18-OHF antibodies or antigen-binding fragments thereof

Abstract

The invention relates to the technical field of immunodetection, in particular to an 18-OHF antibody or an antigen binding fragment thereof, which comprises heavy chains CDR1-CDR3 with amino acid sequences as shown in SEQ ID NO: 1-3 in sequence; the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO: 4, the sequence of the light chain CDR2 is TTS, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO: 5. The 18-OHF antibody or the antigen binding fragment thereof provided by the invention has excellent affinity and sensitivity to 18-OHF, has good specificity, and is only specifically bound with 18-OHF compared with other analogues.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly to an 18-OHF antibody or an antigen-binding fragment thereof. Background Art

[0002] 18-Hydroxycortisol (18-OHF) belongs to hybrid steroids and is the most important and strongest mineralocorticoid in the human body, having the chemical structure characteristics of both aldosterone and cortisol. Most of it binds to transcortin and albumin. Its abnormal expression in the body is of great significance for the clinical diagnosis of primary aldosteronism (PA), Cushing's syndrome, and cardiovascular diseases.

[0003] However, the existing main detection technology for 18-OHF is liquid chromatography-tandem mass spectrometry (LC-MS / MS), which has relatively high detection costs and operation requirements, and has certain limitations on its clinical application. Therefore, there is an urgent need to develop highly sensitive and specific monoclonal antibodies against 18-OHF and establish immunoassay technologies to meet the needs of accurate detection in clinical diagnosis and scientific research. Summary of the Invention

[0004] The present invention encompasses the following technical solutions: According to one aspect of the present invention, it relates to an 18-OHF (18-hydroxycortisol) antibody or an antigen-binding fragment thereof, which comprises heavy chain CDR1 to CDR3 with amino acid sequences shown in SEQ ID NO: 1 to 3 in sequence; and light chain CDR1 with an amino acid sequence shown in SEQ ID NO: 4, light chain CDR2 with the sequence of TTS, and light chain CDR3 with an amino acid sequence shown in SEQ ID NO: 5.

[0005] According to another aspect of the present invention, it relates to an antibody conjugate, which comprises the above-mentioned 18-OHF antibody or an antigen-binding fragment thereof.

[0006] According to still another aspect of the present invention, it relates to a kit, which comprises the above-mentioned 18-OHF antibody or an antigen-binding fragment thereof, or the above-mentioned antibody conjugate.

[0007] According to yet another aspect of the present invention, it relates to the application of the above-mentioned 18-OHF antibody or an antigen-binding fragment thereof, or the above-mentioned antibody conjugate in the preparation of a diagnostic reagent or kit for diseases related to cortisol hormone disorders.

[0008] According to yet another aspect of the present invention, it relates to isolated nucleic acid that can encode the above-mentioned 18-OHF antibody or an antigen-binding fragment thereof.

[0009] According to yet another aspect of the present invention, it relates to a vector containing the nucleic acid as described above.

[0010] According to yet another aspect of the present invention, it relates to a host cell containing the nucleic acid as described above or transformed with the vector as described above.

[0011] The 18 - OHF antibody or its antigen - binding fragment provided by the present invention has excellent affinity and sensitivity for 18 - OHF, good specificity, and specifically binds only to 18 - OHF compared to other analogs. Detailed Embodiments

[0012] Reference will now be provided in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0013] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Through further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used in the specification of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention.

[0014] In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and conventional procedures widely used in the respective fields. At the same time, for a better understanding of the present invention, the definitions and explanations of related terms are provided below.

[0015] The terms "and / or", "or / and", and "and / or" used in the present invention cover any one of two or more related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present invention, this technical solution undoubtedly includes the technical solution connected by "logical AND", and undoubtedly also includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (i.e., the technical solution connected by "logical AND").

[0016] The terms "comprising", "containing", and "including" used in the present invention are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0017] The numerical ranges expressed by endpoints in the present invention include all the numerical values and fractions included within the range, as well as the recited endpoints.

[0018] As used in the present invention, unless otherwise indicated, the singular forms of the articles "a", "an", and "the" include plural referents.

[0019] In the present invention, descriptions such as "a plurality of" and "a variety of", unless otherwise specified, mean greater than or equal to 2 in number.

[0020] In the present invention, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open-ended technical solution containing the listed features.

[0021] In the present invention, "preferably", "more preferably", "most preferably", and "it is advisable" are only used to describe embodiments or examples with better effects. It should be understood that they do not constitute a limitation to the protection scope of the present invention. In the present invention, "optionally", "optional", and "optional" mean having or not having, that is, any one of the two parallel solutions selected from "having" or "not having". If "optional" appears in a technical solution in multiple places, unless otherwise specified and there is no contradiction or mutual restriction relationship, each "optional" is independent of each other.

[0022] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically incorporated by reference. Unless it conflicts with the object of the present invention and / or the technical solution thereof, the cited documents involved in the present invention are incorporated by reference in their entirety and for all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When the present invention involves cited documents, the examples and preferred modes of the relevant technical features cited can also be incorporated by reference into the present invention as a reference, provided that the present invention can be implemented. It should be understood that when the cited content conflicts with the description in the present invention, the present invention shall prevail or be amended adaptively according to the description of the present invention.

[0023] In the present invention, the technical term "antibody" refers to a protein that binds to a specific antigen, and generally refers to all proteins and protein fragments containing complementarity-determining regions (CDR regions), especially full-length antibodies or antibody functional fragments. The term "full-length antibody" includes polyclonal antibodies and monoclonal antibodies. The term "antigen-binding fragment" is a substance that contains a part or all of the CDRs of an antibody, which lacks at least some of the amino acids present in the full-length chain but can still specifically bind to an antigen.

[0024] In the present invention, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an immunoglobulin that are responsible for antigen binding. Each of the variable regions of the heavy and light chains contains three CDRs, and these CDRs are usually defined by the Kabat numbering system known in the art (Kabat et al., Sequences of proteins of immunological interest, 5th Ed, US Department of Health and Human Services, NIH, 1991, and later editions). Here, depending on the situation, the terms "CDR" and "CDRs" are used to refer to regions that contain one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody for the antigen or epitope it recognizes.

[0025] In the present invention, the term "affinity constant" or "K D (M)" or "K D " is characterized by K D (M)= Kd (or koff, the dissociation rate of a specific binding molecule-target molecule interaction) / Ka (or kon, the association rate of a specific binding molecule-target molecule interaction). The smaller the K D (M), the stronger the antigen-antibody affinity. K DValues can be determined by methods well-known in the art, such as by measuring using surface plasmon resonance (SPR) in a Biacore instrument or by ELISA method. SPR is based on an optical principle. When biomolecules bind or dissociate on the surface of a sensor chip, it will cause a change in the refractive index of the chip surface, thereby resulting in a change in the SPR angle. By monitoring this change, the intermolecular interaction can be analyzed in real time. An exemplary SPR process mainly includes the following steps: First, fix the antigen or antibody to be tested on the surface of the sensor chip; Subsequently, use different concentration gradients of the antibody or antigen to be tested as the mobile phase to flow through the chip in turn, and monitor the dynamic changes in the SPR angle during the molecular binding and dissociation process in real time and generate a binding / dissociation curve; Finally, fit the curve data through software to calculate the association rate constant (kon), dissociation rate constant (koff), and affinity constant (K D ), thereby evaluating the antibody affinity. The principle of detecting antibody affinity by ELISA method is to dilute the antibody (or antigen) in gradient in the presence of a small amount of antigen (or antibody), and detect the concentration of the antigen-antibody complex. When the concentration of the complex accounts for half of the total antigen concentration, the corresponding antibody concentration is the affinity constant K D . An exemplary ELISA process mainly includes the following steps: First, coat the antigen to be tested on the enzyme-linked immunosorbent assay (ELISA) plate and block the unbound sites; Subsequently, dilute the antibody to be tested in gradient and add it to the wells for incubation, and wash away the unbound components; Then add the enzyme-labeled secondary antibody for incubation and wash; Add the chromogenic substrate to react and then measure the absorbance (OD value); Finally, by plotting the curve of the OD value versus the antibody concentration, determine the antibody concentration corresponding to the half-saturation point, and calculate the affinity constant based on this to evaluate the affinity.

[0026] The first aspect of the present invention relates to an 18-OHF antibody or an antigen-binding fragment thereof, which comprises heavy-chain CDR1 to CDR3 having amino acid sequences shown in SEQ ID NOs: 1 to 3 in sequence; and light-chain CDR1 having an amino acid sequence shown in SEQ ID NO: 4, light-chain CDR2 having the sequence TTS, and light-chain CDR3 having an amino acid sequence shown in SEQ ID NO: 5.

[0027] In some embodiments, the 18-OHF antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region having an amino acid sequence shown in SEQ ID NO: 6, and a light-chain variable region having an amino acid sequence shown in SEQ ID NO: 7.

[0028] To improve / alter the binding affinity and / or other biological properties of an antibody, amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. These modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding. Variants of the above amino acid sequences should be considered within the scope of the present invention according to the conventional understanding of those skilled in the art. Variants of the 18-OHF antibody or its antigen-binding fragment of the present invention, compared with the CDR sequences of SEQ ID NO:1 to SEQ ID NO:5 and any one of the TTS polypeptides, respectively contain at most 1, 2, or 3 amino acid mutations occurring in the CDR region (if any); or, the variant has a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity relative to the entire sequence of SEQ ID NO:6 or SEQ ID NO:7. Generally, said mutations are conservative amino acid substitutions.

[0029] "Conservative amino acid substitution" is well known in the art and refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. The similarities include, for example, similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the relevant residues. The exemplary principles of amino acid replacement are as follows: 1) Preferential replacement based on physicochemical property similarity Acidic amino acids: ASP (D) and GLU (E) are interchangeable; Basic amino acids: ARG (R), LYS (K), and HIS (H) are interchangeable among each other; Hydrophobic amino acids: ILE (I), LEU (L), VAL (V), and MET (M) are interchangeable; Aromatic amino acids: PHE (F), TYR (Y), and TRP (W) are selectively interchangeable; Polar uncharged amino acids: SER (S) and THR (T) are interchangeable; ASN (N) and GLN (Q) are interchangeable; Small molecule amino acids: ALA (A) and GLY (G) are interchangeable.

[0030] 2) Replacement guided by evolutionary conservation Based on the high-frequency substitution pairs of the Dayhoff / PAM matrix: SER (S) - THR (T); LYS (K) - ARG (R); ILE (I) - VAL (V); ASP (D) - ASN (N) / GLU (E).

[0031] 3) Structural conservation constraints β - sheet region: preferential substitution of VAL (V), ILE (I), THR (T) with each other; α - helix region: substitution of ALA (A), LEU (L), GLU (E) with each other; Turn region: PRO (P) can be replaced by ALA (A) or GLY (G).

[0032] 4) Functional site avoidance principle Key antigen - binding sites (such as CDR regions): avoid substituting residues such as TYR (Y), TRP (W) that participate in hydrogen bonding / hydrophobic interactions; CYS (C) related to disulfide bonds is only allowed to be replaced by SER (S) or ALA (A).

[0033] 5) Treatment of special residues Cysteine (CYS): Unpaired CYS is preferably mutated to SER (S) or ALA (A) to reduce aggregation; Histidine (HIS): Replace with LYS (K) or ARG (R) in pH - sensitive regions; Proline (PRO): Replacement in rigid - structure regions requires verification by molecular dynamics simulation.

[0034] In this field, affinity maturation of antibody / antigen - binding fragments can also be carried out. Currently, affinity maturation methods mainly include directed evolution based on display technology, rational design, and computational simulation - assisted optimization. The specific steps include: (1) Using the phage display platform, constructing an antibody mutant library through random mutagenesis or focused mutagenesis, and enriching high - affinity clones through multiple rounds of antigen - binding screening and amplification cycles; (2) Adopting site - directed saturation mutagenesis technology, designing mutant combinations for key residues in the complementarity - determining regions (CDRs) or framework regions (FRs), and evaluating the binding performance; (3) Applying error - prone PCR to introduce random mutations during amplification, generating a diversity library and obtaining optimized variants through functional screening; (4) With the aid of computer - aided design, predicting key sites affecting affinity through molecular docking, free - energy perturbation (FEP), or machine - learning models to guide directed mutagenesis. In practical applications, multiple strategies are often integrated. For example, first screening potential hotspots through calculation, and then combining in vitro display technology for verification and optimization to achieve efficient and precise affinity improvement.

[0035] In the field of antibody applications, there is also a need to generate antibodies engineered with cysteine to artificially introduce highly reactive thiol groups (-SH). Labeling or coating with thiol groups and maleimide is used to achieve directed coupling. Specifically, one or more residues in the antibody are replaced with cysteine residues. To avoid affecting the specific binding of the antibody to the antigen, the residues being replaced are usually in the constant region of the antibody. By replacing specific amino acid residues with cysteine, reactive thiol groups are generated, enabling specific sites on the antibody to be used for conjugating the antibody to other moieties, such as antibody-label, antibody-solid support, antibody-specific protein, etc.

[0036] The isoelectric point (pI) is the pH value at which a protein has a net charge of zero in solution, and its value is jointly determined by the pKa values of ionizable groups (such as the carboxyl groups of ASP / D and GLU / E, and the amino groups of LYS / K and ARG / R) in the amino acid sequence. In antibody preparation, pI affects solubility (such as the colloidal stability of high-concentration preparations) and purification efficiency (ion-exchange chromatography binding characteristics). In the detection field, aggregation is usually avoided by adjusting the antibody pI to be close to the pH of the reaction buffer, thereby improving the stability of the reagent. Strategies for regulating the antibody isoelectric point include: replacing surface charge residues by site-directed mutagenesis (such as LYS→GLN to lower pI), chemical modification (introducing negative charges by PEGylation), or glycosylation engineering (regulating the sialylation level in the Fc region). For example, replacing basic residues in the CDR region of the antibody with neutral residues can lower the pI from 8.5 to 7.2. In practical applications, computational tools (such as ExPASy ProtParam) need to be combined to predict the pI, and capillary isoelectric focusing electrophoresis is used for verification.

[0037] The 18-OHF antibody or its antigen-binding fragment of the present invention may have a constant region, which can be the constant region of any one of IgG (including any one of IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgA, and IgM. The species source of the constant region can be bovine, equine, porcine, ovine, caprine, rat, mouse, guinea pig, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, alpaca, or human, etc.

[0038] The antigen-binding fragments of the antibodies provided by the present invention can be designed into antibody fragments with specific functions or application advantages, which optimize physical and chemical properties while retaining antigen-binding ability through structural simplification or modification. Common antibody fragments include: Fab fragment (formed by connecting the variable region VH of the heavy chain and the first constant region CH1 with the complete light chain through a disulfide bond, with a molecular weight of about 50 kDa), Fab' fragment (retaining a part of the hinge region on the basis of Fab and containing a free sulfhydryl group, which can form a multivalent structure through chemical coupling), F(ab')2 fragment (formed by connecting two Fab' through a disulfide bond in the hinge region to form a bivalent binding form), Fv fragment (only containing the variable regions VH and VL of the heavy and light chains, relying on non-covalent interactions to maintain structural stability), and scFv fragment (forming a single-chain structure by connecting VH and VL in series through a flexible polypeptide linker, enhancing stability and facilitating genetic engineering operations). The preparation methods are divided into two categories: enzymatic digestion method (such as papain hydrolyzing the IgG hinge region to generate monovalent Fab fragments, or pepsin cleaving the C-terminus of the IgG heavy chain to produce bivalent F(ab')2 fragments, and then treating with reducing agents such as β-mercaptoethanol to obtain Fab' fragments) and recombinant expression technology (directly synthesizing fragments such as scFv through Escherichia coli, yeast or mammalian cell expression systems, screening for high-affinity variants by combining phage display technology, and enhancing thermodynamic stability by introducing additional disulfide bonds or optimizing the linker peptide).

[0039] The functional fragments of the above antibodies can also be obtained by synthesis using an automatic peptide synthesizer on the market.

[0040] The second aspect of the present invention relates to an antibody conjugate, which comprises the 18-OHF antibody or its antigen-binding fragment as described above.

[0041] An antibody conjugate refers to a complex formed by conjugating an antibody with a functional molecule (such as a chemiluminescent label, a fluorescent dye, an enzyme, a radioactive isotope), a solid phase or a protein, etc. This technology is widely used in the fields of biomedical research, diagnosis and treatment. The resulting ones include antibody-solid phase conjugates, antibody-signal substance conjugates, antibody-polypeptide conjugates, etc.

[0042] For an antibody-solid phase conjugate, the "solid phase" refers to the solid support used to immobilize the antibody, and its function is to provide a stable solid-liquid reaction system to achieve specific capture, separation and signal detection of the target substance. The uses of solid phases are well known in the fields of chemistry, biochemistry, pharmacy and molecular biology.

[0043] Solid phases can be divided into various types according to their characteristics, including organic polymers such as polystyrene, polypropylene, glass and silicone rubber, etc.; inorganic materials such as silicon-based materials, metal materials and alumina, etc.; nanomaterials such as nanoparticles and nanofibers, etc.; and composite materials such as magnetic microspheres and nanocomposites.

[0044] The following methods can be used to achieve immobilization on a solid phase: The solid phase is modified or activated to contain functional groups capable of covalently coupling with an antibody or an antibody complex (the antibody can form a complex with a specific molecule, and the specific molecule includes carrier proteins, biotin / avidin, FITC, etc.). The functional groups are selected from one or more of carboxyl, amino, mercapto, and tosyl groups. The antibody or antibody complex can also be non-covalently attached to the solid phase, for example, through ionic or hydrophobic mechanisms.

[0045] In one embodiment, the solid phase can be in the form of a sheet, a prefabricated disk, a cylinder, fibrous or granular, generally allowing proper contact. The size of the solid phase suitable for the method of the present invention can vary according to the selected method. The antibody can bind to only one solid phase (e.g., a porous plate), or can bind to many solid phases (e.g., beads). In one embodiment, the solid phase can be fibrous or granular to allow optimal contact. The size of the solid phase can vary and can be selected according to the method to be performed.

[0046] In the present invention, the shape of the term "particle" (sometimes referred to as "bead" or "microbead" or "microsphere") can generally vary. For example, in a particular embodiment, the particle is spherical. However, it should be understood that other shapes can also be contemplated in the present invention, such as plate-like, rod-like, disk-like, strip-like, tubular, irregular shapes, etc. In addition, the size of the particles can also vary. For example, the average size (e.g., diameter) of the particles can range from about 0.1 nm to 1 mm, such as 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 500 μm.

[0047] In some embodiments, the particles are magnetic beads, preferably nano-magnetic microspheres, such as magnetite magnetic nano-microspheres. Magnetic microspheres refer to colloidal composite materials that can be uniformly dispersed in a certain base liquid, and have characteristics such as superparamagnetism, a relatively high specific surface area, and functional groups that can be modified. The surface functional groups include, but are not limited to, active groups such as carboxyl, amino, hydroxyl, or mercapto groups. The antibody-conjugated magnetic beads mainly achieve directional binding through physical adsorption or chemical coupling. Physical adsorption relies on electrostatic interactions, hydrophobic interactions, and van der Waals forces, and chemical coupling utilizes groups such as amino, carboxyl, hydroxyl, or mercapto groups modified on the surface of the magnetic beads to bind to the charged groups of the antibody, usually incubated in a suitable buffer solution, and non-specific sites are blocked with BSA.

[0048] For an antibody-signal substance conjugate, the signal substance therein refers to any substance capable of generating a signal for direct or indirect detection. Thus, the signal substance can be detected directly or indirectly. For direct detection, the signal substances applicable to the present invention can be selected from any known group of detectable labels. In some embodiments, the signal substances are selected from chromogens, fluorophores, chemiluminescent groups (such as isoluminol and its derivatives, acridinium esters or dioxetanes), electrochemiluminescent compounds, catalysts, enzymes, enzyme substrates, dyes, fluorescent dyes (such as fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and its derivatives). Other examples of the signal substances are: luminescent metal complexes such as ruthenium or europium complexes (such as for ECLIA), enzymes (such as for ELISA) and radioisotopes (such as for RIA). In some embodiments, the signal substances are selected from ABEI and its derivatives, acridinium esters, alkaline phosphatase and horseradish peroxidase. Indirect detection systems include, for example, labeling a specific molecule with a first partner of a bioaffinity binding pair. Examples of suitable binding pairs are: biotin or biotin analogs such as aminobiotin, iminobiotin or desthiobiotin / avidin or streptavidin, sugar / lectin, nucleic acid or nucleic acid analog / complementary nucleic acid.

[0049] Preferably a chemiluminescent signal substance, the chemiluminescent signal substance includes but is not limited to luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxane and its derivatives, rosamide and its derivatives, and peroxyoxalate and its derivatives. Antibodies form antibody-signal substance conjugates through direct coupling and indirect coupling. Direct coupling is the direct connection of the reactive groups of the antibody and the luminescent agent; indirect coupling is the connection between the antibody and the luminescent agent through a "bridge", and the bridge can introduce advantages such as new reactive groups and reduction of steric hindrance. Common coupling methods include carbodiimide, glutaraldehyde method and N-hydroxysuccinimide active ester method, etc.; for example, when the antibody is directly labeled with ABEI, the ABEI ring lactone reacts with the amino residue of lysine on the antibody to form an amide bond. The main process of antibody labeling with ABEI: (1) Prepare the labeling buffer; (2) Antibody labeling: Label the antibody with ABEI and stir the reaction at 25 °C; (3) Dialyze to remove small molecules after labeling.

[0050] For an antibody-polypeptide conjugate, it can be a fusion protein formed by fusing an antibody with a polypeptide that does not affect the antibody structure. The polypeptide can be one or more of 6×His, GST, MBP, FLAG, HA, c-Myc, SUMO, Strep, etc.; it can also be a complex formed by coupling an antibody with other antibodies and specific molecules. The specific molecules include one or more of carrier proteins, biotin / avidin, FITC, etc.

[0051] The third aspect of the present invention relates to a kit, which comprises the 18-OHF antibody or its antigen-binding fragment as described above, or the antibody conjugate as described above.

[0052] The term "kit" in the present invention may refer to any article (e.g., a package or container) comprising at least one device, which comprises a detection agent as described in the present invention. The kit may further comprise instructions for use, supplementary reagents and / or components or assemblies used in the methods or steps described in the present invention.

[0053] As needed, the kit may further contain other components necessary for performing the assay, such as any one or more of the following components: standards, sample pretreatment reagents (such as sample purification and enrichment reagents, lysis solutions, etc.), buffers / diluents (such as phosphate buffer), substrates for signal substances (usually chromogenic reagents, for example, if the signal substance is ABEI, the chromogenic reagent is sodium hydroxide and hydrogen peroxide), preservatives (any one or a mixture of two or more of sodium azide, sodium nitrite, sodium benzoate, Proclin series, potassium sorbate). These components may be packaged separately, or two or more may be premixed for ease of use / preservation. One or more components in the kit may be in liquid form or may be lyophilized under low pressure and exist in the form of solid powders.

[0054] The kits of the present invention include, but are not limited to, enzyme-linked immunosorbent assay kits, chemiluminescent kits, radioimmunoassay kits, fluorescence immunoassay kits, etc.

[0055] In some specific embodiments, the kit is a chemiluminescent kit, which comprises a solid-phase reagent and a label reagent. The solid-phase reagent comprises an antibody-solid phase conjugate, wherein the solid phase is a nano-magnetic microsphere, and the label reagent comprises a label-solid phase conjugate, wherein the label is ABEI and its derivatives. The detection steps of the kit are as follows: The sample to be tested is contacted with the solid-phase reagent and the label reagent simultaneously or successively to form an antigen-antibody immune complex, and the amount of the target substance in the sample to be tested is determined by detecting the amount of the immune complex. The sample to be tested may be blood, urine, cerebrospinal fluid, tissue sample or other liquids containing the analyte.

[0056] The fourth aspect of the present invention relates to the use of the 18-OHF antibody or its antigen-binding fragment, or the antibody conjugate as described above in the preparation of reagents or kits for diagnosing, differentially diagnosing, typing, monitoring treatment or prognosticating cortisol hormone disorder-related diseases.

[0057] In some embodiments, the cortisol hormone disorder-related diseases are selected from: primary aldosteronism, aldosterone deficiency, pseudohypoaldosteronism, Cushing's syndrome, endocrine hypertension, hypoaldosteronism, aldosterone synthase deficiency, and Addison's disease.

[0058] Primary aldosteronism includes five subtypes, namely aldosterone-producing adenoma (APA), idiopathic hyperaldosteronism (or bilateral adrenal hyperplasia, IHA), primary adrenal hyperplasia (or unilateral adrenal hyperplasia, PAH), familial hyperaldosteronism (FH, including types FH-I, II, III, and IV), and adrenocortical carcinoma and other subtypes.

[0059] The subject of the above method or use may refer to a patient or an animal suspected of having a disease related to cortisol hormone disorder, especially a mammal; preferably a primate, more preferably a human.

[0060] The fifth aspect of the present invention relates to an isolated nucleic acid that can encode the 18-OHF antibody or its antigen-binding fragment as described above.

[0061] The term "isolated nucleic acid" in the present invention refers to a deoxyribonucleic acid or ribonucleic acid polymer existing in single-stranded or double-stranded form. The isolated nucleic acid includes RNA genomic sequences, DNA (gDNA and cDNA), or RNA sequences transcribed from DNA. Moreover, unless otherwise specified, the polypeptide also includes natural polynucleotides, sugars, or analogs with base alterations. According to one aspect of the present invention, the polynucleotide is a light-chain polynucleotide.

[0062] The isolated nucleic acid includes a nucleotide sequence encoding the amino acid sequence of the protein complex, and also includes a nucleotide sequence complementary thereto. The complementary sequence includes a completely complementary sequence and a substantially complementary sequence, which refers to a sequence that can hybridize with the nucleotide sequence encoding the amino acid sequence of the protein complex under stringent conditions known in the art.

[0063] Moreover, the nucleotide sequence encoding the amino acid sequence of the protein complex can be altered or mutated. The alterations include additions, deletions, or non-conservative substitutions or conservative substitutions. The polynucleotide encoding the amino acid sequence of the protein complex can be interpreted to include a nucleotide sequence having substantial identity to the isolated nucleic acid. The substantial identity aligns the nucleotide sequence with another random sequence in such a way that they are maximally corresponding. When the aligned sequences are analyzed using common algorithms in the art, the sequences can show a homology of greater than 80%, such as greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0064] In addition, the codons of the nucleic acid corresponding to the 18-OHF antibody or its antigen-binding fragment can be optimized according to the species of the host cell.

[0065] The sixth aspect of the present invention relates to a vector containing the nucleic acid as described above.

[0066] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell by transformation, transduction or transfection, so that the genetic element carried by it can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), pox viruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). In some embodiments, the vectors of the present invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0067] The seventh aspect of the present invention relates to a host cell containing the nucleic acid as described above, or transformed by the vector as described above.

[0068] The selection of the host cell should be based on the specific requirements of antibody expression, including but not limited to prokaryotic or eukaryotic host systems.

[0069] Eukaryotic host cells include filamentous fungi, yeasts, and cells from multicellular organisms (invertebrates and vertebrates), such as plant cells, insect cells or mammalian cells. In some embodiments, host cells or cell lines suitable for expressing the antigen-binding proteins of the present invention include but are not limited to suspension culture-adapted cell lines: such as human embryonic kidney cells HEK293, human embryonic retina cells PER.C6, canine kidney cells MDCK, African green monkey kidney cells COS, myeloma cells such as mouse myeloma cells NS0 and Sp2 / 0, hamster kidney cells BHK-21, and Chinese hamster ovary cells (CHO cells) and CAP / CAP-T cell lines. Fibroblasts can also be used. Human cells can be used, thus allowing the molecule to be modified with the human glycosylation pattern. Alternatively, other eukaryotic cell lines can be employed. The selection of suitable mammalian host cells, as well as the methods for transformation, culture, amplification, screening, and product production and purification, are known in the art.

[0070] It can be demonstrated that prokaryotic cells such as bacterial cells can be used as host cells, which are suitable for expressing the proteins of the present invention or other embodiments. However, since proteins expressed in bacterial cells tend to be in an unfolded form, an incorrectly folded form, or a non-glycosylated form, any proteins produced in bacterial cells must be screened to retain antigen-binding ability. If the molecules expressed by the bacterial cells are produced in a properly folded form, the bacterial cells will be the desired host, or, in an alternative embodiment, the molecules can be expressed in a bacterial host and then refolded. For example, various strains of Escherichia coli used for expression are well-known host cells in the field of biotechnology. Various strains of Bacillus subtilis, Streptomyces, and other Bacillus genera can also be used in this method.

[0071] If desired, yeast cell strains and insect cells known to those skilled in the art, such as Drosophila and Lepidoptera insects and virus expression systems, can also be used as host cells.

[0072] In some embodiments, the nucleic acid is inserted into the cell genome and can be stably expressed. The insertion method can use the vectors described above, or the nucleic acid can be directly transferred into the cell without being ligated to a vector (such as liposome-mediated transfection technology).

[0073] The eighth aspect of the present invention relates to a method for preparing the 18-OHF antibody or its antigen-binding fragment as described above, which includes: culturing the host cell as described above under culture conditions suitable for antibody expression.

[0074] In addition, the method further includes an optional step of separating and recovering the expressed antibody from the host cell or its culture medium.

[0075] The antibodies provided by the present invention can be prepared by recombinant techniques, and specifically, reference can be made to the relevant methods disclosed in US Patent No. 4,816,567. In one embodiment of the present invention, it involves separating and obtaining a nucleic acid fragment encoding the antibody. These nucleic acid molecules contain the amino acid sequences of the light chain variable region (VL) and / or the heavy chain variable region (VH) of the antibody. Further, the nucleic acid can be cloned into one or more vectors, especially expression vectors, for expression in host cells.

[0076] After the antibodies of the present invention are produced, they can be purified from cell culture contents according to standard procedures in the art, which include ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc. Such techniques are within the scope of conventional techniques in the art and do not limit the present invention. Culturing host cells can also be understood as expressing the antibody in animals (especially transgenic animals or nude mice). This involves an expression system using the animal casein promoter, which, when transgenically incorporated into mammals, allows female animals to produce the desired recombinant protein in their milk. The culture broth secreting the antibody can be purified by conventional techniques. For example, purification can be carried out using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. Then, the bound antibody is eluted by a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be immediately frozen, such as at -70 °C, or lyophilized.

[0077] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, priority is given to the guidance provided in the present invention. It can also be carried out according to the experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0078] In the following specific examples, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0079] The main reagents and instruments used in the following examples are sourced as follows: The microplate reader for detection was purchased from BioTek, model: ELX800; The protein separation and purification system was purchased from cytiva, model AKTA Pure; 18 - hydroxycortisol (18 - OHF, Shanghai Zhenzhun, ZS - 20214), 18 - oxocortisol (18 - OXOF, Shanghai Bevonta, product number TB - 131081), 18 - oxocorticosterone (18 - OHB, Shanghai Zhenzhun, number IR - 15291); deoxycortisone (Aladdin, C302974), 11 - deoxycorticosterone (Aladdin, D133971), corticosterone (Aladdin, C104537), progesterone (Aladdin, P106427), aldosterone (Aladdin, product number A299444), 17α - hydroxyprogesterone (Aladdin, H137675), hydrocortisone (Aladdin, H110523), prednisolone (Aladdin, P276607), triamterene (Aladdin, T303644), dexamethasone (Aladdin, D137736), spironolactone (Aladdin, S303875), eplerenone (Aladdin, E129934), amiloride (Aladdin, A131615), fludrocortisone (Aladdin, F340821), mitotane (Aladdin, product number D139898), cortisone (Aladdin, C119445), prednisone (Aladdin, P116562).

[0080] Example 1 Antibody Preparation Steps 1) Gene Retrieval After centrifuging to collect positive clone cells, mRNA was extracted, and cDNA products were obtained through reverse transcription. Using universal primers for the FR1 and FR4 regions of the light / heavy chains, the target fragment was amplified by Taq enzyme, added with an A - tail, and then ligated to the pMD19 - T vector. It was transformed into JM109 competent cells, and 10 positive colonies were selected for sequencing after colony PCR verification.

[0081] 2) Antibody Gene Sequence Analysis and Alignment The gene sequences obtained from the above sequencing were analyzed and aligned in the IMGT antibody database to determine the integrity of the antibody structure. The sequence repeatability was analyzed and compared on the SnapGene software, and sequences were selected for construction and expression.

[0082] 3) Construction of Recombinant Antibody Expression Plasmid Specific upstream and downstream primers for the light and heavy chains were designed according to the sequencing results. The 5' end of the primers carried homologous arms of about 15bp that were homologous to the ligation ends of the linearized vector. The light and heavy chain gene fragments with homologous arms were obtained by PCR amplification, specifically as follows: front homologous arm - VL / VH - rear homologous arm; Construct a recombinant antibody expression vector using pEE 6.1 as the vector. Perform double digestion of pEE 6.1 with HindIII / EcoRI, electrophoretically recover the linear vector, ligate it through a homologous recombination protocol, transform JM109 competent cells, and after verifying the positive by colony PCR, expand the culture and extract the endotoxin-free plasmid to obtain the vector plasmid pL carrying the light chain and the vector plasmid pH carrying the heavy chain.

[0083] 4) Expression and purification After resuscitating Chinese hamster ovary (CHO) cells, place the culture flask in a carbon dioxide shaker (100 rpm, 37 °C, 5% CO2), culture the cells until the density ≥ 3×10 6 cells / mL, expand the culture after subculture, collect the cells, count them and calculate the viability, plate them at a density of 1×10 6 cells / mL, and culture overnight; mix the plasmids in the ratio of pL:pH = 1.5:1, and transfect the mixed plasmids into CHO cells according to the Gibco liposome transfection instructions.

[0084] Purify the above expression supernatant according to the operation instructions of the GE AKTA Pure protein separation and purification system to obtain the expressed antibody.

[0085] The variable region sequence of antibody A is as follows: The heavy chain variable region sequence is shown in SEQ ID NO: 6: QEQLMESGGGLVTPGGILTLTCTASGVTISNSDMNWVRQAPGKELEWIGVINSYGNTYYASWAKSRSTITRNTNLNTVTLVMTSLTAADTATYFCARDFGGWSTDAFDPWGPGTLVTISS; The light chain variable region sequence is shown in SEQ ID NO: 7: AVMTQTPSPVSAAVGGTVIISCQASQSVANNKNLAWYQQKPGQPPKLLIYTTSSLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGGYNSISDGVAFGGGTEVVVK.

[0086] The heavy chain CDR1 of antibody A is shown in SEQ ID NO: 1: GVTISNSD; CDR2 is shown in SEQ ID NO: 2: INSYGNT; CDR3 is shown in SEQ ID NO: 3: ARDFGGWSTDAFDP. The light chain CDR1 is shown in SEQ ID NO: 4: QSVANNKN; CDR2 is shown in: TTS; CDR3 is shown in SEQ ID NO: 5: AGGYNSISDGVA.

[0087] Performance Verification of the Antibody Itself in Example 2 1) Affinity Instruments and Reagents: SPR instrument (Biacore 8K); Chip Selection: S series CM5 / CM7 chips (covalently coupled chips); Amino coupling kit (EDC, NHS, ethanolamine); Buffers: 10×PBS-P+ (200 mM phosphate, 27 mM KCl, 1.37 M NaCl, 0.5% Tween 20), 10 mM sodium acetate (coupling dilution, pH 4 - 5.5), 10 mM Gly-HCl (chip regeneration solution, pH 1.5 - 3.0).

[0088] Experimental Procedures: (1) Immobilization of Antibody A: The ligand was immobilized by direct amino coupling. First, inject EDC / NHS (prepared freshly and mixed 1:1) for 5 - 10 min to activate the carboxyl groups; then dilute Antibody A to 10 μg / ml with sodium acetate buffer at pH 4.5 and inject it into the detection channel until the target coupling level (small molecule analyte, high coupling, 20000 RU) is reached; then inject ethanolamine (pH 8.5) for 5 - 10 min to block the unreacted sites; finally, rinse with 1×PBS-P buffer until the baseline is stable.

[0089] (2) Analyte Detection: Prepare a 10 mM stock solution of 18 - OHF, then dilute it to 100 μM, and further dilute it in 6 gradients by a factor of 2, plus a 0 concentration, for a total of 8 samples. Use the multi - cycle kinetic detection method, inject samples in ascending order of concentration, with a flow rate of 10 μL / min, for 180 s, dissociation time of 600 s, and regeneration with 10 mM Gly-HCl (pH 2.0) for 120 s.

[0090] Use the analysis software for data fitting and analysis to calculate the affinity.

[0091] The experimental results show that the K D (M) of Antibody A reaches 1.4 ± 0.29×10 -10 , which can meet the requirements of immunoassay.

[0092] 2) Specificity and Anti - interference Sample Preparation: To verify the specificity and anti - interference ability of the aforementioned antibody, set the blank group of the analyte as (50 mM PBS), prepare 18 - OHF solutions with a concentration of 10 ng / mL and solutions of different concentrations of analogs using PBS solution. To approximate the clinical detection environment, the determination standard for the concentration of analogs is 10 times the actual concentration in the reported clinical samples.

[0093] The OD value is detected using antibody A according to the following steps. The OD value of the blank group is denoted as B0, the OD value of the analyte 18-OHF is denoted as B1, and the detected OD values of the analogs 18-OXOF, 18-OHB, deoxycortisone, 11-deoxycorticosterone, corticosterone, progesterone, aldosterone, 17α-hydroxyprogesterone, hydrocortisone, prednisolone, triamterene, dexamethasone, spironolactone, eplerenone, amiloride, fludrocortisone, mitotane, cortisone, and prednisone are denoted as B2, B3... B 20 。

[0094] Experimental procedure: The 18-OHF-bovine IgG conjugate is coated on the enzyme-linked immunosorbent assay (ELISA) plate. After washing the plate, 1% OVA is added and incubated at 37 °C for 0.5 h for blocking; after washing the plate, 50 μL of the analyte and 50 μL of the analyte antibody (concentration: 1 μg / mL) are added and incubated at 37 °C for 30 min; after washing the plate, the goat anti-mouse secondary antibody labeled with HRP is added and incubated at 37 °C for 30 min; after washing the plate, the chromogenic solution TMB is added and incubated at room temperature for 10 min, and then the termination solution 3M H2SO4 is added, and the OD value is detected at 450 nm.

[0095] 0.98 < B n / B0 < 1.02 is regarded as unbound, and the result is recorded as "-"; 1.02 ≤ B n / B0 ≤ 2.0 is regarded as having binding, and the result is recorded as "+"; B n / B0 > 2.0 is regarded as having strong binding, and the result is recorded as "++". The experimental results are shown in Table 1. It shows that: the antibody in this scheme specifically binds only to 18-OHF.

[0096] Table 1

[0097] 3) Sensitivity Sample preparation: 18-OHF solutions with concentration gradients of 0, 50, 200, 1000, 5000, 25000, and 50000 pg / mL are prepared separately for standby. The detection steps are as follows: Competitive assay detection steps: 1. Reagents used for detecting the 18-OHF item (1) Buffer: 0.05M Tris buffer.

[0098] (2) Magnetic bead reagent: Coated with an antibody that specifically recognizes the 18-OHF small molecule. The feeding ratio of magnetic beads to antibody is 1 mg:10 μg; the buffer is: citrate buffer.

[0099] (3)ABEI labeling reagent: The 18-OHF-bovine IgG conjugate protein is coupled with ABEI. The feeding ratio of ABEI to antigen is 0.2 mg: 3.6 μg; the buffer is PBS.

[0100] 2. Detection process: The detection is carried out on the chemiluminescence immunoassay analyzer MAGLUMI X6 of Shenzhen New Industries Biomedical Engineering Co., Ltd. The instrument sample addition process parameters are: 40 μL sample + 50 μL Buffer + 20 μL magnetic beads, incubated at 37 °C for 9 min, and 50 μL ABEI is added and incubated at 37 °C for 9 min. After washing 3 times, NaOH and H2O2 are added, and the photomultiplier tube detects the optical signal.

[0101] According to the light intensity detection results, calculate the discrimination of the detection. The calculation formula is the ratio of the light intensity of the 0 value to the light intensities of each gradient. A discrimination > 2.0 is considered that the detection result is credible.

[0102] When antibody A detects a sample of 50 pg / mL, the discrimination from the 0 value sample reaches 2.04. Therefore, the results show that antibody A can accurately detect a sample of 50 pg / mL and meets the detection requirements.

[0103] Table 2

[0104] The above-described embodiments merely represent several implementation manners of the present disclosure. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present disclosure, several modifications and improvements can still be made, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure patent should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. 18-OHF antibody or an antigen-binding fragment thereof, characterized in that, comprising heavy chain CDR1 to CDR3 with amino acid sequences shown in SEQ ID NO: 1 to 3 in sequence; and light chain CDR1 with an amino acid sequence shown in SEQ ID NO: 4, light chain CDR2 with the sequence TTS, and light chain CDR3 with an amino acid sequence shown in SEQ ID NO:

5.

2. The 18-OHF antibody or antigen-binding fragment thereof according to claim 1, wherein comprising a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 6, and a light chain variable region with an amino acid sequence shown in SEQ ID NO:

7.

3. An antibody conjugate, characterized in that, comprising the 18-OHF antibody or its antigen-binding fragment as claimed in claim 1 or 2.

4. Kit, characterized in that, comprising the 18-OHF antibody or its antigen-binding fragment as claimed in claim 1 or 2, or the antibody conjugate as claimed in claim 3.

5. The kit according to claim 4, wherein The kit is a chemiluminescence kit, comprising a solid-phase reagent and a labeling reagent, and the solid-phase reagent or the labeling reagent comprises the 18-OHF antibody or its antigen-binding fragment as claimed in claim 1 or 2, or the antibody conjugate as claimed in claim 3.

6. Use of the 18-OHF antibody or its antigen-binding fragment as claimed in claim 1 or 2, or the antibody conjugate as claimed in claim 3 in the preparation of a reagent or a kit for diagnosing, differentially diagnosing, typing, monitoring treatment or prognosticating a cortisol hormone disorder-related disease.

7. The application according to claim 6, characterized in that, The cortisol hormone disorder-related diseases are selected from: primary aldosteronism, aldosterone deficiency, pseudohypoaldosteronism, Cushing's syndrome, endocrine hypertension, hypoaldosteronism, aldosterone synthase deficiency, and Addison's disease.

8. An isolated nucleic acid, characterized in that, capable of encoding the 18-OHF antibody or its antigen-binding fragment as claimed in claim 1 or 2.

9. Carrier, characterized in that, containing the nucleic acid as claimed in claim 8.

10. A host cell, characterized in that, comprising the nucleic acid as claimed in claim 8, or transformed by the vector as claimed in claim 9.

Images