Aldehyde dehydrogenase-1 as a biomarker for cancer

Abstract

The application provides an aldosterone antigen binding molecule and application thereof, and relates to the technical field of biotechnology.The aldosterone antigen binding molecule is selected from a first antigen binding molecule containing CDR sequences of SEQ ID NO.1 and 2 or a second antigen binding molecule containing CDR sequences of SEQ ID NO.3 and 4.The aldosterone antigen binding molecule can be used in a method or product for detecting aldosterone based on the immunological principle; and the first antigen binding molecule and the second antigen binding molecule can also form an immune complex of a double-antibody sandwich structure with aldosterone, so that the double-antibody sandwich principle is used to detect aldosterone.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an aldosterone antigen-binding molecule and its applications. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Aldosterone (ALD) is a mineralocorticoid synthesized and secreted by the zona glomerulosa cells of the adrenal cortex. It primarily acts on the distal convoluted tubules and collecting ducts of the kidneys, increasing sodium reabsorption and promoting potassium excretion. It also acts on the medullary collecting ducts, promoting hydrogen ion excretion and acidifying urine. Aldosterone maintains fluid balance by regulating the kidneys' reabsorption of sodium.

[0004] Elevated aldosterone levels are often associated with the following clinical symptoms: 1) Primary aldosteronism caused by adrenal hyperplasia; 2) Secondary aldosteronism caused by hypothalamic-pituitary dysfunction and ectopic adrenocorticotropic hormone secretion; 3) Aldosterone levels may also be elevated to some extent during the luteal phase of menstruation and late pregnancy in women; 4) Long-term use of oral contraceptives and estrogen-like drugs can also promote aldosterone secretion. Decreased aldosterone levels are mainly seen in primary adrenal insufficiency (also known as Addison's disease); in addition, certain medications, such as propranolol, methyldopa, reserpine, clonidine, licorice, and heparin, as well as excessive saline infusion, can inhibit aldosterone secretion.

[0005] Currently, ALD testing primarily employs a competitive assay. This method is suitable for antigens with small surface areas that cannot form a sandwich structure. However, competitive assays have some limitations: their signal intensity is negatively correlated with the concentration of the analyte antigen, making signal changes at low concentrations difficult to detect; furthermore, the standard curve for competitive assays is typically S-shaped with a narrow linear range, limiting the detection range; and due to the interaction of multiple components involved, competitive assays carry the risk of cross-reactivity, potentially leading to false positives and reduced specificity.

[0006] The small-molecule double-antibody sandwich assay uses two antibodies to jointly recognize two independent epitopes of the analyte. The detection signal is proportional to the amount of analyte, offering higher sensitivity, specificity, and detection range compared to competitive immunoassays. However, this method faces significant challenges in antibody development. Small molecules have weak immunogenicity, making it difficult to obtain high-affinity, high-specificity antibodies using conventional immunoassay methods. The affinity and specificity of antibodies directly affect the sensitivity and accuracy of detection, and poor antibody quality limits the application of the double-antibody sandwich assay. Furthermore, antibody pairing is difficult, requiring the discovery of a pair of antibodies that can recognize different epitopes of the antigen and cooperate well, which places higher demands on antibody screening and optimization.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The purpose of this invention is to provide an aldosterone antigen-binding molecule and its applications.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] In a first aspect, an aldosterone antigen-binding molecule is provided, the antigen-binding molecule comprising complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and complementarity-determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region; the antigen-binding molecule is selected from a first antigen-binding molecule or a second antigen-binding molecule.

[0011] First antigen-binding molecule: HCDR1, HCDR2, and HCDR3 of the first antigen-binding molecule include amino acid sequences consistent with those of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 1; LCDR1, LCDR2, and LCDR3 include amino acid sequences consistent with those of LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO. 2.

[0012] Second antigen-binding molecule: HCDR1, HCDR2 and HCDR3 of the second antigen-binding molecule include amino acid sequences consistent with those of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 3; LCDR1, LCDR2 and LCDR3 include amino acid sequences consistent with those of LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO. 4.

[0013] Secondly, a biomaterial is also provided, wherein the biomaterial is selected from any one of (i) to (iii):

[0014] (i) A polynucleotide, said polynucleotide comprising a nucleotide sequence encoding the aldosterone antigen-binding molecule described in the first aspect.

[0015] (ii) A vector carrying the polynucleotides mentioned in (i) above.

[0016] (iii) Cells carrying the polynucleotides described in (i) above, or containing the carriers described in (ii) above, or expressing the aldosterone antigen-binding molecule described above.

[0017] Thirdly, a modified aldosterone antigen-binding molecule is also provided, wherein the modified aldosterone antigen-binding molecule is the aldosterone antigen-binding molecule described in the first aspect modified by a modifier.

[0018] Fourthly, an aldosterone antigen-binding molecule composition is also provided, the composition comprising the first antigen-binding molecule of the first aspect and the first antigen-binding molecule of the second aspect.

[0019] Fifthly, the use of the aldosterone antigen-binding molecule described in the first aspect, or the biological material described in the second aspect, or the modified aldosterone antigen-binding molecule described in the third aspect, or the aldosterone antigen-binding molecule composition described in the fourth aspect in any one of (I) to (III) is also provided:

[0020] (I) Detection of aldosterone for non-diagnostic and therapeutic purposes;

[0021] (II) Preparation of products for the detection of aldosterone;

[0022] (III) Prepare a product for the diagnosis of at least one of aldosteronism, endocrine hypertension, hyperkalemia and hyperchloremic metabolic acidosis.

[0023] In a sixth aspect, a kit for detecting aldosterone is also provided, the kit comprising the aldosterone antigen-binding molecule of the first aspect, or the modified aldosterone antigen-binding molecule of the third aspect, or the aldosterone antigen-binding molecule composition of the fourth aspect.

[0024] In a seventh aspect, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the aldosterone antigen-binding molecule of the first aspect, or the biological material of the second aspect, or the modified aldosterone antigen-binding molecule of the third aspect; the pharmaceutical composition further comprises pharmaceutical excipients. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 Image of SDS-PAGE gel electrophoresis of antibody 3302# prepared in Example 3;

[0027] Figure 2 HPLC chromatogram of antibody 3302# prepared in Example 3;

[0028] Figure 3 The linear correlation curves of the detection results of the positive control antibody and ALD 3302# antibody against clinical samples in Example 4 are shown.

[0029] Figure 4 This is an SDS-PAGE gel electrophoresis image of antibody 5638# screened in Example 4.

[0030] Figure 5 HPLC chromatogram of antibody 5638# screened in Example 4;

[0031] Figure 6 The linear correlation curve between the liquid chromatography-mass spectrometry (LC-MS) results and the immunoassay results in Example 5;

[0032] Figure 7 The linear correlation curve between the liquid chromatography-mass spectrometry (LC-MS) results and the immunoassay results after removing abnormal samples in Example 5;

[0033] Figure 8 The graph shows the linear correlation curve between the first-generation and second-generation detection results in Example 5. Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0036] In this document, the term "aldosterone antigen-binding molecule" refers to any molecule capable of specifically binding to aldosterone antigens. Aldosterone antigen-binding molecules may include antibodies against aldosterone antigens as defined herein, or antigen-binding fragments thereof. Aldosterone antigen-binding molecules also encompass immunoglobulin superfamily antibodies (IgSF) or CDR transplantation molecules.

[0037] In this article, aldosterone antigens include aldosterone molecules or aldosterone molecules conjugates with other molecules, such as conjugates with protein carriers, including but not limited to bovine gamma globulin, bovine serum albumin, ovalbumin, peg-like hemocyanin, human serum albumin, thyroglobulin, tetanus toxoid, synthetic polylysine, or purified protein derivatives.

[0038] In this document, "antibody" refers to all proteins and protein fragments containing a complementarity-determining region (CDR). The terms "antibody" and "full-length antibody" include both polyclonal and monoclonal antibodies. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. Non-naturally occurring antibodies are also referred to as "recombinant antibodies" in this document. The term "antibody" is used interchangeably with "immunoglobulin." Antibodies can be monoclonal, polyclonal, dimer, multimer, or multispecific (e.g., bispecific) antibodies.

[0039] An antigen-binding fragment is a fragment containing part or all of an antibody's CDR, lacking at least some amino acids present in the full-length chain but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. These fragments are selected from, but are not limited to, F(ab')2, Fab', Fab, Fv (composed of VH and VL), ScFv (single-chain antibody with VH and VL linked by a linker peptide), dsFv (disulfide-stabilized Fv fragments), bispecific antibodies, nanobodies, and the smallest recognition unit of an antibody. In addition to the functional fragments mentioned above, any fragment with an extended half-life is also included.

[0040] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The variable domain of the heavy chain can be referred to as "VH," and the variable domain of the light chain as "VL." These domains are typically the most variable parts of the antibody and contain antigen-binding sites. Both the heavy and light chain variable regions consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The extent of the backbone region and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well-known in the art, including combinations of methods, can identify CDRs of variable domains. CDRs in each chain are held together closely by FRs to form variable regions. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by linking the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0041] The term "constant region" or "constant domain" of an antibody refers to the constant region of the antibody light chain, either alone or in combination, or the constant region of the antibody heavy chain. The antibody heavy chain has a variable domain (VH), followed by one or more constant domains or regions, such as hinges, CH1, CH2, CH3, and CH4. The CH1 domain is adjacent to the VH domain and is located at the amino terminus of the hinge region of the antibody heavy chain, and does not form a portion of the antibody's Fc region. The hinge region includes the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. The N-terminus of CH2 is typically the CH3 domain, which usually forms the C-terminal portion of the antibody. In some antibody types, such as IgM and IgE, the constant region also includes the CH4 domain. The constant region of an antibody can originate from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as the constant regions of their subclasses and mutant forms.

[0042] This invention does not limit the method of obtaining the aldosterone antigen-binding molecule. In some optional embodiments, the corresponding antibody can be obtained by linking a polynucleotide encoding the aldosterone antigen-binding molecule to a vector and expressing it in cells. The above-mentioned vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a structure capable of expressing the aldosterone antigen-binding molecule. In other optional embodiments, the aldosterone antigen-binding molecule can also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as by an automated peptide synthesizer (e.g., an automated peptide synthesizer sold by Applied BioSystems, etc.); the antigen-binding fragment can also optionally be generated by enzymatic cleavage of the antigen-binding molecule (including the intact antibody), such as by cleavage with pepsin or papain; or by chemical cleavage, such as by chemical reduction of disulfide bonds to obtain the above-mentioned antigen-binding fragment.

[0043] The terms "specific recognition," "selective binding," "selective binding," and "specific binding," or similar expressions, refer to the binding of antigen-binding molecules to pre-defined epitopes on antigens. Typically, antigen-binding molecules bind at a concentration of approximately less than 10-1. -5 M, for example, approximately less than 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller Kd values ​​are required for binding. The Kd value of an antibody can be determined using methods well-established in the art. Other standard assays for evaluating the binding ability of ligands, such as antibodies, to targets are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry analyses.

[0044] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. Polynucleotides encode the aforementioned anti-aldosterone antibody or antigen-binding fragments, optionally encoding the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0045] In this article, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells.

[0046] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, episome plasmids, microcircular DNA, phage particles, and Cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0047] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, and may differ morphologically and / or in genomic DNA. “Transformation” and “transformed cell” include primary test cells and cultures derived therefrom. Cells may be prokaryotic or eukaryotic, with prokaryotic cells including, but not limited to, *Escherichia coli*, *Bacillus*, or *Staphylococcus*. Eukaryotic cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.

[0048] In this document, the term "signaling substance" refers to a substance that can provide a detectable signal that can be directly observed by the naked eye or detected by conventional instruments acceptable in the art. The signaling substance can provide a signal directly, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; or it can provide a signal indirectly through a reaction in which the signaling substance participates, such as catalyzing a specific substrate reaction to produce any of the above signals.

[0049] In this document, unless otherwise stated, any numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order or importance between these entities or behaviors, such as numbers (Ⅰ), (Ⅱ), (Ⅲ).

[0050] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0051] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0052] In this text, the articles “a / an,” “an,” and “the” include plural references unless the context clearly indicates otherwise. For example, “an antibody” refers to one or more antibodies.

[0053] This invention uses ALD-3-CMO-KLH as an immunogen to immunize rabbits, constructs a phage library, and after panning and screening, obtains a high-performance antibody, 3302#. Then, using the ALD and 3302# complex as a screening agent, an antibody, 5638#, is screened from a human phage library. The variable region sequences and CDR sequences of these two antibodies are obtained by sequencing. Based on the variable region sequences of this antibody, in a first aspect, an aldosterone antigen-binding molecule is provided. This antigen-binding molecule contains complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, and complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region. The antigen-binding molecule is selected from either a first antigen-binding molecule or a second antigen-binding molecule.

[0054] First antigen-binding molecule

[0055] The first antigen-binding molecule comprises HCDR1, which includes an amino acid sequence identical to that of HCDR1 in the heavy chain variable region shown in SEQ ID NO.1; HCDR2, which includes an amino acid sequence identical to that of HCDR2 in the heavy chain variable region shown in SEQ ID NO.1; and HCDR3, which includes an amino acid sequence identical to that of HCDR3 in the heavy chain variable region shown in SEQ ID NO.1; and LCDR1, which includes an amino acid sequence identical to that of LCDR1 in the light chain variable region shown in SEQ ID NO.2; LCDR2, which includes an amino acid sequence identical to that of LCDR2 in the light chain variable region shown in SEQ ID NO.2; and LCDR3, which includes an amino acid sequence identical to that of LCDR3 in the light chain variable region shown in SEQ ID NO.2.

[0056] It is understood that the sequences shown in SEQ ID NO. 1 and 2 are only used to define the CDR region. The regions other than the CDR region in the variable regions shown in SEQ ID NO. 1 and 2 are not used to limit the aldosterone antigen-binding molecule provided by this invention. For example, if the antigen-binding molecule in some embodiments contains a backbone region, it may be different from the backbone region in the variable regions shown in SEQ ID NO. 1 and 2. The CDR region in the variable regions shown in SEQ ID NO. 1 and 2 can be divided according to any optional manner known in the art. Optionally, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of the variable regions can be defined by any one or a combination of multiple definition systems such as Kabat, Chothia, IMGT, ABM, or Contact. Taking Kabat, Chothia, IMGT, ABM, or Contact as examples, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 1 are shown in Table 1, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO. 2 are shown in Table 2.

[0057] Table 1

[0058]

[0059]

[0060] Table 2

[0061]

[0062] In an optional implementation, the first antigen-binding molecule, excluding the CDR region, has a remaining sequence (e.g., a backbone region or a constant region) derived from one or more of the following species: rabbit, cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, human, and mutants thereof.

[0063] In an optional implementation, the first antigen-binding molecule contains a backbone region of at least one heavy chain variable region, such as one, two, three, or four heavy chain variable regions.

[0064] In an optional embodiment, the first antigen-binding molecule contains a backbone region of a heavy chain variable region, wherein the backbone region FR1 of the heavy chain variable region comprises an amino acid sequence consistent with FR1 of the heavy chain variable region shown in SEQ ID NO.1; and / or, the backbone region FR2 comprises an amino acid sequence consistent with FR2 of the heavy chain variable region shown in SEQ ID NO.1; and / or, the backbone region FR3 comprises an amino acid sequence consistent with FR3 of the heavy chain variable region shown in SEQ ID NO.1; and / or, the backbone region FR4 comprises an amino acid sequence consistent with FR4 of the heavy chain variable region shown in SEQ ID NO.1.

[0065] In an optional embodiment, the first antigen-binding molecule contains a backbone region of at least one light chain variable region, such as one, two, three or four light chain variable regions.

[0066] In an optional embodiment, the first antigen-binding molecule contains a backbone region of a light chain variable region, wherein the backbone region FR1 of the light chain variable region comprises an amino acid sequence consistent with FR1 of the light chain variable region shown in SEQ ID NO. 2; and / or, the backbone region FR2 comprises an amino acid sequence consistent with FR2 of the light chain variable region shown in SEQ ID NO. 2; and / or, the backbone region FR3 comprises an amino acid sequence consistent with FR3 of the light chain variable region shown in SEQ ID NO. 2; and / or, the backbone region FR4 comprises an amino acid sequence consistent with FR4 of the light chain variable region shown in SEQ ID NO. 2.

[0067] Optionally, the first antigen-binding molecule has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.1 or having at least 80% (e.g., 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 95%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO.1; and / or, the first antigen-binding molecule has an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% (e.g., 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 95%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO.2.

[0068] Optionally, the first antigen-binding molecule has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO.1 and a light chain variable region of the amino acid sequence shown in SEQ ID NO.2.

[0069] Second antigen-binding molecule

[0070] The second antigen-binding molecule comprises HCDR1, which has an amino acid sequence identical to that of HCDR1 in the heavy chain variable region shown in SEQ ID NO.3; HCDR2, which has an amino acid sequence identical to that of HCDR2 in the heavy chain variable region shown in SEQ ID NO.3; and HCDR3, which has an amino acid sequence identical to that of HCDR3 in the heavy chain variable region shown in SEQ ID NO.3; and LCDR1, which has an amino acid sequence identical to that of LCDR1 in the light chain variable region shown in SEQ ID NO.4; LCDR2, which has an amino acid sequence identical to that of LCDR2 in the light chain variable region shown in SEQ ID NO.4; and LCDR3, which has an amino acid sequence identical to that of LCDR3 in the light chain variable region shown in SEQ ID NO.4.

[0071] It is understood that the sequences shown in SEQ ID NO. 3 and 4 are only used to define the CDR region. The regions other than the CDR region in the variable regions shown in SEQ ID NO. 3 and 4 are not used to limit the aldosterone antigen-binding molecule provided by this invention. For example, if the antigen-binding molecule in some embodiments contains a backbone region, it may be different from the backbone region in the variable regions shown in SEQ ID NO. 3 and 4. The CDR region in the variable regions shown in SEQ ID NO. 3 and 4 can be divided according to optional methods known in the art. Optionally, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of the variable regions can be defined by any one or a combination of multiple definition systems such as Kabat, Chothia, IMGT, ABM, or Contact. Taking Kabat, Chothia, IMGT, ABM, or Contact as examples, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 3 are shown in Table 3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO. 4 are shown in Table 4.

[0072] Table 3

[0073]

[0074] Table 4

[0075]

[0076] In an optional implementation, the second antigen-binding molecule, excluding the CDR region, has a remaining sequence (e.g., a backbone region or a constant region) derived from one or more of the following species: rabbit, cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, human, and mutants thereof.

[0077] In an optional embodiment, the second antigen-binding molecule contains a backbone region of at least one heavy chain variable region, such as one, two, three or four heavy chain variable regions.

[0078] In an optional embodiment, the second antigen-binding molecule contains a backbone region of a heavy chain variable region, wherein the backbone region FR1 of the heavy chain variable region comprises an amino acid sequence consistent with FR1 of the heavy chain variable region shown in SEQ ID NO. 3; and / or, the backbone region FR2 comprises an amino acid sequence consistent with FR2 of the heavy chain variable region shown in SEQ ID NO. 3; and / or, the backbone region FR3 comprises an amino acid sequence consistent with FR3 of the heavy chain variable region shown in SEQ ID NO. 3; and / or, the backbone region FR4 comprises an amino acid sequence consistent with FR4 of the heavy chain variable region shown in SEQ ID NO. 3.

[0079] In an optional embodiment, the second antigen-binding molecule contains a backbone region of at least one light chain variable region, such as one, two, three or four light chain variable regions.

[0080] In an optional embodiment, the second antigen-binding molecule contains a backbone region of a light chain variable region, wherein the backbone region FR1 of the light chain variable region comprises an amino acid sequence consistent with FR1 of the light chain variable region shown in SEQ ID NO.4; and / or, the backbone region FR2 comprises an amino acid sequence consistent with FR2 of the light chain variable region shown in SEQ ID NO.4; and / or, the backbone region FR3 comprises an amino acid sequence consistent with FR3 of the light chain variable region shown in SEQ ID NO.4; and / or, the backbone region FR4 comprises an amino acid sequence consistent with FR4 of the light chain variable region shown in SEQ ID NO.4.

[0081] Optionally, the second antigen-binding molecule has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.3 or having at least 80% (e.g., 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 95%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO.3; and / or, the second antigen-binding molecule has an amino acid sequence as shown in SEQ ID NO.4 or having at least 80% (e.g., 80%, 82%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 95%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO.4.

[0082] Optionally, the second antigen-binding molecule has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO.3; and a light chain variable region of the amino acid sequence shown in SEQ ID NO.4.

[0083] In an optional embodiment, the first antigen-binding molecule and the second antigen-binding molecule are respectively independently an antibody or an antigen-binding fragment.

[0084] In optional embodiments, the first antigen-binding molecule and the second antigen-binding molecule independently include F(ab')2, Fab', Fab, Fv, scFv, dsFv, bispecific antibodies, domain antibodies, or antigen-binding fragments composed of one or more of these. Domain antibodies include antibodies composed of a single domain or multiple domains, such as, but not limited to, dimers, triplets, tetramers, and microsomes.

[0085] In an optional implementation, the first antigen-binding molecule and the second antigen-binding molecule each independently contain part or all of the sequence of the constant region.

[0086] In an optional embodiment, the constant region sequence is selected from partial or complete constant regions of any one of IgG, IgA, IgM, IgE, or IgD, including their mutant forms. All of the above-mentioned antibody types encompass all subclasses known in the art; for example, IgG can be one or more of IgG1, IgG2, IgG3, IgG4, and IgG5.

[0087] The aldosterone antigen-binding molecule provided in the first aspect can specifically bind to aldosterone, and the first and second antigen-binding molecules can be used as paired antibodies to detect aldosterone using the double-antibody sandwich principle. Its performance in terms of titer, gradient, specificity, repeatability, stability, linear range, and clinical relevance meets the requirements of the magnetic microparticle chemiluminescent aldosterone sandwich assay kit.

[0088] Secondly, a biomaterial is also provided, wherein the biomaterial is selected from any one of (i) to (iii):

[0089] (i) A polynucleotide, said polynucleotide comprising a nucleotide sequence encoding the aldosterone antigen-binding molecule described in the first aspect.

[0090] In an optional embodiment, the polynucleotide contains at least one fragment of a nucleotide sequence as shown in SEQ ID NO. 5 to 8.

[0091] In an optional embodiment, the polynucleotide contains a nucleotide sequence as shown in SEQ ID NO.5, which encodes the heavy chain variable region shown in SEQ ID NO.1.

[0092] In an optional embodiment, the polynucleotide contains a nucleotide sequence as shown in SEQ ID NO. 6, which encodes the light chain variable region shown in SEQ ID NO. 2.

[0093] In an optional embodiment, the polynucleotide contains a nucleotide sequence as shown in SEQ ID NO.7, which encodes the heavy chain variable region shown in SEQ ID NO.3.

[0094] In an optional embodiment, the polynucleotide contains a nucleotide sequence as shown in SEQ ID NO. 8, which encodes the light chain variable region shown in SEQ ID NO. 4.

[0095] (ii) A vector carrying the polynucleotides mentioned in (i) above.

[0096] (iii) Cells carrying the polynucleotides described in (i) above, or containing the carriers described in (ii) above, or expressing the aldosterone antigen-binding molecule described above.

[0097] Thirdly, a modified aldosterone antigen-binding molecule is also provided, wherein the modified aldosterone antigen-binding molecule is the aldosterone antigen-binding molecule described in the first aspect modified by a modifier.

[0098] In an optional embodiment, the modification includes chemical modification, such as modification with polyethylene glycol (PEG).

[0099] In an optional embodiment, the modification includes connection to a signaling object and / or a solid support.

[0100] In optional embodiments, the signaling substance includes, but is not limited to, one or more of the following: enzymes, fluorescent molecular markers, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, colloidal selenium, colloidal silver, colloidal carbon, quantum dots, biotin, streptavidin, radionuclides, radioactive contrast agents, paramagnetic ions, metals, and photosensitizers.

[0101] In optional embodiments, the solid-phase support includes, but is not limited to, microtubes, columns, microparticles, or carrier membranes; more specifically, it can be, but is not limited to, enzyme-labeled wells, immunochromatographic test strips, or magnetic microparticles.

[0102] Fourthly, an aldosterone antigen-binding molecule composition is also provided, the composition comprising the first antigen-binding molecule of the first aspect and the first antigen-binding molecule of the second aspect.

[0103] In an optional embodiment, the first antigen-binding molecule of the aldosterone antigen-binding molecule composition is a modified aldosterone antigen-binding molecule, and / or, the second antigen-binding molecule of the aldosterone antigen-binding molecule composition is a modified aldosterone antigen-binding molecule.

[0104] In an optional embodiment, the first antigen-binding molecule is linked to a solid-phase carrier.

[0105] In an optional embodiment, the second antigen-binding molecule is linked to a signaling agent.

[0106] Fifthly, the use of the aldosterone antigen-binding molecule described in the first aspect, or the biological material described in the second aspect, or the modified aldosterone antigen-binding molecule described in the third aspect, or the aldosterone antigen-binding molecule composition described in the fourth aspect, in any one of (I) to (VI) is also provided:

[0107] (I) Detection of aldosterone for non-diagnostic and treatment purposes.

[0108] (II) Application in the preparation of products for the detection of aldosterone.

[0109] (III) Prepare products for the diagnosis of at least one of aldosteronism, endocrine hypertension, hyperkalemia, and hyperchloremic metabolic acidosis. Spontaneous overproduction of aldosterone leads to primary aldosteronism (PA), the most common cause of secondary endocrine hypertension; decreased aldosterone secretion leads to hyperkalemia and hyperchloremic metabolic acidosis. Therefore, antigen-binding molecules conjugated with aldosterone antigens can be used to prepare products for the diagnosis of the above-mentioned diseases.

[0110] (IV) Reducing aldosterone levels in subjects for non-diagnostic and non-therapeutic purposes.

[0111] (V) Preparation of aldosterone blockers.

[0112] (VI) Prepare a drug for treating at least one of aldosteronism, endocrine hypertension, hyperkalemia and hyperchloremic metabolic acidosis.

[0113] In an optional embodiment, the first antigen-binding molecule is applied to any one of (Ⅳ) to (Ⅵ) above.

[0114] Sixthly, a kit for detecting aldosterone is also provided, the kit comprising the aldosterone antigen-binding molecule described in the first aspect, or the modified aldosterone antigen-binding molecule described in the third aspect, or the aldosterone antigen-binding molecule composition described in the fourth aspect. The kit comprises one or more reagents, and when the kit contains one reagent, the kit is equivalent to the reagent.

[0115] In an optional implementation, the kit is used for immunoassay and contains reagents for immunoassay.

[0116] In optional embodiments, the immunoassay includes chemiluminescence detection, immunochromatographic detection, ELISA detection, immunomagnetic particle detection, immunofluorescence detection, or immunoblotting detection.

[0117] In optional embodiments, the kit further contains reagents and / or consumables for detection, including but not limited to one or more of primers, probes, buffers, dyes, diluents, washing solutions, chromogenic solutions, lysis buffers, negative controls, positive controls, and blank controls; consumables include but are not limited to solid-phase carriers, such as magnetic beads, enzyme-labeled wells, or nitrocellulose membranes. Those skilled in the art can select the reagent composition of the kit according to the specific detection method, and this invention does not limit this selection.

[0118] In an optional embodiment, the immunoassay utilizes the double-antibody sandwich principle to detect aldosterone. In the double-antibody sandwich immunoassay, the first antigen-binding molecule acts as a capture agent for aldosterone, forming a first antigen-binding molecule-aldosterone immune complex, and the second antigen-binding molecule is used to bind to the first antigen-binding molecule-aldosterone immune complex.

[0119] In an optional embodiment, the first antigen-binding molecule is coated on a solid-phase carrier, and the second antigen-binding molecule is labeled with a signal.

[0120] In a seventh aspect, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the aldosterone antigen-binding molecule of the first aspect, or the biological material of the second aspect, or the modified aldosterone antigen-binding molecule of the third aspect; the pharmaceutical composition further comprises pharmaceutical excipients. The pharmaceutical excipients include, but are not limited to, one or more of buffer components, solvents, stabilizers, protectants, and pH adjusters.

[0121] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0122] Example 1 Antibody Library Preparation

[0123] (I) Antigen preparation:

[0124] ALD-conjugated keyhole limpet hemocyanin (KLH) antigen ALD-3-CMO-KLH and ALD-conjugated bovine serum albumin (BSA) antigen ALD-3-CMO-BSA were prepared separately.

[0125] The preparation route of ALD-3-CMO-BSA is shown in equation (Ⅰ):

[0126]

[0127] The preparation route of ALD-3-CMO-KLH is the same as that of formula (Ⅰ), except that BSA is replaced with KLH.

[0128] (II) Immunized Animals:

[0129] 1. Rabbit Immunization: Dilute ALD-3-CMO-KLH antigen with PBS (0.01M, pH 7.4) and emulsify thoroughly with Freund's adjuvant at a volume ratio of 1:1. The immunization dose is 1 mg / rabbit for the first immunization and 0.5 mg / rabbit for the second immunization, administered via multiple subcutaneous injections throughout the body. The interval between the first and second immunizations is 30 days, and the interval between the second and third immunizations is also 30 days. The maximum immunization dose per rabbit should not exceed 0.8 mL.

[0130] 2. Determination of titer and gradient: The ALD-3-CMO-BSA antigen coated with the antigen was detected by plate chemiluminescence assay. The titer and gradient were determined and the results are shown in Table 5. A rabbit phage library was constructed.

[0131] Table 5. Detection efficacy

[0132] 1 / 500 406541 203453 316482 1 / 1K 409593 180872 293058 1 / 2K 354056 157507 252744 1 / 4K 340123 139575 227185 1 / 8K 324131 125659 207181 1 / 16K 315337 102003 165008 1 / 32K 250840 61363 108158 1 / 64K 167333 35231 66319

[0133] Table 6 Gradient Measurement Results

[0134]

[0135] The results above show that animal #272 has the best titer and gradient.

[0136] (III) Nucleic acid extraction and library preparation:

[0137] RNA was extracted from the spleen of rabbit #272, and cDNA was reverse transcribed. The variable regions of the light and heavy chains were amplified by PCR and extended by overlapping PCR. The light and heavy chains were spliced ​​together with the pCOMb3xss enzyme chain vector.

[0138] 1. PCR reaction system and reaction parameters:

[0139] PCR reaction system (50 μL system): VL mixed template: 50 ng; VH mixed template: 50 ng; upstream primer 2933 (10 μM):

[0140] 1.5 μL; downstream primer 2934 (10 μM): 1.5 μL; 2×primerstar Max: 25 μL; high-pressure purified water: make up to 50 μL.

[0141] PCR reaction parameter settings: as shown in Table 7.

[0142] Table 7 PCR reaction parameters

[0143]

[0144] 2. Enzyme digestion reaction system (50μL system, scFv: 50μL / vial × 5 vials; pcomb3xss: 50μL / vial × 8 vials):

[0145] Antibody digestion system: scFv: 2 μg; sfiI (5 U / μL): 2.5 μL; 10×bufferG: 5 μL; high-pressure purified water: make up to 50 μL.

[0146] Vector digestion system: Pcomb3xss: 4 μg; sfiI (5 U / μL): 2.5 μL; 10×buffer G: 5 μL; high-pressure purified water: make up to 50 μL.

[0147] 3. Ligation reaction, system (20 μL):

[0148] Vector: 0.4 μg; scFv: 246 ng; ligase: 1 μL; 5× ligase buffer: 10 μL; autoclaved water: make up to 20 μL.

[0149] Connection parameters: 37℃, overnight (14-18h), high-efficiency electroporation transformation of E. coli TG1. The next day, plate colony count was performed.

[0150] (iv) Calculation of storage capacity and electroconversion efficiency:

[0151] After electroporation, the volume of the library = colony count × dilution gradient / coating amount × 30 mL, in CFU;

[0152] Electroconversion efficiency = colony count × dilution gradient / coating volume × 30 mL / amount of nucleic acid added (μg) cfu / μg.

[0153] Electroconversion efficiency 3.4 × 10 9 cfu / μg. Gene library volume ≥10 8 CFU.

[0154] (V) Preparation of phage antibody library:

[0155] Add 5×10 to the electroconversion library 12 ~4×10 13PFU helper phage M13KO7 (1:20 ratio) was incubated at 37°C for 30 min. Then, it was cultured at 37°C and 250 rpm for 1 h. (The helper phage should be vortexed before use.) The mixture was centrifuged at 4500 rpm for 10 min, the supernatant was discarded, and 120 mL of AK was added. The mixture was then incubated overnight at 30°C and 250 rpm.

[0156] (vi) Titer determination:

[0157] 1. Centrifuge the overnight cultured bacteria at 8000 rpm for 10 min and collect the supernatant. Transfer 40 mL of the supernatant to three 50 mL conical centrifuge tubes containing 10 mL of 5×PEG 8000 / NaCl. Mix thoroughly and incubate on ice or at 4°C for 20 min. Centrifuge at 8000 rpm for 10 min at 4°C and discard the supernatant. Centrifuge at 8000 rpm for 2 min at 4°C and remove any excess liquid.

[0158] Add 2 mL of 1×PBS (gibco) to each tube to resuspend the precipitate. Centrifuge at 8000 rpm for 5 min, transfer the supernatant (phage antibody library) to a new centrifuge tube, add DMSO at a ratio of 6%, mix well, aliquot into 1.5 mL centrifuge tubes, and store at -20℃.

[0159] 2. Take 20 μL of bacteriophage + 180 μL of 2YT and perform a 10-fold serial dilution: 10 1 ~10 10 Add 180 μL of pre-prepared TG1 with OD600 = 0.4–0.6, and incubate at 37°C for 30 min. Take 10 μL of each... 9 ~10 10 100 μL of the bacterial suspension was spread onto Amp+2YT plates. After the plates dried, they were incubated overnight at 37°C with the plates inverted. The phage titer was measured to be 7.5 × 10⁻⁶ on the second day. 12 The statistical formula for titer is: Titer = 2 × A x ×10x+1pfu / mL; (x: dilution factor, A) x (Number of clones at x-fold dilution).

[0160] Example 2 Antibody Panning

[0161] (I) Main steps of antibody washing and screening:

[0162] The day before, take the immunotherapy tubes and coat them with 1 mL of ALD-3-CMO-BSA immunogen (20 μg for the first round, 10 μg for the second round, and 5 μg for the third round) according to the washing protocol. The next day, discard the coating solution, wash the immunotherapy tubes three times with 0.05% PBST, add 5 mL of casein, and block at 37°C for 1 hour. Add 1 mL of centrifuged phage to the immunotherapy tubes and incubate at 37°C, 70 rpm for 2 hours. Discard any unbound phage in the immunotherapy tubes, add 4 mL of 0.05% PBST to each tube, and wash the tubes (6 times for the first round, 8 times for the second round, and 10 times for the third round, increasing the number of washes each time). Gently tap for 30 seconds after each wash, then wash twice with 1×PBS. Add 1 mL of ALD-3-CMO elution buffer to each tube (5 μg for the first round, 10 μg for the second round, and 20 μg for the third round), and incubate at 37°C, 70 rpm for 60 minutes. Transfer the dissociated phages into a new 1.5 mL centrifuge tube.

[0163] Quickly add 1 mL of eluted phage to 5 mL of TG1, then add 1 mL of TG1 to the corresponding immunoassay tube and incubate at 37°C for 30 min. (Mix the bacterial cells in the immunoassay tube and centrifuge tube, mix well, and take 10 μL for dilution and titer measurement. (Take 10 μL of bacterial solution for 10-fold serial dilution, 10...) 1 ~10 5 20 μL of phage + 180 μL of 2YT (spread to 100 μL, calculate the discharge volume), add 6 μL of 2% Amp+, incubate at 37℃, 250 rpm for 1 h. Add M13K07 (MOI = 5), incubate at 37℃ for 30 min. Add 10 mL of 2% Amp+. + GLU-2YT (containing @%), incubated at 37℃ and 250 rpm for 1 h. Centrifuged at 4500 rpm for 10 min at room temperature, the supernatant was discarded, and 5 mL of Amp was added. + K + -2 YT, 30℃, 250 rpm overnight incubation. The following day, the output was 2.7 × 10⁻⁶. 6 Outbound quantity = 6 × A x ×10x + 1 pfu / mL (x: dilution factor, A) x (Number of clones at x-fold dilution).

[0164] (II) Screening for phage monoclonal antibodies:

[0165] Add 400 μL / well of 2YT-AG (final concentration 50 μg / mL Amp+, 2% glucose) to each well of a 96-well medium-well plate. Pick single colonies and label them as PC (positive control) and NC (negative control). Remove the pipette tip and seal the plate with film. Incubate overnight at 37°C and 250 rpm. Prepare 2YT-0.1% AG liquid medium, aliquot 100 μL / well into each 96-well deep-well plate, and add 5 μL of the picked overnight single colony culture. Activate by incubating at 37°C and 250 rpm for 2–3 hours (OD value approximately 2). Prepare 2YT+helper phage (MOI = 5) mixture, add 20 μL / well to each deep-well plate. Incubate at 37°C for 30 minutes. Incubate at 37℃ and 220 rpm for 1.5 h, then add 300 μL of 2YT-AK (final concentration 100 μg / mL Amp+, 50 μg / mL Kan+) to each well and incubate overnight at 30℃ and 250 rpm for 16–18 h. The next day, centrifuge at 4000 rpm and 4℃ for 15 minutes, and collect the supernatant for ELISA detection.

[0166] (III) Phage monoclonal indirect and competitive ELISA detection:

[0167] Take a chemiluminescence plate, dilute ALD-3-CMO-BSA to 0.25 μg / mL with CB, add 100 μL / well and coat overnight at 4°C. The next day, wash the plate twice with PBST, blot dry, and add 150 μL / well of casein using a multipipeline. Mix well by micro-shaking, block at room temperature for 2 hours, and then blot dry with absorbent paper. For the competitive reaction, after blocking with casein, add calibrators and antibodies for a competitive reaction. First, use an indirect method to determine the antibody titer (affinity), and select those with affinity for the competitive reaction.

[0168] Indirect reaction: Take 50 μL of phage supernatant per well and add it to the antigen-coated plate. Mix well by micro-shaking and incubate at 37°C for 0.5 h. Wash with PBST 5 times and blot dry. Add 100 μL of anti-M13-HRP (horseradish peroxidase-labeled antibody specifically binding to the M13 phage capsid protein) (1 / 5k) per well. Incubate at 37°C for 30 min. Wash plate: Wash manually with PBST 5 times. Blot dry. Add 100 μL of luminescent substrate A + substrate B and mix well. Detect using a LUMO chemiluminescence analyzer. Detect immediately and after 5 min. Results after 5 min are considered accurate.

[0169] Competitive response: ALD monoclonal competitive screening strategy: Initial screening of superior clones: using only 2-point evaluation (very low, very high), using 0 pg / mL and 1500 pg / mL calibrators; Screening of clones with low crossover rate and retesting superior clones: selecting clones with better calibrators for retesting, increasing the calibrator gradient: 0, 40, 200, 1000 pg / mL, selecting the better clones at 0 / 40 points.

[0170] According to the indirect luminescence method, the phage was diluted with diluent to a luminescence value around 5w. Different calibrators were used at 50 μL / well for initial screening of superior clones: only two-point evaluation (very low, very high) was used, with 0 pg / mL and 1500 pg / mL calibrators. The plates were mixed by micro-shaking and incubated at 37°C for 0.5 h. The plates were washed manually with PBST buffer 5 times. The plates were then blotted dry. 100 μL / well of the enzyme conjugate anti-M13-HRP (1 / 5k) was added. The plates were incubated at 37°C for 30 min. The plates were washed manually with PBST buffer 5 times. The plates were blotted dry. For color development, 100 μL / well of luminescent substrate A and substrate B were added and mixed. The plates were detected using a LUMO luminescence analyzer. Immediate and 5-minute results were performed. The 5-minute result was considered the definitive result. Select the best clones for retesting with calibrators, increasing the calibrator concentration gradient to 0, 40, 200, and 1000 pg / mL, and choosing the 0 / 40 point clones as the best; other parameters are the same as above. The test results are shown in Table 8.

[0171] Table 8. Results of the competition method test

[0172]

[0173] (iv) Antibody sequencing: 20 dominant phage strains were selected for sequencing.

[0174] Example 3: Construction of full-length antibody

[0175] (I) Antibody Construction:

[0176] 1. VL and VH target fragment amplification and recovery: Different primers for the variable region of the rabbit monoclonal antibody were selected based on the sequencing results. PCR settings were established with the primer pairing scheme and optimal annealing temperature as follows: PCR reaction system (25 μL system): Template DNA: 2.5 μL; Upstream primer (10 μM): 0.75 μL; Downstream primer (10 μM): 0.75 μL; 2×primerstar Max: 12.5 μL; Autoclaved water: 8.5 μL. PCR reaction parameters were set as shown in Table 7. Nucleic acid was purified using a gel after PCR.

[0177] 2. Homologous recombination reaction: Homologous recombination reaction system (10 μL system): Vector: 50 ng; Fragment: 40 ng; 2× homologous recombination enzyme: 5 μL; Autoclaved water: to make up to 10 μL. React at 50℃ for 1 h, then incubate at 4℃. Transform into Top10 E. coli. Spread on plates and incubate upside down at 37℃ for at least 12 hours.

[0178] 3. Recombinant screening: Pick a single colony and incubate it in 3 mL of LB liquid medium (Amp+) at 37℃ with shaking at 220 rpm for 3 h.

[0179] Prepare the PCR reaction solution (50 μL system): primer on the upper side: 1.5 μL; primer on the lower side: 1.5 μL; 2×Taq MixPLUS: 25 μL; high-pressure purified water: 20 μL; aliquot into 15 μL containers and add 1 μL of bacterial culture. PCR reaction parameters are set as shown in Table 7. After the PCR reaction, agarose gel electrophoresis is performed for detection. Positive clones are sequenced, and plasmids are extracted from correctly sequenced clones.

[0180] (II) Antibody Expression and Purification:

[0181] 1. Expression of dominant antibody #IgG:

[0182] (1) Plasmid extraction: Use the MN kit to extract plasmids (intermediate extraction).

[0183] (2) Transient plasmid transfection: On day 3 of 293F cell culture, aseptic samples were taken for cell counting, and the cell density reached (3.0–5.0) × 10⁻⁶. 6 Cells / mL, viability ≥90%, discard an appropriate amount of cell suspension and add fresh culture medium to 500 mL, adjusting the cell density to 2.0 × 10⁶ cells / mL. 6 Cells / mL were cultured in a shaker at 37°C, 5% CO2, and 130 rpm (25 mm amplitude).

[0184] (3) On day 2 of 293F cell culture, aseptic samples were taken for cell counting, and the cell density reached (3.0–5.0) × 10⁻⁶. 6 Cells / mL, viability ≥90%, discard an appropriate amount of cell suspension and add fresh culture medium to 500 mL, adjusting the cell density to 3.0 × 10⁶ cells / mL. 6 Cells / mL were cultured in a shaker at 37°C, 5% CO2, and 130 rpm (25 mm amplitude).

[0185] Preparation for transfection with a single 200mL cell suspension: In a clean bench, take two sterile 50mL centrifuge tubes, label them A and B respectively. According to the calculation results in Table 9, add the light chain and heavy chain plasmids to tube A, and gently shake to mix. Add 2mg / mL PEI to tube B, and gently shake to mix. Incubate solutions A and B separately at room temperature for 5 minutes. Then, use a pipette to add solution B to solution A to prepare the AB mixture. Gently shake to mix, and incubate at room temperature for 10 minutes. After incubation at room temperature, use a pipette to slowly add the AB mixture to a 1L shake flask, gently shaking to mix thoroughly. After completion, incubate in a shaker at 37℃, 5% CO2, and 130rpm (amplitude 25mm).

[0186] Table 9. Volume of each component added to 200mL cell suspension

[0187]

[0188] 2. Antibody purification (SPA method):

[0189] Pass 250 mL of the supernatant sample to be purified through a 400-mesh nylon mesh and determine the IgG concentration using an A280 (0.02 M PBS pH 7.4 as blank buffer). Filter the culture supernatant through a 0.45 μm filter membrane and load it for later use. Take 20 μL of the filtered sample and dilute it to 2 mg / mL with 0.02 M PBS pH 7.4. The experiment used an AKTA purification system with a SPA column, following equilibration, loading, rinsing, and elution steps. Dialyze the eluted sample to PBS buffer, concentrate using an ultrafiltration tube, filter the antibody through a 0.22 μm filter membrane, and dilute with 0.01 M PBS pH 7.2 to a final antibody concentration of 5.0 ± 0.2 mg / mL.

[0190] (III) Antibody Evaluation: The method was the same as in Part (III) of Example 2, except that the phage was replaced with purified IgG antibody. A strain 3302# was selected, and its titer, gradient, stability, reproducibility, and clinical relevance met the requirements of the kit. The SDS-PAGE gel electrophoresis results of antibody 3302# are as follows: Figure 1 As shown, the heavy chain has a density of 53±3 KD, the light chain has a density of 22±3 KD, and the purity is >97%. The HPLC chromatogram is shown below. Figure 2 As shown in the figure. The results indicate that the antibody has high purity and no dimers.

[0191] 1. Titer and gradient: The control antibody sequence is shown in Table 27, and the results are shown in Table 10.

[0192] Table 10. Antibody 3302# titer and gradient

[0193] Conclusion: The titer of ALD 3302# stable monoclonal antibody was comparable to the control, and the gradients of calibrators and quality control samples were superior to the control. 2. Crossover rate, the results are shown in Table 11:

[0194] Table 11 Crossover Rate

[0195] - Original mixed serum sample 5mg / mL 0.05 4369429 34.16 - 1 cholesterol 5mg / mL 0.05 4136253 40.90 0.00％ 2 Creatinine 500mg / mL 5 4554631 29.25 0.00％ 3 Portugal 1g / 10mL 1 4316805 35.63 0.00％ 4 Vitamin C 6mg / mL 0.06 2842136 93.29 0.00％ 5 Urea 4g / dL 0.4 4401918 33.27 0.00％ 6 Boric acid 2g / 10mL 2 4382598 33.80 0.00％ 7 Acetaminophen 20mg / mL 0.2 4325762 35.37 0.00％ 8 salicylic acid 59.9 mg / mL 0.599 4519243 30.16 0.00％ 9 valproic acid 99％ 1％ 4072412 42.85 0.00％ 10 tartaric acid 1g / 10mL 1 4452712 31.91 0.00％ 11 Propranolol 2300mg / mL 23 4313239 35.73 0.00％ 12 Metoprolol 1.28 mg / mL 0.0128 4234196 37.99 0.00％ 13 Aminopterin 8860mg / mL 88.6 4288362 36.43 0.00％ 14 Spironolactone 600mg / mL 6 3387398 67.51 0.00％ 15 tetracycline 1.51 mg / mL 0.0151 4258345 37.29 0.00％ 16 Nifedipine 40mg / mL 0.4 4279922 36.67 0.00％ 17 Furosemide 5.99 mg / mL 0.0599 4363706 34.32 0.00％ 18 Linopril 327mg / mL 3.27 4220645 38.38 0.00％ 19 Valsartan 11mg / mL 0.11 4321269 35.50 0.00％ 20 uric acid 100mg / mL 1 4335001 35.12 0.00％ 21 Pregnenolone 10mg / mL 0.1 1234071 242.13 0.00％ 22 Estriol 1mg / mL 0.01 4312420 35.75 0.00％ 24 estradiol 10mg / mL 0.1 3549065 61.02 0.00％ 25 Enapuri 424mg / mL 4.24 4182582 39.51 0.00％ 26 testosterone 1mg / mL 0.01 3032214 83.53 0.00％ 27 androstenedione 5mg / mL 0.05 108792 926.74 0.00％ 28 Estrone 2mg / mL 0.02 3000481 85.10 0.00％ 29 17α-hydroxyprogesterone 10mg / mL 0.1 228734 672.92 0.00％ 30 Methylprednisolone 10mg / mL 0.1 1947270 155.57 0.00％ 31 cortisol 5mg / mL 0.05 1221799 244.17 0.00％ 32 Prednisone 16mg / mL 0.16 113845 909.93 0.00％ 33 Dexamethasone 10mg / mL 0.1 3459902 64.54 0.00％ 34 Cortisone 10mg / mL 0.1 46112 1281.15 0.00％

[0196] Conclusion: The cross-contamination rate of all 34 cross-contamination samples was 0.

[0197] 3. Stability, the results are shown in Tables 12 and 13:

[0198] Table 12 Detection Values

[0199]

[0200]

[0201] Table 13 Signal Value Decrease

[0202] Calibrator -1％ -2％ Bole Quality Control -1％ 2％ Endocrine quality control -3％ 0％ Urine matrix -3％ 2％ EDTA matrix -4％ 0％ serum matrix -2％ 2％

[0203] Conclusion: After 7 days of thermal acceleration at 37℃, the gradient change was small, and the signal value decrease was less than 5%, meeting the requirements. 4. Repeatability, the results are shown in Table 14:

[0204] Table 14

[0205]

[0206]

[0207] Conclusion: With ALD 3302# antibody coated at 0.008 μg / T, the N1 concentration variation was 2%, the N2 concentration variation was 2%, and the N3 concentration variation was 3%, all within 3%.

[0208] 5. Clinical relevance:

[0209] Clinical samples were tested using antibody 3302#, and the results are shown in Figure 15. A linear relationship curve was plotted between the results of the control antibody test and the antibody 3302# results, as shown in the figure. Figure 3 As shown in the figure. The results showed that antibody 3302# had a 99% correlation with the positive control antibody.

[0210] Table 15 Clinical Sample Detection Results

[0211]

[0212]

[0213] (iv) Sequence information of antibody 3302#:

[0214] 1. Heavy chain variable region:

[0215] amino acid sequence:

[0216] QEQLEESGGRLVTPGTPLTLTCTASGFSLKNYDMSWVRQAPGKGLEWIGAMNANGKIYYATW ARGRFTISKTSTTVELRIASPTTEDTATYFCARTWPGDISDVSLWGQGTLVTISS (SEQ ID NO. 1).

[0217] Nucleotide sequence:

[0218] CAGGAGCAGCTGGAGGAATCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAAGAACTACGACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGCGCCATGAATGCTAACGGTAAAATATATTAC GCGACTTGGGCGAGAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGAACTGAGAATCGCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAACTTGGCCTGGTGATATTAGTGATGTGAGCTTGTGGGGCCAAGGCACCCTGGTCACCATCTCTTCA(SEQ ID NO.5).

[0219] The CDR sequences are shown in Table 16:

[0220] Table 16

[0221]

[0222] 2. Light chain variable region:

[0223] amino acid sequence:

[0224] DIVLTQTPASVEVAVGGTVSISCQSSQSVYANNLLSWYQQKPGQPPKLLIYKIFTLASGVPSRFK GSGSGTRFTLTISDLECDDAATYYCAGSYWGGIATFGGGTKLEIK (SEQ ID NO. 2). .

[0225] Nucleotide sequence:

[0226] GACATTGTGCTGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCACAGTCAGCATCAGTTGCCAGTCCAGTCAGAGTGTTTATGCTAACAATCTCTTATCCTGGTATCAGCAGAAACCAGGCCAGCCTCCCAAGCTCTTGATATATAAGATTTTCACTCT GGCATCTGGGGTCCCATCCGGTTCAAAGGCAGTGGATCTGGGACACGGTTCACTCTCACCATCAGCGACCTGGAATGTGACGATGCTGCCACTTACTACTGTGCAGGCAGTTATTGGGGTGGGATTGCTACTTTCGGCGGAGGGACCAAACTGGAAATCAAA(SEQ ID NO.6).

[0227] The CDR sequences are shown in Table 17:

[0228] Table 17

[0229]

[0230] Example 4: Screening for paired antibodies against antibody 3302# using the double-antibody sandwich method for ALD detection.

[0231] (I) Preparation of antigen-antibody complexes:

[0232] Antibody 3302# and ALD were mixed at a molar ratio of 1:50, reacted at 25°C for 3 hours, and then aliquoted and stored at -20°C.

[0233] (II) Cleaning the elderly's warehouse:

[0234] 1. Preparation of the elderly library: (1) Collect 581 samples of elderly (≥60 years old) and process them with PBMC; (2) RNA extraction and reverse transcription; (3) Gene amplification of the variable region of the light and heavy chains; (4) scFv preparation (κ:λ=3:1); (5) Enzyme digestion and ligation; (6) Competent cells preparation and electroporation library construction. The resulting library was named the elderly library.

[0235] 2. Add 20 μg of the ALD antibody complex (positive sieve) prepared in step (I) and 5 μg of 3302# antibody (negative sieve) to an immunoassay tube, and make up to 1 mL of coating buffer. Incubate overnight at 4°C. The next day, discard the coating buffer, wash with PBST, and block with casein blocking buffer for 1 hour at 37°C. Add 1×10⁻⁶ ppm of the antibody complex to the positive sieve tube. 12Phages were incubated at 37°C for 2 hours, washed with PBST, and then 1 mL of pH 2.2 GLY-HCl was added for 7 min. Tris base was then used for neutralization. For negative sieving, 1 mL of the phage was added to a negative sieve tube and incubated at 37°C for 1 hour at 70 rpm. 1 mL of the negative sieve phage was added to 5 mL of TG1 and incubated at 37°C for 30 min. The cells in the immunoassay tube and centrifuge tube were mixed, and 100 μL of the sample was retained for library analysis. 6 μL of Amp was added, and the mixture was incubated at 37°C for 1 hour at 250 rpm. M13KO7 (MOI = 5) was added and incubated at 37°C for 30 min. 10 mL of 2% AG-2YT was added and the mixture was incubated at 37°C for 1 hour at 250 rpm. The mixture was centrifuged at 4500 rpm for 10 min at room temperature, the supernatant was discarded, and 5 mL of AK-2YT (Amp+ and Kan+) was added and incubated overnight at 30°C for 250 rpm. The next day, the sample was centrifuged at 8000 rpm for 10 min, and the supernatant was extracted at room temperature. 370 μL of DMSO was added to prepare a 7% DMSO phage supernatant, and 100 μL was used for titer determination. The second and third rounds used ALD complexes (positive sieve: 10–5 μg each) and 3302# antibody (negative sieve: 10–20 μg each).

[0236] (III) Screening for phage monoclonal antibodies:

[0237] Add 400 μL / well of 2YT-AG (final concentration 50 μg / mL) and 2% glucose to each well of a 96-well plate. Pick single colonies and label them as PC (positive control) and NC (negative control). Remove the pipette tip and seal the plate. Incubate overnight at 37°C and 250 rpm. Prepare 2YT-0.1% AG liquid medium, aliquot 100 μL / well into each 96-well plate, and add 5 μL of the picked overnight single colony culture. Activate by incubating at 37°C and 250 rpm for 2–3 h (OD value around 2). Prepare 2YT+helper phage (MOI = 5) mixture, add 20 μL / well to each well of the plate. Incubate statically at 37°C for 30 minutes. Incubate at 37°C and 220 rpm for 1.5 h, then add 300 μL of 2YT-AK (final concentration 100 μg / mL) to each well. Incubate overnight at 30℃ and 250 rpm for 16–18 h (Amp+, 50 μg / mL Kan+). The next day, centrifuge at 4000 rpm and 4℃ for 15 minutes, and collect the supernatant for ELISA detection.

[0238] (iv) Phage monoclonal ELISA detection (indirect method):

[0239] Take a chemiluminescence plate, dilute the antigen-antibody complex (positive sieve) and 3302# (negative sieve) antibody to 0.25 μg / mL with CB, add 100 μL / well and coat overnight at 4°C. The next day, wash the plate twice with PBST, blot dry, and add 150 μL / well of casein using a multipipe. Mix well by micro-shaking, block at room temperature for 2 hours, and then blot dry the liquid in the wells.

[0240] Indirect reaction: Take 50 μL of phage supernatant per well and add it to the positive and negative sieve coating plates, respectively. Mix well by micro-shaking and incubate at 37°C for 0.5 h. Wash with PBST 5 times. Pat dry. Add 100 μL of anti-M13-HRP (1 / 5k) enzyme conjugate per well. Incubate at 37°C for 30 min. Wash plate: Wash manually with PBST 5 times. Pat dry. Add 100 μL of luminescent substrate A + substrate B and mix well. Detect using a LUMO chemiluminescence analyzer. Detect immediately and after 5 min. The result after 5 min is considered the most accurate.

[0241] (V) Screening strategy for monoclonal complex antibodies: Select clones with better indirect methods for retesting. Use antibody 3302# as the coating antibody and add antigen gradients: 0, 40, 200, 1000 pg / mL. Select clones with better gradients.

[0242] According to the indirect method luminescence value, the phage was diluted with diluent to a luminescence value around 5w. Using 3302# as the coating antibody, 50 μL of antigen was added per well. Detection was performed using 0, 40, 200, and 1000 pg / mL antigen. The mixture was shaken in a micro-oscillator and incubated at 37°C for 0.5 h. The plate was manually washed with PBST buffer 5 times. The plate was then patted dry. 50 μL of phage was added, and the plate was incubated at 37°C for 30 min. 100 μL of the enzyme conjugate anti-M13-HRP (1 / 5k) was added per well. The plate was incubated at 37°C for 30 min. The plate was washed manually with PBST buffer 5 times. The plate was then patted dry. For color development, 100 μL of luminescent substrate A + substrate B was added and mixed. Detection was performed using a LUMO luminescence analyzer. Immediate and 5-minute tests were performed. The 5-minute result was considered the most accurate. Clones with the best gradient were selected.

[0243] (vi) Antibody sequencing: Twenty dominant phage strains were screened and sequenced. Subsequently, 14 full-length human anti-antibody strains were constructed (with sequencing repetitive sequences removed) in the same manner as in Example 3 for further evaluation.

[0244] (VII) Antibody Evaluation: The reaction model was antibody plate (coated with 3302#) + antigen + antibody + anti-human-136#-HRP. Specifically, 1 μg / mL of 3302# was added to each well, along with 50 μL of antigen at concentrations of 0, 40, 200, or 1000 pg / mL. The plate was shaken and mixed thoroughly in a micro-oscillator at 37°C for 0.5 h. The plate was manually washed with PBST buffer 5 times. The plate was then blotted dry. The purified antibody was added at 1 μg / mL in 50 μL, and the plate was incubated at 37°C for 30 min. The enzyme conjugate anti-human-136#-HRP (1 / 5k) was added at 100 μL per well. The plate was incubated at 37°C for 30 min. The plate was then manually washed with PBST buffer 5 times. The plate was blotted dry. Colorimetric Development: 100 μL of luminescent substrate A and substrate B were added and mixed. The plate was then detected using a LUMO chemiluminescence analyzer. Immediate and 5-minute detection were performed. The 5-minute result shall be used as the standard.

[0245] Table 18

[0246]

[0247] Finally, one anti-complex antibody, 5638#, was selected. Its titer, gradient, stability, reproducibility, and clinical relevance met the kit requirements for ALD determination using a sandwich method with coated antibody 3302#. The SDS-PAGE gel electrophoresis results of antibody 5638# are shown below. Figure 4 As shown, the heavy chain weight in the reducing gel was 53±3 KD, the light chain weight was 22±3 KD, and the purity was >97%. The HPLC chromatogram is shown below. Figure 5 As shown in the figure. The results indicate that the antibody has high purity and no dimers.

[0248] (viii) Sequence information of antibody 5638#:

[0249] 1. Heavy chain variable region:

[0250] amino acid sequence:

[0251] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYA DSVKGRFTISSRDNAKNSLYLQMNSLRAEDTALYYCAKDIGFENYYYYGMDVWGQGTTVTVSS (SEQ ID NO. 3).

[0252] Nucleotide sequence:

[0253] GAAGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATCGGGTTCGAGAACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO. 7).

[0254] CDR sequences, as shown in Table 19:

[0255] Table 19

[0256]

[0257]

[0258] 2. Light chain variable region:

[0259] Amino acid sequence:

[0260] RPVLTQPPSVSVAPGKTATITCGGNNIGTKGVHWYQQKPGQAPLLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSGSDQYVFGSGTELTVL (SEQ ID NO. 4).

[0261] Nucleotide sequence:

[0262] CGGCCCGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCACGATTACCTGTGGGGGAAACAACATTGGAACTAAAGGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTCTGTTGGTCATCTATTATGATAGCGACCGGCCCTC CGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTATTGTCAGGTGTGGGATAGTGGTAGTGATCAATATGTTTTCGGAAGTGGGACCGAGCTGACCGTCCTA(SEQ ID NO.8).

[0263] The CDR sequences are shown in Table 20:

[0264] Table 20

[0265]

[0266] Example 5: Performance Evaluation of Paired Antibodies

[0267] The magnetic microparticle ALD (aldosterone) detection kit (magnetic microparticle chemiluminescence method) adopts the principle of small molecule double antibody sandwich method: the capture antibody is antibody 3302#, and the detection antibody is antibody 5638#. Specifically, antibody 3302# is coated with magnetic microparticles, and horseradish peroxidase-labeled antibody 5638# is used to form an antibody-antigen-enzyme-labeled antibody complex through an immune reaction. This complex catalyzes the luminescent substrate solution to emit photons, and the luminescence intensity is proportional to the content of the analyte. The antibody evaluation method is described in Part (III) of Example 3.

[0268] (I) Valence and Gradient:

[0269] Table 21

[0270]

[0271]

[0272] Conclusion: The combination of ALD 3302# and HRP-ALD-5638# resulted in better signal strength, background, and gradient than the control, meeting the requirements.

[0273] (ii) Crossover rate

[0274] Table 22

[0275]

[0276]

[0277] Conclusion: Through testing of 33 cross-components, the interference of the sample test results was less than 10%, which meets the usage requirements.

[0278] 3. Stability

[0279] Table 23

[0280]

[0281] Conclusion: After 10 days of accelerated heat treatment at 37℃, there was no significant difference in serum plasma signal values, but there were differences between high and low values. The decrease in low signal value was <15%, and the decrease in high signal value was <10%, which met the requirements.

[0282] (iv) Repeatability

[0283] Table 24

[0284] Q1 mean 1,104,106 1,222,935 1,139,417 CV 1.60％ 2.57％ 3.69％ Q2 mean 10,927,695 12,861,061 12,189,339 CV 1.19％ 1.78％ 1.46％ Q3 mean 37,529,275 43,981,855 40,872,956 CV 1.32％ 2.78％ 1.58％

[0285] Conclusion: The results show that the variation of Q1 signal value is 1.60%, 2.57%, and 3.69%, Q2 signal value is 1.19%, 1.78%, and 1.46%, and Q3 signal value is 1.32%, 2.78%, and 1.58%. The repeatability meets the requirements of the ALD project.

[0286] (V) Clinical relevance: The first generation uses antibody 3302# competitive assay, and the third generation immunoassay uses the magnetic microparticle ALD (aldosterone) detection kit (magnetic microparticle chemiluminescence assay) from Example 5:

[0287] Table 25

[0288]

[0289]

[0290] Table 26

[0291]

[0292]

[0293]

[0294]

[0295] Experimental results are as follows Figures 6-8As shown in Tables 25 and 26, the results indicate that for 51-point serum clinical samples, the third-generation method had one abnormally high-value sample compared to liquid chromatography-mass spectrometry (LC-MS). After removing the abnormal sample, the correlation between the third-generation method and LC-MS was >95%, which meets the current project team's requirements. For 398-point serum clinical samples plus EDTA samples, the first-generation method had approximately 90% correlation with LC-MS, which also meets the project team's requirements, but the third-generation method was superior.

[0296] In conclusion, after evaluating the performance of ALD-coated antibody 3302# and HRP-ALD-5638# enzyme-labeled antibody in terms of titer, gradient, repeatability, stability, cross-linking rate, and clinical relevance, they all meet the requirements.

[0297] Table 27 Antibody sequences of control antibodies

[0298]

[0299] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An aldosterone antigen-binding molecule, characterized in that, The antigen-binding molecule contains complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region; the antigen-binding molecule is selected from a first antigen-binding molecule or a second antigen-binding molecule. First antigen-binding molecule: HCDR1, HCDR2, and HCDR3 of the first antigen-binding molecule have amino acid sequences identical to those of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO.1; LCDR1, LCDR2, and LCDR3 have amino acid sequences identical to those of LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO.

2. Second antigen-binding molecule: HCDR1, HCDR2, and HCDR3 of the second antigen-binding molecule have amino acid sequences consistent with those of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO.3; LCDR1, LCDR2, and LCDR3 have amino acid sequences consistent with those of LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO.

4. The aldosterone antigen-binding molecules HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the systems Kabat, Chothia, IMGT, ABM, or Contact.

2. The aldosterone antigen-binding molecule according to claim 1, characterized in that, The first antigen-binding molecule is defined according to the IMGT specification as follows: the amino acid sequence of HCDR1 is shown in SEQ ID NO.13, the amino acid sequence of HCDR2 is shown in SEQ ID NO.18, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.21; the amino acid sequence of LCDR1 is shown in SEQ ID NO.24, the amino acid sequence of LCDR2 is KIF, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.

27. Alternatively, the second antigen-binding molecule may be defined as follows: the amino acid sequence of HCDR1 is shown in SEQ ID NO. 33, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 38, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 41; the amino acid sequence of LCDR1 is shown in SEQ ID NO. 44, the amino acid sequence of LCDR2 is YDS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.

47.

3. The aldosterone antigen-binding molecule according to claim 1 or 2, characterized in that, The aldosterone antigen-binding molecule is an antibody or an antigen-binding fragment.

4. The aldosterone antigen-binding molecule according to claim 1 or 2, characterized in that, The antigen-binding molecule, excluding the CDR region, is derived from one or more species including rabbit, cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, human, and their mutants.

5. The aldosterone antigen-binding molecule according to claim 1 or 2, characterized in that, At least one backbone region of the heavy chain variable region includes an amino acid sequence consistent with the backbone region of the heavy chain variable region shown in SEQ ID NO.1 or 3.

6. The aldosterone antigen-binding molecule according to claim 1 or 2, characterized in that, At least one backbone region of the light chain variable region includes an amino acid sequence consistent with the backbone region of the light chain variable region shown in SEQ ID NO.2 or 4.

7. The aldosterone antigen-binding molecule according to claim 1 or 2, characterized in that, The first antigen-binding molecule has an amino acid sequence as shown in SEQ ID NO.1 or a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1; and / or, the first antigen-binding molecule has an amino acid sequence as shown in SEQ ID NO.2 or a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.

2.

8. The aldosterone antigen-binding molecule according to claim 1 or 2, characterized in that, The second antigen-binding molecule has an amino acid sequence as shown in SEQ ID NO.3 or a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.3; and / or, the second antigen-binding molecule has an amino acid sequence as shown in SEQ ID NO.4 or a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.

4.

9. The antigen-binding molecule according to claim 3, characterized in that, The antigen-binding molecules include F(ab')2, Fab', Fab, Fv, scFv, dsFv, or antigen-binding fragments composed of one or more of these.

10. The aldosterone antigen-binding molecule according to claim 9, characterized in that, The antigen-binding molecule also contains some or all of a constant region; The constant region sequence is selected from a portion or all of the constant region sequence of any one of IgG, IgA, IgM, IgE or IgD.

11. A biomaterial, characterized in that, The biomaterial is selected from any one of (i) to (iii): (i) A polynucleotide, including a nucleotide sequence encoding an aldosterone antigen-binding molecule as described in any one of claims 1 to 10; (ii) A vector carrying the polynucleotide in (i); (iii) A cell that carries the polynucleotide of (i), or contains the carrier of (ii), or expresses the aldosterone antigen-binding molecule.

12. The biomaterial according to claim 11, characterized in that, The polynucleotide contains at least one fragment of nucleotide sequence as shown in SEQ ID NO. 5-8.

13. A modified aldosterone antigen-binding molecule, characterized in that, It is an aldosterone antigen-binding molecule according to any one of claims 1 to 10 modified by a modifier; the modifier is a substance capable of detecting signals.

14. An aldosterone antigen-binding molecular composition, characterized in that, It comprises: the first antigen-binding molecule as described in any one of claims 1 to 10 and the second antigen-binding molecule as described in any one of claims 1 to 10.

15. The use of the aldosterone antigen-binding molecule according to any one of claims 1 to 10, or the biomaterial according to claim 11, or the modified aldosterone antigen-binding molecule according to claim 13, or the aldosterone antigen-binding molecule composition according to claim 14 in (I) or (II): (I) Aldosterone testing for non-diagnostic and non-therapeutic purposes; (II) Preparation of products for the detection of aldosterone.

16. A kit for detecting aldosterone, characterized in that, Compositions comprising the aldosterone antigen-binding molecule according to any one of claims 1 to 10, the modified aldosterone antigen-binding molecule according to claim 13, or the aldosterone antigen-binding molecule composition according to claim 14.

17. The kit according to claim 16, characterized in that, The kit is for immunoassay and contains reagents for immunoassay.

18. The kit according to claim 17, characterized in that, The immunoassays include chemiluminescence detection, immunochromatographic detection, ELISA detection, immunomagnetic particle detection, immunofluorescence detection, or immunoblotting detection.

19. The kit according to claim 17, characterized in that, In the immunoassay, the first antigen-binding molecule is used to capture aldosterone to form a first antigen-binding molecule-aldosterone immune complex, and the second antigen-binding molecule is used to bind to the first antigen-binding molecule-aldosterone immune complex.

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