents the concentration of free aldosterone in the clinical samples (pg / mL), and the y-axis represents the detected luminescence value. The R-value of the standard curve is... 2 =0.9534, linear detection range 0-1000pg / mL, the sample concentration calculation formula is derived as: y=5519.6x+4296.6. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.

[0026] Example 1: Preparation of rabbit monoclonal antibody against aldosterone

[0027] 1) Preparation of immunogens Aldosterone-KLH was prepared with an immunogen purity of over 90%, meeting the purity requirements for preparing monoclonal antibodies.

[0028] 2) Animal immunization The aldosterone-KLH prepared above was emulsified with complete Freund's adjuvant at a 1:1 volume ratio and administered subcutaneously to approximately 2 kg New Zealand white rabbits at a dose of 800 μg / rabbit. A second immunization was performed two weeks later, emulsified with incomplete Freund's adjuvant at a 1:1 volume ratio, at a dose of 400 μg / rabbit. Tail blood was collected after both immunizations and serum titers were determined using a serially diluted ELISA method. The OD value at an ELISA titer of 128000 was used as the baseline. 450 A titer greater than 1.0 is used as the standard. Based on the results, it is determined whether to collect PBMCs or continue immunization. Rabbits with the highest antibody titers are selected for PBMC collection.

[0029] 3) PBMC isolation, specific B cell sorting, and clonal recombination The rabbit was placed supine on the operating table. The fur around the heart was trimmed, and the skin was disinfected with alcohol. The area with the most prominent heartbeat was selected and punctured with a 50mL syringe. Blood flowed into the syringe immediately after the needle entered the heart. The needle was quickly withdrawn after obtaining the required amount of blood. The whole blood in the syringe was transferred into a sterile 50mL tube and mixed with an equal amount of PBS. The mixture was then slowly added dropwise to the lymphocyte separation medium. The mixture was centrifuged at 400×g for 30 minutes at room temperature. After centrifugation, the liquid surface separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (i.e., a mononuclear cell layer), a separation medium layer, and a red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium, thus obtaining rabbit PBMCs.

[0030] Antigen-specific B cells were further sorted from rabbit PBMCs and cultured. Positive clones were selected from the B cell supernatant using antigen-coated ELISA plates. Cells from positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. The full-length light and heavy chain sequences of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones. Rabbit monoclonal antibody expression vectors were constructed using clonal recombination methods, and the sequences were confirmed by sequencing. The results of the amplified full-length PCR products are shown below. Figure 1 .

[0031] 4) Preparation and purification of monoclonal antibodies To obtain multiple rabbit monoclonal antibodies recognizing aldosterone molecules, the heavy and light chain genes of rabbit monoclonal antibodies were loaded into an expression vector. The plasmid was transfected into KEK293 cells, and after 120-144 hours, the culture supernatant contained recombinant rabbit monoclonal antibodies recognizing aldosterone molecules. The cell suspension was collected, the supernatant was obtained by centrifugation, and the antibody was purified by affinity chromatography. The concentration of the purified monoclonal antibody was determined by the BCA method, and then aliquoted, lyophilized, and named rabbit monoclonal antibody mAb4.

[0032] Example 2 Identification of aldosterone rabbit monoclonal antibody

[0033] 1) Specificity identification of rabbit monoclonal antibodies Indirect ELSA was used for detection. The microplate was coated with crosslinking protein and aldosterone-BSA at a concentration of 1 μg / mL and incubated overnight at 4°C. The microplate was then blocked with PBST containing 1% BSA. 10 μg / mL of PBST was added. 4 The purified rabbit monoclonal antibody was diluted 10 times and reacted at 37°C for 50 min. The plate was washed 3 times with PBST, HRP-goat anti-rabbit IgG secondary antibody was added, and the plate was reacted at 37°C for 50 min. The plate was washed 5 times with PBST, TMB was added for color development for 10 min, stop solution was added, and the A450 was measured by microplate reader.

[0034] Figure 2 The results showed that the cross-linking protein reacted negatively with rabbit monoclonal antibody mAb4, and OD 450All values ​​were less than 0.3; the aldosterone-BSA antibody showed a positive reaction with mAb4 antibody, and the OD value was much higher than that of the cross protein, indicating that the rabbit monoclonal antibody for aldosterone in this application specifically recognizes aldosterone.

[0035] 2) Determination of affinity constant of rabbit monoclonal antibody

[0036] Affinity constant (Ka) was determined using a non-competitive ELISA method.

[0037] Coating: Dilute the antigen with carbonate buffer to concentrations of 1, 0.5, 0.1, and 0.05 μg / mL, add 100 μL / well to a 96-well microplate for coating, and incubate at 4°C for 24 h.

[0038] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h.

[0039] Add monoclonal antibody: Wash the plate 4 times with PBST, serially dilute the rabbit monoclonal antibody with carbonate buffer starting at 100 μg / mL, add 100 μL to each well, and incubate at 37°C for 2 h.

[0040] Add enzyme-labeled secondary antibody: Wash the plate 4 times with PBST, add 100 μL of HRP enzyme-labeled goat anti-rabbit Ig secondary antibody diluted 1:10000 to each well, and incubate at 37℃ for 30 min.

[0041] Color development and termination: Wash the plate 4 times with PBST, add 100 μL of substrate color development solution to each well, and react at 37℃ in the dark for 15 min; add 50 μL of 1.0 mol / L H2SO4 stop solution to each well to terminate the reaction.

[0042] Detection: The absorbance value at a wavelength of 450 nm was measured (A). 450 nm).

[0043] An sigmoid curve was plotted with the logarithm of antibody concentration on the x-axis and OD value on the y-axis. The calculated affinity constant Ka for the aldosterone monoclonal antibody mAb4 was 2.5 × 10⁻⁶. 9 L / mol.

[0044] 3) Sandwich antibody pairing To select the optimal combination of coating and detection antibodies, mAb4 was coated onto an ELISA plate and incubated overnight at 4°C. The next day, the plate was removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed three times with PBST. 100 μL of aldosterone (20 ng / mL) was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, the plate was removed, washed three times with PBST, and HRP-labeled complex monoclonal antibody was added as the detection antibody, incubated at 37°C for 1 hour. The plate was washed five times with PBST, TMB substrate was added, and the plate was incubated at 37°C for 10 minutes. Finally, stop solution was added, and the OD was measured using an ELISA reader. 450 Readings. Based on the OD value of the sample and the background value of the negative control, the most ideal antibody pair was selected. The pairing screening results are shown in Table 1.

[0045] Table 1. Results of antibody pairing assay

[0046] As shown in Table 1, rabbit monoclonal antibody mAb17, used as the coating antibody, was effective in determining OD on an ELISA reader. 450 A reading greater than 3 indicates that the pairing combination formed by the antibody is feasible and that the antibody has good overall performance.

[0047] Example 3: Analysis of the gene and amino acid sequence of the variable region of a monoclonal antibody Using the recombinant plasmid of the antibody as a DNA template, sequencing primers for the light chain variable region and heavy chain variable region were designed based on the vector sequences at the 5' ends of the light and heavy chains on the template. Sequencing was performed using an ABI 3730 sequencer. The nucleotide sequences of the light and heavy chain variable regions of the rabbit monoclonal antibody were obtained by sequencing.

[0048] Using the internet and the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the nucleotide sequences of the light chain variable region and the heavy chain variable region were sequenced and analyzed. The amino acid sequence of the light chain variable region of rabbit monoclonal antibody mAb4 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.

[0049] The VL is 113 amino acids long. The number of amino acids in the four domains of its FR are 26, 17, 36 and 11, respectively. The number of amino acids in the three domains of LCDR are 6, 3 and 14, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-102aa, respectively. Their amino acid sequences are QSIGSS (SEQ ID NO.1), DVS (SEQ ID NO.2) and QGYYWSSYTTYGNI (SEQ ID NO.3), respectively.

[0050] The VH is 119 amino acids in length. The number of amino acids in the four domains of its FR are 25, 17, 39 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 7 and 12, respectively. HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 97aa-108aa, respectively. Their amino acid sequences are GIDLNTNA (SEQ ID NO.4), IDSGGTW (SEQ ID NO.5) and ARSYRYWWYFNL (SEQ ID NO.6), respectively.

[0051] Example 4: Aldosterone monoclonal antibody used for magnetic microparticle chemiluminescent immunoassay.

[0052] 1. Detection Principle and Method A magnetic microparticle chemiluminescence immunoassay technique based on the double-antibody sandwich method was employed. Biotin-labeled mAb4 was immobilized with SA magnetic beads, and ALP was conjugated to an aldosterone sandwich method rabbit monoclonal antibody. Simultaneously, aldosterone, ALP substrate, and corresponding buffer components were placed on a Cosmetic fully automated magnetic microparticle chemiluminescence analyzer, and the instrument program was set for detection. A positive result was interpreted as a signal-to-noise ratio (SNR) greater than 2.0. The magnitude of the luminescence value reflects the amount of bound enzyme-labeled antibody and is directly proportional to the concentration of aldosterone in the sample. A standard curve was plotted based on the measured luminescence values ​​of the standards, as shown below. Figure 3 The concentration of aldosterone in the sample to be tested can be obtained from the standard curve.

[0053] 2. Composition of the magnetic microparticle chemiluminescence detection kit for aldosterone detection

[0054] 1) SA magnetic beads bound to biotin mAb4: Take 50 μL of magnetic beads into a 0.5 mL centrifuge tube, place it on a magnetic rack, and remove the supernatant after 1 min; wash the magnetic beads 3 times with 0.5 mL of antibody dilution buffer; add a certain amount of biotin-labeled mAb4 and mix at room temperature for 60 min; after magnetic separation, resuspend in magnetic storage buffer at a working concentration of 0.5 mg / mL.

[0055] 2) ALP conjugation with complex antibody: First, the 2-IT antibody is reduced; then, the ALP-SMCC intermediate is formed; finally, the ALP-SMCC is conjugated with the reducing antibody. After conjugation, the ALP is diluted to the working concentration using ALP preservation buffer.

[0056] 3) Washing buffer: is standard pH 7.4 PBST containing 0.05% Proclin 300, prepared as a 20-fold concentrate.

[0057] 4) Chemiluminescent colorimetric solution: purchased from Aivid Biotechnology.

[0058] 5) Sample diluent: PBST containing 1% BSA and 0.05% Proclin 300, filtered for sterilization.

[0059] 6) Standard: Aldosterone small molecule, diluted to 5 μg / mL with PBS containing 1% BSA, 5% sucrose, 10% glycerol and 0.05% Proclin 300 as diluent, filtered for sterilization and aseptically dispensed.

[0060] 3. Detection of aldosterone in clinical samples Clinical samples with different aldosterone concentrations were processed. mAb4 was used as the coating antibody, and the complex antibody was used as the detection antibody. The above detection method was applied to detect clinical samples of different concentrations. The results are shown below. Figure 4 .

[0061] Based on the results, the rabbit monoclonal antibody described in this application, when used in a magnetic microparticle chemiluminescent immunoassay reagent, showed good clinical correlation within a sample range of 0-1000 pg / mL.

[0062] In summary, when the aldosterone mAb4 monoclonal antibody of this application is applied to immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence methods, the double-antibody sandwich chemiluminescence platform detects aldosterone standard antigen with a detection sensitivity of less than 10 pg / mL. When the magnetochemiluminescence method detects clinical samples, the clinical concordance rate is >0.95 in the sample range of 0-1000 pg / mL, which is significantly higher than that of traditional competitive detection methods. Moreover, the process is simple and breaks through the limitations of traditional competitive methods.

[0063] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An aldosterone monoclonal antibody mAb4 or its antigen-binding fragment, characterized in that, Includes the light chain variable region VL and the heavy chain variable region VH. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.1 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

1. The amino acid sequence of LCDR2 is DVS or a variant of DVS with one amino acid substitution, deletion, or addition. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.3; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is the sequence shown in SEQ ID NO.4 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

4. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.5 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

6.

2. The aldosterone monoclonal antibody mAb4 or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region VL comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO. 7, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids; the heavy chain variable region VH comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO. 8, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids.

3. The aldosterone monoclonal antibody mAb4 or its antigen-binding fragment according to claim 2, characterized in that, The light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.

8.

4. The aldosterone monoclonal antibody mAb4 or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

5. A biomaterial, characterized in that, The biomaterial is selected from polynucleotides, carriers, or cells. The polynucleotide encodes the aldosterone monoclonal antibody mAb4 or its antigen-binding fragment as described in any one of claims 1-4; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is capable of expressing the aldosterone monoclonal antibody mAb4 or its antigen-binding fragment as described in any one of claims 1-4.

6. The method for preparing the aldosterone monoclonal antibody mAb4 or its antigen-binding fragment according to any one of claims 1-4, characterized in that, The method includes culturing the cells as described in claim 5, wherein the cells are prepared by transforming the cells with a polynucleotide encoding aldosterone monoclonal antibody mAb4 or an antigen-binding fragment thereof, the polynucleotide including a heavy chain expression plasmid and a light chain expression plasmid, and the transformation includes co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

7. The application of the aldosterone monoclonal antibody mAb4 or its antigen-binding fragment according to any one of claims 1-4, or the biomaterial according to claim 5, characterized in that, The application is selected from one or more of the following: 1) Detection of aldosterone for purposes other than disease diagnosis and treatment; 2) Prepare immunoassay products for detecting aldosterone; 3) Used for purifying aldosterone; 4) Prepare products for purifying aldosterone.

8. An aldosterone immunoassay product, characterized in that, The immunoassay product comprises the aldosterone monoclonal antibody mAb4 as described in any one of claims 1-4 or its antigen-binding fragment, or the biological material as described in claim 5.

9. The aldosterone immunoassay product according to claim 8, characterized in that, The aldosterone immunoassay products are reagents or kits for detection using enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunohistochemical staining, flow cytometry, immunoprecipitation, and magnetic microparticle chemiluminescence immunoassay.

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