Anti-aldosterone antibody and application thereof

Abstract

The invention discloses an anti-aldosterone antibody and application thereof, and relates to the field of antibodies. The anti-aldosterone antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for aldosterone detection, and has good affinity or activity.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an anti-aldosterone antibody and its application. Background Technology

[0002] Aldosterone is a steroid hormone (a member of the mineralocorticoid family) produced by the adrenal cortex. It primarily acts on the kidneys to reabsorb sodium and water, thus maintaining stable blood pressure. Overall, aldosterone is a hormone that enhances the kidneys' reabsorption of ions and water, and is part of the renin-angiotensin system.

[0003] Aldosterone primarily acts on the distal convoluted tubule and collecting duct. The cells of these tubules and ducts contain mineralocorticoid receptors that bind to aldosterone. These receptors are linked to a segment of deoxyribonucleic acid (DNA) with a hormonal response. When aldosterone binds to the MR receptor, this DNA undergoes specific transcription, producing messenger RNA (mRNA), which is then translated into a specific protein. This protein increases the permeability of the tubular membrane to sodium ions and increases the activity of the sodium-potassium pump on the basolateral membrane (its primary function). It also stimulates ATP hydrolysis to produce phosphate ions, which are supplied to the pump for phosphorylation. Phosphorylation alters the pump's conformation, reducing its permeability to sodium ions. This allows sodium ions and water in the urine to be reabsorbed back into the bloodstream, while potassium ions are excreted into the urine. Simultaneously, it stimulates increased ATP production in the mitochondria to provide the energy required for phosphorylation of the sodium-potassium pump during active sodium ion reabsorption.

[0004] Aldosterone plays a crucial role in maintaining intravascular volume and electrolyte balance. Studies have found that aldosterone measurement can be used to investigate cardiovascular diseases, cirrhosis, kidney diseases (adrenal incidental tumors, adrenal carcinoma, Addison's disease, congenital adrenal hyperplasia, renal artery stenosis, and renal tubular access disease, etc.), insulin resistance, and diabetes. Accurate aldosterone measurement is essential for the screening, diagnosis, and subtype classification of primary aldosteronism. Primary aldosteronism is a serious and frequently missed secondary hypertension, mainly caused by aldosterone-producing adenomas or idiopathic aldosteronism caused by bilateral adrenal hyperplasia. The prevalence of primary aldosteronism ranges from 3% to 32%, and excessive aldosterone secretion is accompanied by severe damage to target organs such as the heart and brain. Therefore, early identification of primary aldosteronism is crucial, which depends on accurate aldosterone measurement.

[0005] Currently, the main detection methods for aldosterone include chemiluminescence immunoassay, which is an immunological detection method based on the specific reaction between antigen and antibody, and using luminescent substances (such as peroxidase, isoluminol, etc.) to amplify and display the detected signal. Similar immunological detection methods include radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), and latex-enhanced immunoturbidimetric assay.

[0006] All of the above immunological detection methods require antibodies against aldosterone. Therefore, there is a strong demand in this field for anti-aldosterone antibodies with good performance. Summary of the Invention

[0007] This application provides an antibody against aldosterone, which provides an important source of raw materials for the detection of aldosterone and has good activity or affinity.

[0008] To achieve the above objectives, according to one aspect of the present invention, an anti-aldosterone antibody is provided, the antibody comprising three complementary determining regions having a heavy chain variable region having any one of the amino acid sequences SEQ ID NO:21, 22, 23 and three complementary determining regions having a light chain variable region having any one of the amino acid sequences SEQ ID NO:27, 28, 29.

[0009] To achieve the above objective, according to a second aspect of the present invention, an anti-aldosterone antibody is provided, the antibody comprising the following complementarity-determining region:

[0010] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1;

[0011] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2;

[0012] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3 or 17;

[0013] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4;

[0014] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5;

[0015] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6 or 18.

[0016] To achieve the above objectives, according to a third aspect of the present invention, an anti-aldosterone antibody is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID NO:21, 22, 23; and the amino acid sequence of the light chain variable region is shown as any one of SEQ ID NO:27, 28, 29.

[0017] To achieve the above objectives, according to a fourth aspect of the present invention, an anti-aldosterone antibody is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 24, 25, 26; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 30, 31, 32.

[0018] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the antibodies described above.

[0019] To achieve the above objectives, according to a sixth aspect of the present invention, a reagent or kit is provided, the reagent or kit comprising the antibody or antibody conjugate described above.

[0020] To achieve the above objectives, according to a seventh aspect of the present invention, the use of the above-described antibody and antibody conjugate in the preparation of aldosterone detection products is provided.

[0021] To achieve the above objectives, the present invention also provides a nucleic acid, a vector, a cell, and a method for preparing the above-mentioned antibody. Detailed Implementation

[0022] In a first aspect, embodiments of the present invention provide an anti-aldosterone antibody, the antibody comprising three complementary determinant regions having a heavy chain variable region having any one of the amino acid sequences SEQ ID NO:21, 22, 23 and three complementary determinant regions having a light chain variable region having any one of the amino acid sequences SEQ ID NO:27, 28, 29.

[0023] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to those of HCDR1, HCDR2, and HCDR3 in the same heavy chain variable region defined in the antibody described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to those of LCDR1, LCDR2, and LCDR3 in the same light chain variable region defined in the antibody described in the first aspect.

[0024] For example, HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:21; and LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:27.

[0025] In this invention, the term "antibody" is used in the broadest sense, and may include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments, as long as they exhibit the desired biological activity.

[0026] Antigen-binding fragments typically possess the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the aforementioned antigen-binding fragments can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0027] Antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0028] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0029] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0030] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below; definitions vary slightly in different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0031] Table 1: CDR Definition 1

[0032] CDR Kabat AbM2 IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0033] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0034] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0035] 3If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0036] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0037] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0038] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0039] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0040] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0041] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0042] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0043] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0044] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0045] Secondly, embodiments of the present invention provide an antibody against aldosterone, the antibody comprising the following complementarity-determining regions:

[0046] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1.

[0047] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2.

[0048] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3 or 17.

[0049] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4.

[0050] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5.

[0051] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6 or 18.

[0052] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0053] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0054] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0055] In an optional embodiment, the antibody described in the first or second aspect further comprises HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0056] In an optional embodiment, the HFR1 includes / such as SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it;

[0057] The HFR2 includes / is such as SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it;

[0058] The HFR3 includes / is, for example, SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it;

[0059] The HFR4 includes / is, for example, SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it;

[0060] The LFR1 includes / such as SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it;

[0061] The LFR2 includes / is, for example, SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it;

[0062] The LFR3 includes / is, for example, SEQ ID NO:13 or an amino acid sequence having at least 80% identity with it; and

[0063] The LFR4 includes / such as SEQ ID NO:14 or an amino acid sequence having at least 80% identity with it.

[0064] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the anti-aldosterone antibody provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) mentioned above.

[0065] In an optional embodiment, the HFR1 includes / as shown in SEQ ID NO:19, an amino acid sequence.

[0066] In an optional embodiment, the LFR1 includes / as shown in SEQ ID NO:20, an amino acid sequence.

[0067] In an optional embodiment, the antibody has a KD < 2.65 × 10⁻⁶.-8 M has an affinity for aldosterone.

[0068] In an optional embodiment, the antibody has a KD ≤ 10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M or KD≤10 -12 M binds to aldosterone with its affinity.

[0069] In an optional embodiment, the antibody has a KD ≤ 2.27 × 10⁻⁶. -9 M binds to aldosterone with its affinity.

[0070] There are many methods for determining antibody affinity (KD), which can be categorized into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods based on their detection principles. Common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biomembrane optical interferometry (BLI); and common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA).

[0071] In an optional implementation, KD is measured using a kinetic detection method; alternatively, surface plasmon resonance, for example, by using a method such as The system's biosensor system.

[0072] Thirdly, embodiments of the present invention provide an anti-aldosterone antibody comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID NO:21, 22, 23, and the amino acid sequence of the light chain variable region is shown as any one of SEQ ID NO:27, 28, 29.

[0073] In an optional implementation, the heavy chain variable region and the light chain variable region described in the first or third aspect above are selected from any combination of the following:

[0074] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:21 SEQ ID NO:27 2 SEQ ID NO:23 SEQ ID NO:27 3 SEQ ID NO:22 SEQ ID NO:27 4 SEQ ID NO:21 SEQ ID NO:29 5 SEQ ID NO:21 SEQ ID NO:28 .

[0075] In optional embodiments, the antibodies described in the first, second, and third aspects above further include a constant region.

[0076] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0077] In an optional implementation, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0078] In an optional embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0079] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0080] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0081] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0082] In an optional implementation, the species source of the constant region is mice.

[0083] In this paper, the partitioning of the variable and constant regions is based on the IMGT partitioning method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:housemouse(Mus musculus)IGHC,IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org .Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. theinternational ImMunoGenetics information http: / / www.imgt.orgCreated: 16 / 03 / 2011. Version: 17 / 01 / 2020. The variable regions delineated by different methods may differ in some amino acids from the C-terminus of the variable region delineated by IMGT or the N-terminus of the constant region. Variable regions or constant regions delineated by other methods known in the art are also within the scope of protection of this invention.

[0084] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region sequence (CL) is as shown in SEQ ID NO:16.

[0085] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant region (SEQ ID NO: 15 or 16).

[0086] In an optional embodiment, the antibody includes any one of F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0087] Fourthly, the present invention provides an antibody against aldosterone, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO:24, 25, 26, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:30, 31, 32.

[0088] In an optional embodiment, the antibody described in the first, second, third, or fourth aspect above includes any combination of the heavy and light chains:

[0089] combination Heavy chain Light chain 1 SEQ ID NO:24 SEQ ID NO:30 2 SEQ ID NO:26 SEQ ID NO:30 3 SEQ ID NO:25 SEQ ID NO:30 4 SEQ ID NO:24 SEQ ID NO:32 5 SEQ ID NO:24 SEQ ID NO:31

[0090] Fifthly, the present invention provides an antibody conjugate comprising the antibodies described above.

[0091] In an optional embodiment, the antibody conjugate further includes biotin or a biotin derivative conjugated with the antibody.

[0092] In an optional embodiment, the antibody conjugate may further include a marker or purification tag conjugated to the antibody.

[0093] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0094] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0095] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0096] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0097] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0098] In optional embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0099] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0100] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0101] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0102] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0103] In an optional embodiment, the colloidal metal is colloidal gold.

[0104] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0105] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0106] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0107] In a sixth aspect, the present invention provides a reagent or kit comprising the antibody or antibody conjugate described above.

[0108] As previously stated, the antibodies in some embodiments or examples of this invention can effectively bind to aldosterone. Therefore, reagents or kits containing the aldosterone antibodies can effectively perform qualitative or quantitative detection of aldosterone. The reagents or kits provided by this invention can be used, for example, for detections involving the specific binding properties of aldosterone and its antibodies, such as immunoblotting and immunoprecipitation. As previously stated, the antibodies in some embodiments or examples of this invention have higher binding activity or affinity to aldosterone; therefore, reagents or kits containing the antibodies have higher detection sensitivity or specificity.

[0109] In a seventh aspect, the present invention provides a method for detecting aldosterone, comprising: a) contacting the antibody, antibody-conjugate, reagent or kit described above with aldosterone in a sample to be tested under conditions sufficient to cause an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample;

[0110] In an optional embodiment, the immune complex further includes a second antibody that binds to the antibody.

[0111] In an optional embodiment, the immune complex further includes a second antibody that is bound to aldosterone.

[0112] Eighthly, the present invention provides the use of the above-mentioned anti-aldosterone antibody and antibody conjugate in the preparation of products for detecting aldosterone.

[0113] It should be noted that the products of this invention include, but are not limited to, reagents, kits, test strips, or reagent plates.

[0114] Ninthly, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0115] In a tenth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0116] In the eleventh aspect, the present invention provides cells containing the above-described carrier.

[0117] In a twelfth aspect, the present invention provides a method for preparing an anti-aldosterone antibody, comprising: culturing cells as described above.

[0118] Based on the amino acid sequence of the anti-aldosterone antibody disclosed in this invention, those skilled in the art will readily conceive of preparing the anti-aldosterone antibody using genetic engineering or other techniques (chemical synthesis, recombinant expression), such as isolating and purifying the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the anti-aldosterone antibody of this invention, it falls within the protection scope of this invention.

[0119] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0121] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0122] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0123] Example 1: Antibody Discovery of Monoclonal Antibodies

[0124] 1. Animal immunization

[0125] Aldosterone antigen (from Phypeng Biotechnology) was mixed with an equal volume of Freund's complete adjuvant to obtain an oily emulsion. This emulsion was injected subcutaneously at multiple sites into BALB / c mice at a dose of 0.2 ml per mouse. Fourteen days later, mice were immunized intraperitoneally with the same antigen and adjuvant. Immunization continued until the fourth injection, and tail blood was collected for titer testing. When the titer met the fusion requirements, a booster immunization was administered intraperitoneally three days before fusion, using the same dose of antigen mixed with an equal volume of 0.9% sodium chloride injection.

[0126] 2. Preparation of hybridoma cell lines

[0127] On the third day after booster immunization of mice, spleens were removed under sterile conditions. Mouse tumor cells and immune spleen cells were mixed at a ratio of 1:10, fused, and cultured. The culture medium was changed twice on the sixth day. On the seventh day after fusion, the cell supernatant was collected for antibody detection to screen for hybridoma cell lines secreting specific antibodies. One such line was selected and named Anti-ALD10F11.

[0128] Example 2: Preparation of Monoclonal Antibodies

[0129] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TM The RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen Pharmaceuticals. Primer synthesis and gene sequencing were performed by Invitrogen.

[0130] 1. Antibody sequence acquisition

[0131] (1) Antibody gene preparation

[0132] mRNA was extracted from the Anti-ALD 10F11 hybridoma cell line, and DNA products were obtained by RT-PCR. The product was then inserted into the pMD-18T vector after being subjected to an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After the cells grew, four clones of the Heavy Chain and Light Chain genes were collected and sent to a gene sequencing company for sequencing.

[0133] (2) Sequence analysis of antibody variable region genes

[0134] The gene sequences obtained from the sequencing were analyzed in the Kabat antibody database and VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was 318 bp, with a 57 bp leader peptide sequence preceding it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 363 bp, belonging to the VH1 gene family, with a 57 bp leader peptide sequence preceding it.

[0135] (3) Construction of recombinant antibody expression plasmid

[0136] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The 0.70kb Light Chain gene fragment and the 1.41kb Heavy Chain gene fragment were amplified by PCR.

[0137] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0138] 2. Recombinant antibody production

[0139] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. Cells were then incubated at 35°C (120 rpm) with 8% CO2 for 13 days. The samples were collected by centrifugation. The supernatant was purified using a protein A affinity chromatography column to obtain purified antibodies.

[0140] The resulting antibody was named Anti-ALD 10F11Rmb1. Mutations were performed on Anti-ALD 10F11Rmb1 to obtain a mutant antibody. The heavy chain (H) and light chain (L) sequences of the above antibody are shown in the table below:

[0141] Table 2: Antibody Sequences

[0142] Antibody name Heavy chain Light chain Anti-ALD 10F11Rmb1 SEQ ID NO:24 SEQ ID NO:30 Anti-ALD 10F11Rmb2 SEQ ID NO:26 SEQ ID NO:30 Anti-ALD 10F11Rmb3 SEQ ID NO:25 SEQ ID NO:30 Anti-ALD 10F11Rmb4 SEQ ID NO:24 SEQ ID NO:32 Anti-ALD 10F11Rmb5 SEQ ID NO:24 SEQ ID NO:31

[0143] Example 2: Antibody Performance Detection

[0144] 1. Affinity Analysis

[0145] The antibody was pre-diluted and purified, while the aldosterone antigen (from Phytobio) was serially diluted. Using a CM5 chip pre-conjugated with goat anti-mouse IgG, the binding and dissociation curves of the antigen and antibody were tested on a Biacore 8K+ device. The instrument automatically fitted and obtained the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0146] Table 3: Affinity Data

[0147]

[0148]

[0149] 2. Activity identification

[0150] Dilute aldosterone antigen (from Feipeng Biotechnology) to 3ug / ml with coating buffer (main component NaHCO3), 100uL per well, overnight at 4℃; the next day, wash twice with washing buffer (main component Na2HPO4 + NaCl), pat dry; add blocking buffer (20% BSA + 80% PBS), 120uL per well, incubate at 37℃ for 1h, pat dry; add diluted purified antibody and control antibody, 100uL / well, incubate at 37℃ for 30min; wash 5 times with washing buffer, pat dry; add goat anti-mouse IgG-HRP, 100uL per well, incubate at 37℃ for 30min; wash 5 times with washing buffer, pat dry; add chromogenic solution A (50uL / well), add chromogenic solution B (50uL / well), incubate for 10min; add stop solution, 50uL / well; read OD value at 450nm (reference 630nm) on a microplate reader.

[0151] Notes: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0152] Table 4: Activity Data

[0153] Concentration (ng / ml) 250 125 62.5 31.25 15.63 0 Comparison 1.202 0.817 0.461 0.193 0.112 0.014 Anti-ALD 10F11RMb1 1.818 1.136 0.855 0.452 0.207 0.028 Anti-ALD 10F11RMb2 1.733 1.125 0.832 0.473 0.249 0.023 Anti-ALD 10F11RMb3 1.877 1.234 0.797 0.452 0.282 0.024 Anti-ALD 10F11RMb4 1.764 1.143 0.798 0.394 0.204 0.028 Anti-ALD 10F11RMb5 1.886 1.143 0.872 0.401 0.271 0.025

[0154] 3. Stability assessment

[0155] The above-mentioned antibody was placed at 4℃ (refrigerator), -80℃ (refrigerator), and 37℃ (incubator) for 21 days. Samples were taken at 7 days, 14 days, and 21 days for observation, and the activity of the 21-day sample was tested. Table 5 below shows the OD results of the enzyme immunoassay for the antibody Anti-ALD 10F11Rmb3 after 21 days.

[0156] Table 5: Stability Data

[0157] Sample concentration (ng / ml) 125 62.5 0 4℃, 21-day sample 1.233 0.779 0.011 -80℃, 21-day sample 1.254 0.765 0.012 37℃, 21-day sample 1.236 0.734 0.014

[0158] Conclusion: The results showed that no significant changes in protein state were observed in the antibodies after 21 days under the three testing conditions, and the activity did not decrease with increasing testing temperature, indicating that the antibodies were stable.

[0159] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0160] The partial amino acid sequences involved in this application are shown in Table 6:

[0161]

[0162]

Claims

1. An anti-aldosterone antibody, said antibody comprising three complementary determinant regions having a heavy chain variable region having any one of the amino acid sequences SEQ ID NO:21, 22, 23 and three complementary determinant regions having a light chain variable region having any one of the amino acid sequences SEQ ID NO:27, 28, 29.

2. The antibody according to claim 1, characterized in that, The complementary determination region of the variable region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

3. An antibody against aldosterone, characterized in that, The antibody includes the following complementarity-determining regions: HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1; HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2; HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3 or 17; LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4; LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5; LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6 or 18; Optionally, the HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it; The HFR2 comprises SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it; The HFR4 comprises SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it; The LFR1 includes SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it; The LFR2 comprises SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it; The LFR3 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:13; and The LFR4 comprises SEQ ID NO:14 or an amino acid sequence having at least 80% identity with it; Optionally, the antibody has a KD < 2.65 × 10⁻⁶. -8 M binds to aldosterone with its affinity.

4. An anti-aldosterone antibody comprising a heavy chain variable region and / or a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 21, 22, and 23; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 27, 28, and 29. Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations: ； Optionally, the antibody further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Optionally, the species source of the constant region is mice; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% identity with it; Optionally, the light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity with it; Optionally, the antibody comprises any one of F(ab')2, Fab', Fab, Fv, and scFv.

5. An anti-aldosterone antibody, comprising a heavy chain and / or a light chain, characterized in that, The amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 24, 25, 26; the amino acid sequence of the light chain is shown in any one of SEQ ID NO: 30, 31, 32.

6. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further includes biotin or a biotin derivative conjugated with the antibody; Optionally, the antibody conjugate may further include a marker or purification tag conjugated to the antibody; Optionally, the marker is selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers; Optionally, the antibody conjugate may further include a solid-phase carrier conjugated to the antibody.

7. A reagent or kit, characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1 to 5 or the antibody conjugate as described in claim 6.

8. Use of the antibody according to any one of claims 1 to 5, or the antibody conjugate according to claim 6, in the preparation of products for detecting aldosterone; Optionally, the uses include: a) Under conditions sufficient to induce an antibody / antigen binding reaction, the antibody of any one of claims 1 to 5, the antibody conjugate of claim 6, or the reagent or kit of claim 7 is contacted with aldosterone in the sample to be tested to form an immune complex; and b) Detect the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample; Optionally, the immune complex further includes a second antibody, which binds to the antibody; Optionally, the immune complex further includes a second antibody that is bound to aldosterone.

9. A nucleic acid, a vector, a cell, or a method for preparing an antibody according to any one of claims 1 to 5, wherein the nucleic acid encodes an antibody according to any one of claims 1 to 5; the vector contains a nucleic acid encoding an antibody according to any one of claims 1 to 5; the cell contains the aforementioned nucleic acid or vector; and the method comprises the aforementioned cell.

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