18-OHF antibody or antigen-binding fragment thereof

Abstract

The present invention relates to the field of immunoassay technology, and specifically to an 18-OHF antibody or antigen-binding fragment thereof, comprising heavy chain CDR1 to CDR3 having amino acid sequences as shown in SEQ ID NOs: 1 to 3, respectively; a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a light chain CDR2 having a TTS sequence, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 5. The 18-OHF antibody or antigen-binding fragment thereof provided by the present invention has excellent affinity and sensitivity for 18-OHF, and good specificity. Compared with other analogs, it specifically binds only to 18-OHF.

Description

Technical Field

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

[0002] 18-Hydroxycortisol (18-OHF) is a hybrid steroid and the most important and potent mineralocorticoid in the human body. It possesses the chemical structures of both aldosterone and cortisol, and is primarily bound to transcortisol protein and albumin. Its abnormal expression in the body is of great significance in the clinical diagnosis of primary aldosteronism (PA), Cushing's syndrome, and cardiovascular disease.

[0003] However, the current primary detection technology for 18-OHF, liquid chromatography-tandem mass spectrometry (LC-MS / MS), is costly and requires high operation, limiting its clinical application. Therefore, there is an urgent need to develop highly sensitive and specific monoclonal antibodies against 18-OHF and establish immunoassay technology to meet the demand for precise detection in clinical diagnosis and scientific research. Summary of the Invention

[0004] The present invention covers the following technical solutions:

[0005] 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 having amino acid sequences as shown in SEQ ID NOs: 1 to 3;

[0006] And a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO:4, a light chain CDR2 with a sequence of TTS, and a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO:5.

[0007] According to another aspect of the present invention, it relates to an antibody conjugate comprising the 18-OHF antibody or an antigen-binding fragment thereof as described above.

[0008] According to yet another aspect of the present invention, it relates to a kit comprising the above-mentioned 18-OHF antibody or antigen-binding fragment thereof, or the above-mentioned antibody conjugate.

[0009] According to another aspect of the present invention, it relates to the use of the above-mentioned 18-OHF antibody or 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 disorder.

[0010] According to another aspect of the present invention, it relates to an isolated nucleic acid that can encode the 18-OHF antibody or antigen-binding fragment thereof as described above.

[0011] According to yet another aspect of the present invention, it relates to a vector comprising the nucleic acid as described above.

[0012] According to another aspect of the present invention, it relates to a host cell comprising the nucleic acid as described above, or transformed by the vector as described above.

[0013] The 18-OHF antibody or antigen-binding fragment thereof provided by the present invention has excellent affinity and sensitivity to 18-OHF and good specificity. Compared with other analogs, it only specifically binds to 18-OHF. DETAILED DESCRIPTION

[0014] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0015] 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 belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0016] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0017] The terms "and / or", "or / and", and "and / or" used in the present invention include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of 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, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0018] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0019] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0020] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0021] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0022] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0023] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0024] All documents mentioned in the present invention are cited as references in the present invention, just as each document is cited as a reference separately. Unless they conflict with the purpose of the invention and / or technical solution of the present invention, the cited documents involved in the present invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into the present invention as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in the present invention, the present invention shall prevail or be modified adaptively based on the description of the present invention.

[0025] As used herein, the technical term "antibody" refers to a protein that binds to a specific antigen. It broadly refers to all proteins and protein fragments containing the complementarity determining regions (CDRs), particularly full-length antibodies or functional antibody fragments. The term "full-length antibody" includes both polyclonal and monoclonal antibodies. The term "antigen-binding fragment" refers to a substance that contains part or all of an antibody's CDRs but lacks at least some of the amino acids present in the full-length antibody chain, yet is still capable of specifically binding to the antigen.

[0026] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, which are generally defined using 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 versions). Herein, the terms "CDR" and "CDRs," depending on the context, are used to refer to a region comprising one or more, or even all, of the amino acid residues that contribute primarily to the binding affinity of an antibody to its recognized antigen or epitope.

[0027] In the present invention, the term "affinity constant" or "K D (M)" or "K D " is represented 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). K D The smaller the (M), the stronger the affinity between antigen and antibody. DThe value can be determined by methods well known in the art, such as using surface plasmon resonance (SPR) to measure in a Biacore instrument or ELISA method. SPR is based on optical principles. When biomolecules bind or dissociate on the surface of a sensor chip, it causes a change in the refractive index of the chip surface, thereby causing a change in the SPR angle. By monitoring this change, the interaction between molecules is analyzed in real time. An exemplary SPR process mainly includes the following steps: first, the antigen or antibody to be tested is fixed to the surface of the sensor chip; then, the antibody or antigen to be tested with different concentration gradients is passed through the chip in sequence as a mobile phase, and the dynamic changes of the SPR angle during the molecular binding and dissociation process are monitored in real time and a binding / dissociation curve is generated; finally, the curve data is fitted by software to calculate the association rate constant (kon), dissociation rate constant (koff) and affinity constant (K D ) to evaluate antibody affinity. The principle of ELISA method for detecting antibody affinity is to dilute the antibody (or antigen) in a gradient in the presence of a small amount of antigen (or antibody) and detect the concentration of the antigen-antibody complex. When the complex concentration accounts for half of the total antigen concentration, the corresponding antibody concentration is the affinity constant K. D The exemplary ELISA process mainly includes the following steps: first, the antigen to be tested is coated on the enzyme-labeled plate and the unbound sites are blocked; then, the antibody to be tested is diluted in a gradient and added to the wells for incubation to wash away the unbound components; then, the enzyme-labeled secondary antibody is added for incubation and washing; after adding the colorimetric substrate, the absorbance (OD value) is measured after the reaction; finally, by plotting the relationship between the OD value and the antibody concentration, the antibody concentration corresponding to the half-saturation point is determined, and the affinity constant is calculated to evaluate the affinity.

[0028] The first aspect of the present invention relates to an 18-OHF antibody or an antigen-binding fragment thereof, comprising heavy chain CDR1 to CDR3 having amino acid sequences as shown in SEQ ID NOs: 1 to 3;

[0029] And a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO:4, a light chain CDR2 with a sequence of TTS, and a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO:5.

[0030] In some embodiments, the 18-OHF antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 6, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 7.

[0031] 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, insertions, and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding. Variants of the above amino acid sequences are considered within the scope of the present invention, as would be understood by those skilled in the art. Variants of the 18-OHF antibodies or antigen-binding fragments thereof of the present invention comprise CDR sequences comprising up to 1, 2, or 3 amino acid mutations in the CDR regions (if present) compared to any of SEQ ID NOs: 1 to 5 and the TTS polypeptide, respectively; or, alternatively, the variants have a sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the entire sequence of SEQ ID NO: 6 or SEQ ID NO: 7. Typically, the mutations are conservative amino acid substitutions.

[0032] "Conservative amino acid substitutions" are well known in the art and refer to the replacement of one amino acid with another having similar structural and / or chemical properties. Such similarity includes, for example, similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. Exemplary amino acid substitution principles are as follows:

[0033] 1) Prioritize replacement based on similarity in physical and chemical properties

[0034] Acidic amino acids: ASP (D) and GLU (E) interchangeably;

[0035] Basic amino acids: ARG (R), LYS (K), HIS (H) interchangeably;

[0036] Hydrophobic amino acids: ILE (I), LEU (L), VAL (V), MET (M) interchangeably;

[0037] Aromatic amino acids: PHE (F), TYR (Y), TRP (W) selective substitution;

[0038] Polar uncharged amino acids: SER (S) and THR (T) interchange; ASN (N) and GLN (Q) interchange;

[0039] Small molecule amino acids: ALA (A) and GLY (G) interchangeably.

[0040] 2) Evolutionary conservation guides substitution

[0041] High-frequency substitution pairs based on Dayhoff / PAM matrix:

[0042] SER (S) - THR (T);

[0043] LYS(K)-ARG(R);

[0044] ILE (I) - VAL (V);

[0045] ASP(D)-ASN(N) / GLU(E).

[0046] 3) Structural conservative constraints

[0047] β-pleated region: preferential substitution of VAL (V), ILE (I), and THR (T);

[0048] α-helical region: ALA (A), LEU (L), and GLU (E) interchangeably;

[0049] Corner area: PRO (P) can be replaced by ALA (A) or GLY (G).

[0050] 4) Functional site avoidance principle

[0051] Key antigen binding sites (such as CDR regions): avoid replacing residues such as TYR (Y) and TRP (W) that participate in hydrogen bonds / hydrophobic interactions;

[0052] Disulfide bond-related CYS (C) is only allowed to be replaced by SER (S) or ALA (A).

[0053] 5) Special residue treatment

[0054] Cysteine ​​(CYS): Unpaired CYS is preferentially mutated to SER (S) or ALA (A) to reduce aggregation;

[0055] Histidine (HIS): pH-sensitive region replaced with LYS (K) or ARG (R);

[0056] Proline (PRO): Replacement of rigid structural regions needs to be verified in combination with molecular dynamics simulation.

[0057] In this field, affinity maturation of antibody / antigen binding fragments can also be performed. Current affinity maturation methods mainly include directed evolution based on display technology, rational design, and computational simulation-assisted optimization. The specific steps include: (1) using a phage display platform to construct an antibody mutation library through random or focused mutagenesis, and then enriching high-affinity clones through multiple rounds of antigen binding screening and amplification cycles; (2) using site-directed saturation mutagenesis technology to design mutation combinations for key residues in the complementarity determining region (CDR) or framework region (FR) and evaluate binding performance; (3) using error-prone PCR to introduce random mutations during the amplification process, generate a diversity library, and obtain optimized variants through functional screening; (4) using computer-aided design, molecular docking, free energy perturbation (FEP) or machine learning models are used to predict key sites affecting affinity and guide directed mutagenesis. In practical applications, multiple strategies are often integrated, such as first using computational screening for potential hotspots, and then combining in vitro display technology for verification and optimization, to achieve efficient and accurate affinity improvement.

[0058] In the field of antibody applications, there is also a need to produce antibodies engineered with cysteine ​​residues to artificially introduce highly reactive thiol groups (-SH). This is achieved by labeling or coating with thiol groups and maleimides to achieve directed conjugation. 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 replaced residues are typically in the constant region of the antibody. By replacing specific amino acid residues with cysteine ​​residues, reactive thiol groups are generated, allowing specific sites on the antibody to be conjugated to other moieties, such as antibody-label, antibody-solid phase support, or antibody-specific protein.

[0059] The isoelectric point (pI) is the pH at which a protein's net charge in solution is zero. Its value is determined by the pKa values ​​of ionizable groups in the amino acid sequence (e.g., carboxyl groups in ASP / D and GLU / E, and amino groups in LYS / K and ARG / R). In antibody production, the pI influences solubility (e.g., colloidal stability of high-concentration preparations) and purification efficiency (i.e., binding properties in ion exchange chromatography). In the detection field, the pI of antibodies is often adjusted to approach the pH of the reaction buffer to prevent aggregation and improve reagent stability. Strategies for manipulating the isoelectric point of antibodies include site-directed mutagenesis to replace surface-charged residues (e.g., LYS→GLN to lower pI), chemical modification (PEGylation to introduce negative charges), or glycosylation engineering (modulating the sialylation level in the Fc region). For example, replacing basic residues in the CDR region of an antibody with neutral residues can reduce the pI from 8.5 to 7.2. In practical applications, pI prediction requires the use of computational tools (e.g., ExPASyProtParam) and verification by capillary isoelectric focusing electrophoresis.

[0060] The 18-OHF antibodies or antigen-binding fragments thereof of the present invention may comprise a constant region, which may be that of IgG (including any of IgG1, IgG2, IgG3, and IgG4), IgE, IgD, IgA, or IgM. The constant region may be derived from bovine, horse, porcine, sheep, goat, rat, mouse, guinea pig, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, alpaca, or human.

[0061] The antigen-binding fragments of the antibodies provided herein can be designed to have specific functions or application advantages. Through structural simplification or modification, they retain antigen-binding ability while optimizing physicochemical properties. Common antibody fragments include: Fab fragments (comprising the heavy chain variable region (VH) and the first constant region (CH1) linked to an intact light chain via disulfide bonds, with a molecular weight of approximately 50 kDa); Fab' fragments (based on Fab, retaining a portion of the hinge region and containing free sulfhydryl groups, allowing for multivalent structures through chemical coupling); F(ab')2 fragments (comprising two Fab' fragments linked by a hinge region disulfide bond to form a bivalent binding structure); Fv fragments (comprising only the heavy and light chain variable regions (VH and VL), relying on non-covalent interactions to maintain structural stability); and scFv fragments (comprising the VH and VL chains linked via a flexible polypeptide linker to form a single-chain structure, enhancing stability and facilitating genetic engineering). Preparation methods are divided into two categories: enzymatic cleavage (such as papain hydrolysis of the IgG hinge region to generate a monovalent Fab fragment, or pepsin cleavage of the IgG heavy chain C-terminus to produce a bivalent F(ab')2 fragment, which is then treated with a reducing agent such as β-mercaptoethanol to obtain a Fab' fragment) and recombinant expression technology (direct synthesis of fragments such as scFv through Escherichia coli, yeast or mammalian cell expression systems, combined with phage display technology to screen high-affinity variants, and improve thermodynamic stability by introducing additional disulfide bonds or optimizing the connecting peptide).

[0062] The functional fragments of the above antibodies can also be synthesized using commercially available automatic peptide synthesizers.

[0063] The second aspect of the present invention relates to an antibody conjugate comprising the 18-OHF antibody or antigen-binding fragment thereof as described above.

[0064] Antibody conjugates are complexes formed by coupling antibodies to functional molecules (such as chemiluminescent markers, fluorescent dyes, enzymes, radioisotopes), solid phases, or proteins. This technology is widely used in biomedical research, diagnosis, and therapy. These conjugates include antibody-solid phase conjugates, antibody-signaling agent conjugates, and antibody-peptide conjugates.

[0065] For antibody-solid phase conjugates, "solid phase" refers to the solid support used to immobilize the antibody, which provides a stable solid-liquid reaction system for specific capture, separation, and signal detection of the target. The uses of solid phases are well known in the fields of chemistry, biochemistry, pharmacology, and molecular biology.

[0066] Solid phases can be divided into many types according to their characteristics, including organic polymers such as polystyrene, polypropylene, glass and silicone rubber; inorganic materials such as silicon-based materials, metal materials and alumina; nanomaterials such as nanoparticles and nanofibers; and composite materials such as magnetic microspheres and nanocomposites.

[0067] Immobilization on a solid phase can be achieved using methods that modify or activate the solid phase to include functional groups capable of covalently coupling the antibody or antibody complex (the antibody can form a complex with a specific molecule, such as a carrier protein, biotin / avidin, or FITC). The functional group can be selected from one or more of carboxyl, amino, sulfhydryl, and tosyl groups. The antibody or antibody complex can also be non-covalently attached to the solid phase, for example, via ionic or hydrophobic mechanisms.

[0068] In one embodiment, the solid phase can be a sheet, preformed disk, cylinder, fibrous or granular, generally allowing for proper contact. The size of the solid phase suitable for the method of the present invention can vary depending on the method selected. The antibody can be bound to only one solid phase (e.g., a multi-well plate) or can be bound to many solid phases (e.g., beads). In one embodiment, the solid phase can be fibrous or granular to allow for optimal contact. The size of the solid phase can vary and can be selected based on the method to be performed.

[0069] As used herein, the shape of the term "particles" (sometimes referred to as "beads," "microbeads," or "microspheres") can generally vary. For example, in one particular embodiment, the particles are spherical. However, it should be understood that other shapes, such as plates, rods, disks, strips, tubes, irregular shapes, and the like, are also contemplated by the present invention. Furthermore, the size of the particles can also vary. For example, the average particle size (e.g., diameter) can range from approximately 0.1 nm to 1 mm, preferably, 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, or 500 μm.

[0070] In some embodiments, the particles are magnetic spheres, preferably nanomagnetic microspheres, such as ferroferric oxide magnetic nanospheres. Magnetic microspheres refer to colloidal composite materials that can be uniformly dispersed in a certain base liquid, and have properties such as superparamagnetism, a high specific surface area, and modifiable functional groups. Surface functional groups include but are not limited to active groups such as carboxyl, amino, hydroxyl or sulfhydryl groups. Antibody-coupled magnetic beads mainly achieve directional binding through physical adsorption or chemical coupling. Physical adsorption relies on electrostatic effects, hydrophobic effects and van der Waals forces, and chemical coupling utilizes amino, carboxyl, hydroxyl or sulfhydryl groups modified on the surface of magnetic beads to bind to charged groups of antibodies. They are usually incubated in a suitable buffer and non-specific sites are blocked with BSA.

[0071] In antibody-signal substance conjugates, the signal substance 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, signal substances suitable for use in the present invention can be selected from any known group of detectable labels. In some embodiments, the signal substance is selected from chromogens, fluorescent groups, chemiluminescent groups (e.g., isoluminol and its derivatives, acridinium esters, or dioxetanes), electrochemiluminescent compounds, catalysts, enzymes, enzyme substrates, dyes, fluorescent dyes (e.g., fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine, and their derivatives). Other examples of signal substances include luminescent metal complexes such as ruthenium or europium complexes (e.g., for ECLIA), enzymes (e.g., for ELISA), and radioactive isotopes (e.g., for RIA). In some embodiments, the signal substance is selected from ABEI and its derivatives, acridinium esters, alkaline phosphatase, and horseradish peroxidase. Indirect detection systems include, for example, labeling a specific molecule with the first partner of a bioaffinity binding pair. Examples of suitable binding pairs are: biotin or a biotin analogue such as aminobiotin, iminobiotin or desthiobiotin / avidin or streptavidin, sugar / lectin, nucleic acid or nucleic acid analogue / complementary nucleic acid.

[0072] Preferably, the chemiluminescent signal substance is a chemiluminescent signal substance, which includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, lophan and its derivatives, and peroxalate and its derivatives. The antibody forms an antibody-signal substance conjugate through direct coupling and indirect coupling. Direct coupling is the direct connection between the reactive group of the antibody and the reactive group of the luminescent agent; indirect coupling is the connection between the antibody and the luminescent agent through a "bridge", which has the advantages of introducing new active groups and reducing spatial hindrance. Common coupling methods include carbodiimide, glutaraldehyde method and N-hydroxysuccinimide active ester method; for example, the antibody is directly labeled with ABEI, and 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 is as follows: (1) Preparation of labeling buffer; (2) Antibody labeling: antibody is labeled with ABEI, and the reaction is stirred at 25°C; (3) Dialysis to remove small molecules after labeling.

[0073] For antibody-polypeptide conjugates, it can be a fusion protein formed by the fusion of an antibody and 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 the coupling of an antibody with other antibodies and specific molecules. The specific molecules include one or more of carrier proteins, biotin / avidin, FITC, etc.

[0074] A third aspect of the present invention relates to a kit comprising the 18-OHF antibody or antigen-binding fragment thereof as described above, or the antibody conjugate as described above.

[0075] The term "kit" as used herein refers to any article of manufacture (e.g., a package or container) comprising at least one device, which includes a detection agent as described herein. The kit may further include instructions for use, supplementary reagents, and / or components or assemblies for use in the methods described herein or steps thereof.

[0076] If necessary, the kit may also include other components necessary for performing the assay, such as any one or more of the following: standards, sample pretreatment reagents (e.g., sample purification and enrichment reagents, lysis buffer, etc.), buffers / diluents (e.g., phosphate buffer), substrates for the signal substance (typically a chromogenic reagent; for example, if the signal substance is ABEI, the chromogenic reagent is sodium hydroxide and hydrogen peroxide), and preservatives (sodium azide, sodium nitrite, sodium benzoate, Proclin series, potassium sorbate, or a mixture of two or more). These components can be packaged separately or premixed for ease of use / storage. One or more components in the kit may be liquid or lyophilized into a solid powder form.

[0077] The kits of the present invention include, but are not limited to, enzyme-linked immunosorbent assay kits, chemiluminescence kits, radioimmunoassay kits, fluorescent immunoassay kits, and the like.

[0078] In some specific embodiments, the kit is a chemiluminescent kit comprising a solid-phase reagent and a marker reagent. The solid-phase reagent comprises an antibody-solid-phase conjugate, wherein the solid phase is nanomagnetic microspheres, and the marker reagent comprises a marker-solid-phase conjugate, wherein the marker is ABEI or 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 marker reagent simultaneously or sequentially 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 can be blood, urine, cerebrospinal fluid, tissue sample, or other liquid containing the analyte.

[0079] The fourth aspect of the present invention relates to the use of an 18-OHF antibody or an antigen-binding fragment thereof, or the antibody conjugate as described above, in the preparation of a reagent or kit for diagnosing, differentially diagnosing, typing, monitoring treatment or evaluating the prognosis of a disease associated with a cortisol disorder.

[0080] In some embodiments, the disease associated with cortisol hormone disorder is selected from the group consisting of primary aldosteronism, hypoaldosteronism, pseudohypoaldosteronism, Cushing's syndrome, endocrine hypertension, hypoaldosteronism, aldosterone synthase deficiency, and Addison's disease.

[0081] Primary aldosteronism includes five subtypes: aldosterone-producing adenoma (APA), idiopathic aldosteronism (also known as bilateral adrenal hyperplasia, idiopathic hyperaldosteronism, IHA), primary adrenal hyperplasia (also known as unilateral adrenal hyperplasia, primary adrenal hyperplasia, PAH), familial hyperaldosteronism (FH, including FH-i, II, III, and IV), and adrenocortical carcinoma.

[0082] The subject of the above method or use may refer to a patient or an animal suspected of having a disease related to progesterone disorder, particularly a mammal; preferably a primate, more preferably a human.

[0083] A fifth aspect of the present invention relates to an isolated nucleic acid encoding an 18-OHF antibody or an antigen-binding fragment thereof as described above.

[0084] The term "isolated nucleic acid" as used herein refers to a deoxyribonucleic acid or ribonucleic acid polymer 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. Furthermore, unless otherwise specified, the polypeptide also includes naturally occurring polynucleotides, sugar- or base-modified analogs. According to one aspect of the present invention, the polynucleotide is a light chain polynucleotide.

[0085] 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.

[0086] Furthermore, the nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such alterations include additions, deletions, or non-conservative or conservative substitutions. A polynucleotide encoding the amino acid sequence of the protein complex may be interpreted as comprising a nucleotide sequence that is substantially identical to the isolated nucleic acid. Such substantial identity is when the nucleotide sequence is aligned with another random sequence in a manner that maximizes their correspondence, and when the aligned sequences are analyzed using algorithms commonly used in the art, the sequences may exhibit greater than 80% homology, for example, greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0087] 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 host cell species.

[0088] The sixth aspect of the present invention relates to a vector comprising the nucleic acid as described above.

[0089] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. 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), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). In some embodiments, the vector of the present invention contains 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.).

[0090] The seventh aspect of the present invention relates to a host cell comprising the nucleic acid as described above, or transformed with the vector as described above.

[0091] The choice of host cell should be based on the specific requirements for antibody expression and includes but is not limited to prokaryotic or eukaryotic host systems.

[0092] Eukaryotic host cells encompass filamentous fungi, yeast, 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 retinal 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 may also be used. Human cells may be used, thereby allowing molecules to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells, as well as methods for transformation, culture, amplification, screening, and product production and purification, are known in the art.

[0093] It can be shown that prokaryotic cells, such as bacterial cells, can be used as host cells suitable for expressing proteins or other embodiments of the present invention. However, since proteins expressed in bacterial cells tend to be in an unfolded or improperly folded or non-glycosylated form, any protein produced in bacterial cells must be screened to retain antigen binding capacity. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell will be the desired host, or, in an alternative embodiment, the molecule can be expressed in a bacterial host and subsequently refolded. For example, various strains of E. coli used for expression are well-known host cells in the field of biotechnology. Various strains of Bacillus subtilis, Streptomyces, other Bacillus species, etc. can also be used in this method.

[0094] Yeast cell strains known to those skilled in the art, as well as insect cells, such as Drosophila and Lepidoptera insects, and viral expression systems, can also be used as host cells if desired.

[0095] In some embodiments, the nucleic acid is inserted into the cell genome and stably expressed. Insertion can be performed using a vector as described above, or the nucleic acid can be directly transferred into the cell without being linked to a vector (e.g., liposome-mediated transfection).

[0096] The eighth aspect of the present invention relates to a method for preparing the above-mentioned 18-OHF antibody or antigen-binding fragment thereof, comprising: culturing the above-mentioned host cell under culture conditions suitable for antibody expression.

[0097] In addition, the method may further comprise the optional step of isolating and recovering the expressed antibody from the host cells or their culture medium.

[0098] The antibodies provided herein can be produced using recombinant techniques, for example, the methods disclosed in U.S. Patent No. 4,816,567. In one embodiment, the present invention involves isolating and obtaining nucleic acid fragments encoding the antibodies. These nucleic acid molecules comprise the amino acid sequences of the light chain variable region (VL) and / or heavy chain variable region (VH) of the antibody. Furthermore, the nucleic acids can be cloned into one or more vectors, particularly expression vectors, for expression in host cells.

[0099] After production of the antibodies of the present invention, they can be purified from the cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Such techniques are within the conventional art and do not limit the present invention. Culturing host cells can also be understood as expressing the antibodies in animals (particularly transgenic animals or nude mice). This involves an expression system utilizing an animal casein promoter, which, when transgenically incorporated into a mammal, allows the female animal to produce the desired recombinant protein in its milk. The culture medium containing the secreted antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing an adjusted buffer. Nonspecifically bound components are washed away. Bound antibodies are then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and multimers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, for example, at -70°C, or lyophilized.

[0100] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0101] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0102] The sources of the main reagents and instruments used in the following examples are as follows:

[0103] The microplate reader used for detection was purchased from BioTek, model: ELX800;

[0104] The protein separation and purification system was purchased from cytiva, model AKTA Pure;

[0105] 18-hydroxycortisol (18-OHF, Shanghai Zhenzhun, ZS-20214), 18-oxycortisol (18-OXOF, Shanghai Bevanta, product number TB-131081), 18-oxycorticosterone (18-OHB, Shanghai Zhenzhun, product number IR-15291); deoxycortisone (Aladdin, C302974), 11-deoxycorticosterone (Aladdin, D133971), corticosterone (Aladdin, C104537), progesterone (Aladdin, P106427), aldosterone (Aladdin, product number A299444), 17a-hydroxyprogesterone (Aladdin, H1 37675), 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), and prednisone (Aladdin, P116562).

[0106] Example 1 Antibody Preparation Steps

[0107] 1) Gene retrieval

[0108] After collecting the positive clone cells by centrifugation, mRNA was extracted and cDNA products were obtained by reverse transcription. The target fragments were amplified by Taq enzyme using universal primers for the light / heavy chain FR1 and FR4 regions, and then ligated to the pMD19-T vector. The fragments were transformed into JM109 competent cells. After colony PCR verification, 10 positive colonies were selected for sequencing.

[0109] 2) Antibody gene sequence analysis and comparison

[0110] The gene sequences obtained from the sequencing were analyzed and compared with the IMGT antibody database to confirm the integrity of the antibody structure. Sequence reproducibility was analyzed using SnapGene software, and sequences were selected for construction and expression.

[0111] 3) Construction of recombinant antibody expression plasmid

[0112] Based on the sequencing results, specific upstream and downstream primers for the light and heavy chains were designed. The 5' end of the primers contained approximately 15 bp of homology arms that ligated the terminal end of the linearized vector. PCR amplification was performed to obtain light and heavy chain gene fragments with homology arms, as follows: front homology arm - VL / VH - back homology arm;

[0113] The recombinant antibody expression vector was constructed using pEE 6.1. pEE 6.1 was double-digested with HindIII / EcoRI, and the linear vector was recovered by electrophoresis. The vector was ligated by homologous recombination and transformed into JM109 competent cells. After positive colony PCR verification, the culture was expanded to extract the endotoxin-free plasmid to obtain the vector plasmid pL carrying the light chain and the vector plasmid pH carrying the heavy chain.

[0114] 4) Expression and purification

[0115] After the Chinese hamster ovary (CHO) cells were revived, the culture flask was placed in a carbon dioxide shaker (100 rpm, 37°C, 5% CO2) and the cells were cultured to a density of ≥3×10 6 cells / mL, and after subculture, the cells were expanded and cultured, and the cells were collected, counted, and the activity was calculated. 6 cells / mL density and cultured overnight; the plasmids were mixed at a ratio of pL:pH=1.5:1, and the mixed plasmids were transfected into CHO cells according to the Gibco liposome transfection instructions.

[0116] The expression supernatant was purified according to the instructions of the GE AKTA Pure protein separation and purification system to obtain the expressed antibody.

[0117] The variable region sequence of Antibody A is as follows:

[0118] The heavy chain variable region sequence is shown in SEQ ID NO: 6:

[0119] QEQLMESGGGLVTPGGILTLTCTASGVTISNSDMNWVRQAPGKELEWIGVINSYGNTYYASWAKSRSTITRNTNLNTVTLVMTSLTAADTATYFCARDFGGWSTDAFDPWGPGTLVTISS;

[0120] The light chain variable region sequence is shown in SEQ ID NO: 7:

[0121] AVMTQTPSPVSAAVGGTVIISCQASQSVANNKNLAWYQQKPGQPPKLLIYTTSSLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYCAGGYNSISDGVAFGGGTEVVVK.

[0122] The heavy chain CDR1 of antibody A is shown in SEQ ID NO: 1: GVTISNSD; CDR2 is shown in SEQ ID NO: 2: INSYGNT; and 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 SEQ ID NO: TTS; and CDR3 is shown in SEQ ID NO: 5: AGGYNSISDGVA.

[0123] Example 2 Performance Verification of the Antibody Itself

[0124] 1) Affinity

[0125] Instruments and reagents: SPR instrument (Biacore 8K); Chip selection: S-series CM5 / CM7 chip (covalent coupling chip); Amino coupling kit (EDC, NHS, ethanolamine); Buffer: 10× PBS-P+ (200 mM phosphate, 27 mM KCl, 1.37 M NaCl, 0.5% Tween 20), 10 mM sodium acetate (coupling diluent, pH 4-5.5), 10 mM Gly-HCl (chip regeneration solution, pH 1.5-3.0).

[0126] Experimental steps:

[0127] (1) Antibody A immobilization: The ligand was immobilized using the amino direct coupling method. First, EDC / NHS (prepared and used immediately, 1:1 mixture) was injected for 5-10 minutes to activate the carboxyl group. Then, the antibody A was diluted to 10 μg / ml with sodium acetate buffer at pH 4.5 and injected into the detection channel to the target coupling level (small molecule analyte, high coupling, 20,000 RU). Then, ethanolamine (pH 8.5) was injected for 5-10 minutes to block the unreacted sites. Finally, the solution was rinsed with 1× PBS-P buffer until the baseline was stable.

[0128] (2) Analyte detection: 18-OHF was prepared as a 10 mM stock solution, then diluted to 100 μM, and then diluted in 2-fold steps for 6 steps, plus 0 concentration for a total of 8 steps. A multi-cycle kinetic detection method was used, with injections from low to high concentrations at a flow rate of 10 μL / min for 180 seconds, a dissociation time of 600 seconds, and regeneration with 10 mM Gly-HCl (pH 2.0) for 120 seconds.

[0129] Use analysis software to fit and analyze data and calculate affinity.

[0130] The experimental results showed that the K D (M) reached 1.4±0.29×10 -10 , which can meet the requirements of immune detection.

[0131] 2) Specificity and anti-interference

[0132] Sample preparation: To verify the specificity and anti-interference ability of the aforementioned antibodies, a blank group (50 mM PBS) was set as the test object. 18-OHF with a concentration of 10 ng / mL and analog solutions of varying concentrations were prepared in PBS solution for later use. To approximate the clinical testing environment, the analog concentration was determined to be 10 times the actual concentration in reported clinical samples.

[0133] Antibody A was used to detect OD values ​​according to the following steps. The OD value of the blank group was recorded as B0, the OD value of the test substance 18-OHF was recorded as B1, and the OD values ​​of the analogs 18-OXOF, 18-OHB, deoxycortisone, 11-deoxycorticosterone, corticosterone, progesterone, aldosterone, 17a-hydroxyprogesterone, hydrocortisone, prednisolone, triamterene, dexamethasone, spironolactone, eplerenone, amiloride, fludrocortisone, mitotane, cortisone, and prednisone were recorded as B2, B3...B 20 .

[0134] Experimental steps:

[0135] 18-OHF-bovine IgG conjugate was coated on an ELISA plate. After washing, 1% OVA was added and the plate was blocked at 37°C for 0.5 h. After washing, 50 μL of the test substance and 50 μL of the test antibody (concentration of 1 μg / mL) were added and incubated at 37°C for 30 min. After washing, HRP-labeled goat anti-mouse secondary antibody was added and incubated at 37°C for 30 min. After washing, TMB colorimetric solution was added and incubated at room temperature for 10 min. Then, 3 M H2SO4 stop solution was added and the OD value was detected at 450 nm.

[0136] 0.98<B n / B0<1.02 is considered as unbound and the result is recorded as “-”, 1.02≤B n / B0≤2.0 is considered as binding, and the result is recorded as "+", B n A value of / B0>2.0 was considered as strong binding and the result was recorded as "++". The experimental results are shown in Table 1. It shows that the antibody in this scheme only specifically binds to 18-OHF.

[0137] Table 1

[0138]

[0139] 3) Sensitivity

[0140] Sample preparation:

[0141] Prepare 18-OHF solutions with concentration gradients of 0, 50, 200, 1000, 5000, 25000, and 50000 pg / mL for later use. The detection steps are as follows:

[0142] Competition assay steps:

[0143] 1. Reagents used for 18-OHF detection

[0144] (1) Buffer: 0.05 M Tris buffer.

[0145] (2) Magnetic ball reagent: coated with an antibody that specifically recognizes the 18-OHF small molecule. The ratio of magnetic ball and antibody is 1 mg:10 μg; the buffer is citric acid buffer.

[0146] (3) ABEI labeling reagent: 18-OHF-bovine IgG conjugate protein is coupled with ABEI, the ABEI and antigen feeding ratio is: 0.2 mg: 3.6 μg; the buffer is: PBS.

[0147] 2. Detection process:

[0148] Detection was performed on a MAGLUMI X6 chemiluminescent immunoassay analyzer from Shenzhen New Industry Biotechnology Co., Ltd. Instrument loading parameters were as follows: 40 μL sample + 50 μL buffer + 20 μL magnetic beads, incubation at 37°C for 9 min, addition of 50 μL ABEI and incubation at 37°C for 9 min. After three washes, NaOH and H₂O₂ were added, and light signals were detected using a photomultiplier tube.

[0149] The detection discrimination is calculated based on the light intensity detection results. The calculation formula is the ratio of the zero-value light intensity to the light intensity of each gradient. A discrimination greater than 2.0 is considered a reliable detection result.

[0150] When antibody A detects 50 pg / mL samples, the discrimination between it and the 0 value sample is 2.04. Therefore, the results show that antibody A can accurately detect 50 pg / mL samples and meet the detection requirements.

[0151] Table 2

[0152]

[0153] The above-described embodiments merely represent several implementation methods of the present disclosure, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the scope of the present disclosure, all of which fall within the scope of protection of the present disclosure. Therefore, the scope of protection of the patent disclosed herein shall be based on the appended claims, and the specification may be used to interpret the content of the claims.

Claims

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

5.

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

7.

3. An antibody conjugate, characterized in that The method comprises the 18-OHF antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A kit, characterized in that The method comprises the 18-OHF antibody or antigen-binding fragment thereof according to claim 1 or 2, or the antibody conjugate according to claim 3.

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

6. Use of the 18-OHF antibody or antigen-binding fragment thereof according to claim 1 or 2, or the antibody conjugate according to claim 3, in the preparation of a reagent or kit for diagnosing or prognosing a disease associated with a cortisol hormone disorder; the disease associated with a cortisol hormone disorder is selected from the group consisting of primary aldosteronism and endocrine hypertension.

7. Use of the 18-OHF antibody or antigen-binding fragment thereof according to claim 1 or 2, or the antibody conjugate according to claim 3, in the preparation of a reagent or kit for differential diagnosis or typing of a disease associated with cortisol hormone disorder; the disease associated with cortisol hormone disorder is selected from the group consisting of primary aldosteronism and endocrine hypertension.

8. An isolated nucleic acid, characterized in that The protein can encode the 18-OHF antibody or antigen-binding fragment thereof according to claim 1 or 2.

9. A carrier, characterized in that Containing the nucleic acid according to claim 8.

10. A host cell, characterized in that Contains the nucleic acid of claim 8, or is transformed by the vector of claim 9.

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