An aldosterone sandwich method antibody mAb7 or antigen-binding fragment thereof, and a preparation method and application thereof

Abstract

The application belongs to the technical field of immunoassay, and discloses an aldosterone sandwich method antibody mAb7 or an antigen binding fragment thereof, and a preparation method and application thereof. The aldosterone sandwich method antibody mAb7 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 sandwich method antibody mAb7 is used to detect an aldosterone standard antigen, and the detection sensitivity is lower than 10 pg / mL. The clinical sample is detected by a magnetic chemiluminescence method, and in a sample range of 0-1000 pg / mL, the correlation with R clinical comparison is good, and the aldosterone sandwich method antibody mAb7 has important significance in the diagnosis and treatment of primary hypertension and other aldosterone abnormality related diseases.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, specifically to an aldosterone sandwich antibody mAb7 or its antigen-binding fragment, its preparation method, and its application. Background Technology

[0002] Aldosterone is a steroid hormone synthesized and secreted by the zona glomerulosa of the adrenal cortex. It belongs to the mineralocorticoid family and has the chemical formula CHO5. It mainly acts on the kidneys to regulate water and electrolyte balance and stabilize blood pressure. It plays an absolutely central role in maintaining the balance of blood volume, blood pressure and electrolytes (especially sodium and potassium) in the human body.

[0003] Physiologically, aldosterone maintains electrolyte homeostasis and fluid volume by promoting sodium reabsorption in the distal convoluted tubule and collecting duct of the kidney, while simultaneously increasing potassium and hydrogen ion excretion. Aldosterone secretion is primarily regulated by the renin-angiotensin system. When blood pressure decreases or sodium levels drop, juxtaglomerular cells in the kidney secrete renin, activating angiotensin II and stimulating aldosterone synthesis. Furthermore, elevated serum potassium or decreased serum sodium levels can directly promote aldosterone secretion, and certain medications, such as angiotensin-converting enzyme inhibitors, or physiological states such as pregnancy may also affect its levels.

[0004] Aldosterone abnormalities are associated with a variety of diseases. Excessive secretion can lead to primary aldosteronism, such as that caused by adrenal adenoma, manifesting as hypertension and hypokalemia. Insufficient secretion is seen in adrenal insufficiency, which may result in hyperkalemia and hyponatremia. Clinical testing often involves screening using plasma aldosterone concentration and the aldosterone / renin activity ratio.

[0005] Currently, mass spectrometry is the "gold standard" for aldosterone detection. However, mass spectrometry requires complex and time-consuming sample pretreatment procedures, and clinical applications demand high levels of automation, making large-scale application difficult. Therefore, immunoassay is commonly used in clinical practice. Immunoassay is simple to operate and rapid, making it a feasible alternative to mass spectrometry for small molecules. Because small molecules lack antigenic epitopes, competitive immunoassays are commonly used in clinical practice. Competitive immunoassays suffer from low sensitivity, poor specificity, a high risk of false positives, and complex processes.

[0006] Therefore, there is an urgent need to develop highly specific and sensitive monoclonal antibodies for the aldosterone sandwich assay to achieve the detection of aldosterone using a double-antibody sandwich method. Summary of the Invention

[0007] This application provides a high-specificity, high-affinity aldosterone sandwich antibody mAb7 or its antigen-binding fragment, as well as its preparation method and applications. Antibody mAb7 or its antigen-binding fragment can successfully enable rapid detection of aldosterone in immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence methods, which is of great significance in the diagnosis and treatment of essential hypertension and other aldosterone-related diseases.

[0008] In one aspect, this application provides an aldosterone sandwich antibody mAb7 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 DTS (SEQ ID NO.2) or a variant with one amino acid substitution, deletion, or addition compared to DTS. 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 aldosterone sandwich antibody mAb7 or its antigen-binding fragment can specifically bind to 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 sandwich antibody mAb7 or its antigen-binding fragment as described in the first aspect; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is capable of expressing the aldosterone sandwich antibody mAb7 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 sandwich antibody mAb7 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 sandwich antibody mAb7 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 the application of the aldosterone sandwich antibody mAb7 or its antigen-binding fragment as described in the first aspect, or the biological material as 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 monitor adaptive changes in water and electrolyte metabolism hormones, including aldosterone, in athletes during extreme training or ordinary people entering high-altitude environments; it can be used to assess the effects of ketogenic diets, intermittent fasting, and diets with different sodium / potassium intake ratios on the aldosterone-renin system, and to explain its physiological effects from a hormonal perspective; it can also be used to study chronic changes in the human endocrine system, including aldosterone, under long-term exposure to environmental stressors such as noise, high temperature, and specific chemicals, etc.

[0018] Fifthly, this application also provides an aldosterone immunoassay product, the immunoassay product comprising the aldosterone sandwich antibody mAb7 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 sandwich antibody mAb7 or its antigen-binding fragment of this application can bind to aldosterone with high specificity and high affinity, with an affinity constant Ka reaching 6 × 10⁻⁶. 8 L / mol. The aldosterone sandwich antibody mAb7 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 detects aldosterone standard antigens with a sensitivity of less than 10 pg / mL. Magnetochemiluminescence detection of clinical samples within the 0-1000 pg / mL sample range shows good correlation with clinical R levels, which is of great significance in the diagnosis and treatment of essential hypertension and other aldosterone-related diseases. Attached Figure Description

[0021] Figure 1 This is an electrophoresis diagram of the full-length amplification products of the mAb7 heavy and light chains, where M is the DNA molecular weight marker.

[0022] Figure 2 The mAb7 sandwich ELISA method was used to detect cross-linked proteins and target antigen samples. The vertical axis represents the detected OD value.

[0023] Figure 3 The standard curve was prepared using mAb7 magnetic microparticle chemiluminescence immunoassay for the detection of aldosterone standard antigen. 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 =0.9945, linear detection range 0-1000pg / mL, the sample concentration calculation formula is derived as: y=402.43x-6673.

[0024] Figure 4 The standard curve was used to detect clinical samples using mAb7 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 =0.9663, linear detection range 0-1000pg / mL, the sample concentration calculation formula is derived as: y=1830.6x-54519. 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 using the aldosterone sandwich method 1) Preparation of immunogens The aldosterone complex was prepared with an immunogen purity of over 90%, meeting the purity requirements for preparing monoclonal antibodies.

[0027] 2) Animal immunization The aldosterone complex 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 OD450 at an ELISA titer of 128,000 was considered to be greater than 1.0. Based on the results, it was determined whether to collect PBMCs or continue immunization. Rabbits with the highest antibody titers were selected for PBMC collection.

[0028] 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.

[0029] 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 .

[0030] 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 mAb7.

[0031] Example 2: Identification of Rabbit Monoclonal Antibodies Using the Aldosterone Sandwich Method 1) Specificity identification of rabbit monoclonal antibodies Indirect ELSA was used for detection. The ELISA plate was coated with cross-linked protein and aldosterone complex antigen at a concentration of 1 μg / mL and incubated overnight at 4°C. The plate 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.

[0032] Figure 2The results showed that the cross protein reacted negatively with the rabbit monoclonal antibody mAb7, with OD450 values ​​less than 0.1; the aldosterone complex reacted positively with the mAb7 antibody, with OD values ​​much higher than those of the cross protein, indicating that the aldosterone sandwich method rabbit monoclonal antibody of this application specifically recognizes the aldosterone complex.

[0033] 2) Determination of affinity constant of rabbit monoclonal antibody Affinity constant (Ka) was determined using a non-competitive ELISA method.

[0034] 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.

[0035] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Detection: The absorbance value at a wavelength of 450 nm (A450nm) was measured.

[0040] Plotting the logarithm of antibody concentration on the x-axis and OD value on the y-axis, an S-shaped curve was obtained. The calculated affinity constant Ka for the aldosterone sandwich monoclonal antibody mAb7 was 6 × 10⁻⁶. 8 L / mol.

[0041] 3) Sandwich antibody pairing To select the optimal combination of coating and detection antibodies, aldosterone-binding protein 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 rabbit monoclonal antibody mAb7 was added as the detection antibody. The plate was incubated at 37°C for 1 hour. After washing five times with PBST, TMB substrate was added, and the plate was incubated at 37°C for 10 minutes. Stop solution was added, and the OD450 reading was measured using a microplate reader. Based on the OD values ​​of the samples and the background value of the negative control, the most ideal antibody pair was selected. The pairing screening results are shown in Table 1.

[0042] Table 1. Results of antibody pairing assay

[0043] Therefore, the antibody mAb7 involved in this application is optimal for sandwich assay.

[0044] 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.

[0045] 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 mAb7 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.

[0046] The VL is 110 amino acids long. The number of amino acids in the four domains of its FR are 26, 16, 36 and 11, respectively. The number of amino acids in the three domains of LCDR are 6, 3 and 12, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 49aa-51aa and 88aa-99aa, respectively. Their amino acid sequences are QNIGSD (SEQ ID NO.1), DTS (SEQ ID NO.2) and QCSIVNNNYIGA (SEQ ID NO.3), respectively.

[0047] The VH is 114 amino acids long. The number of amino acids in the four domains of its FR are 25, 17, 38 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 7 and 8, respectively. HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 96aa-103aa, respectively. Their amino acid sequences are GFSLSTYW (SEQ ID NO.4), IDTGGSA (SEQ ID NO.5) and ARFPNSDL (SEQ ID NO.6), respectively.

[0048] Example 4: Aldosterone sandwich method rabbit monoclonal antibody used for magnetic microparticle chemiluminescent immunoassay.

[0049] 1. Detection Principle and Method A magnetic microparticle chemiluminescence immunoassay technique based on the double-antibody sandwich method was employed. Biotin-labeled aldosterone-binding protein was immobilized with SA magnetic beads, and ALP was conjugated to a rabbit monoclonal antibody using the aldosterone sandwich method. Simultaneously, aldosterone, ALP substrate, and corresponding buffer components were placed on a Cosma 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.

[0050] 2. Composition of the magnetic microparticle chemiluminescence detection kit for aldosterone detection

[0051] 1) SA magnetic beads bound to biotin-aldosterone primary antibody: 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 aldosterone primary antibody and mix by rotating at room temperature for 60 min; after magnetic separation, resuspend in magnetic preservation buffer at a working concentration of 0.5 mg / mL.

[0052] 2) mAb7 conjugation to ALP: First, the mAb7 antibody is reduced using 2-IT; then, an ALP-SMCC intermediate is formed; finally, ALP-SMCC is conjugated to the reducing antibody. After conjugation, the ALP is diluted to the working concentration using ALP preservation buffer.

[0053] 3) Washing buffer: is standard pH 7.4 PBST containing 0.05% Proclin 300, prepared as a 20-fold concentrate.

[0054] 4) Chemiluminescent colorimetric solution: purchased from Aivid Biotechnology.

[0055] 5) Sample diluent: PBST containing 1% BSA and 0.05% Proclin 300, filtered for sterilization.

[0056] 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.

[0057] 3. Detection of aldosterone in clinical samples Clinical samples with different aldosterone concentrations were processed. Using aldosterone primary antibody as the coating antibody and mAb7 antibody as the detection antibody, the above detection method was used to detect clinical samples of different concentrations. The results are shown below. Figure 4 .

[0058] Based on the results, the rabbit monoclonal antibody described in this application, used in a magnetic microparticle chemiluminescent immunoassay reagent, showed good correlation with clinical results within a sample range of 0-1000 ng / mL.

[0059] In summary, when the aldosterone sandwich rabbit monoclonal antibody mAb7 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 sensitivity of less than 10 pg / mL. When detecting clinical samples by magnetochemiluminescence, the clinical composite rate is >0.96 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.

[0060] 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 sandwich antibody mAb7 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 shown in SEQ ID NO.

1. The amino acid sequence of LCDR2 is DTS. The amino acid sequence of LCDR3 is shown in SEQ ID NO.3; The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO.

4. The amino acid sequence of HCDR2 is shown in SEQ ID NO.

5. The amino acid sequence of HCDR3 is shown in SEQ ID NO.6; Furthermore, the aldosterone sandwich antibody mAb7 or its antigen-binding fragment can specifically bind to aldosterone.

2. The aldosterone sandwich antibody mAb7 or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.

8.

3. The aldosterone sandwich antibody mAb7 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, and dsFv.

4. A biomaterial, characterized in that, The biomaterial is selected from polynucleotides, carriers, or cells. The polynucleotide encodes the aldosterone sandwich antibody mAb7 or its antigen-binding fragment as described in any one of claims 1-3; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the carrier, or is capable of expressing the aldosterone sandwich antibody mAb7 or its antigen-binding fragment as described in any one of claims 1-3.

5. The method for preparing the aldosterone sandwich antibody mAb7 or its antigen-binding fragment according to any one of claims 1-3, characterized in that, The method includes culturing cells as described in claim 4, wherein the cells are prepared by transforming cells with a polynucleotide encoding an aldosterone sandwich antibody mAb7 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.

6. The application of the aldosterone sandwich antibody mAb7 or its antigen-binding fragment according to any one of claims 1-3, or the biomaterial according to claim 4, 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.

7. An aldosterone immunoassay product, characterized in that, The immunoassay product comprises the aldosterone sandwich antibody mAb7 or its antigen-binding fragment as described in any one of claims 1-3, or the biological material as described in claim 4.

8. The aldosterone immunoassay product according to claim 7, characterized in that, The aldosterone immunoassay products are reagents or kits for enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunohistochemical staining, flow cytometry, immunoprecipitation, and magnetic microparticle chemiluminescence immunoassay.

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