CN120399069A

CN120399069A - 18-OHB antibody or antigen-binding fragment thereof

Info

Publication number: CN120399069A
Application number: CN202510922849.2A
Authority: CN
Inventors: 高莉; 戚永跃; 刘坤; 郑慧广; 李志川; 杜凯; 何海华; 李婷华; 饶微
Original assignee: Shenzhen New Industries Biomedical Engineering Co Ltd
Current assignee: Shenzhen New Industries Biomedical Engineering Co Ltd
Priority date: 2025-07-04
Filing date: 2025-07-04
Publication date: 2025-08-01
Anticipated expiration: 2045-07-04
Also published as: CN120399069B

Abstract

The invention relates to the technical field of immunodetection, in particular to an 18-OHB antibody or an antigen binding fragment thereof, which comprises a heavy chain complementarity determining region with an amino acid sequence shown in SEQ ID NO. 1-3 and a light chain complementarity determining region with an amino acid sequence shown in SEQ ID NO. 4-6. The 18-OHB antibody or antigen binding fragment thereof provided by the invention has excellent affinity, anti-interference capability and sensitivity to 18-OHB.

Description

18-OHB antibodies or antigen binding fragments thereof

Technical Field

The invention relates to the technical field of immunodetection, in particular to an 18-OHB antibody or an antigen binding fragment thereof.

Background

18-Hydroxycortione (18-Hydroxycorticosterone, 18-OHB) is a key steroid hormone intermediate, and its abnormal levels are closely related to diagnosis and differential diagnosis of endocrine disorders, particularly primary aldosteronism (Primary Aldosteronism, PA) and subtypes thereof (e.g., aldosteronism APA). However, structural analogues such as 18-hydroxycortione and corticosterone (Corticosterone), aldosterone (Aldosterone) have high similarity in molecular configuration, so that the existing detection method generally faces the challenge of insufficient specificity, and false positive results or obvious quantitative errors are easily caused in clinical detection.

Therefore, there is an urgent need in the art to develop a specific antibody with high affinity, extremely low cross-reactivity and good stability to 18-hydroxycortione to support the need for highly reliable detection of primary aldosteronism, accurate typing diagnosis of disease mechanism studies and related clinical studies.

Disclosure of Invention

According to one aspect of the invention, the invention relates to an 18-OHB (18-hydroxycortione) antibody or antigen binding fragment thereof, which comprises a heavy chain complementarity determining region with amino acid sequences shown in SEQ ID NO. 1-3 and a light chain complementarity determining region with amino acid sequences shown in SEQ ID NO. 4-6.

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

According to a further aspect of the invention, it relates to a kit comprising an 18-OHB antibody or antigen binding fragment thereof as described above, or an antibody conjugate as described above.

According to a further aspect of the invention, it relates to the use of an 18-OHB antibody or antigen-binding fragment thereof as described above, or an antibody conjugate as described above, for the preparation of a diagnostic reagent or kit for a disease associated with a cortisol hormone disorder.

According to a further aspect of the invention, it relates to an isolated nucleic acid capable of encoding an 18-OHB antibody or antigen binding fragment thereof as described above.

According to a further aspect of the invention, it relates to a vector comprising a nucleic acid as described above.

According to a further aspect of the invention, it relates to a host cell comprising a nucleic acid as described above, or transformed with a vector as described above.

The 18-OHB antibody or antigen binding fragment thereof provided by the invention has excellent affinity, anti-interference capability and sensitivity to 18-OHB.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, 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 invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

The term "and/or", "and/or" as used herein includes a selection of any one of two or more of the listed items and also includes any and all combinations of the listed items, including any two or more of the listed items, or all combinations of the listed items. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present invention, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

In the present invention, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present invention. In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present invention, all references to which this invention pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present invention, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present invention by reference, but are not limited to being able to implement the present invention. It should be understood that when a reference is made to the description of the invention in conflict with the description, the invention is modified in light of or adaptive to the description of the invention.

In the present invention, the term "antibody" refers to a protein that binds to a specific antigen, and broadly refers to all proteins and protein fragments, particularly full length antibodies or antibody functional fragments, that comprise complementarity determining regions (CDR regions). The term "full length antibody" includes polyclonal antibodies as well as monoclonal antibodies, and the term "antigen binding fragment" is a substance comprising a portion or all of the CDRs of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen.

In the present invention, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the antibody variable region responsible for antigen binding. Three CDRs are contained in each of the variable regions of the heavy and light chains, and are generally 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 versions). Here, the terms "CDR" and "CDRs" are used to refer to regions comprising one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody to its recognized antigen or epitope, depending on the circumstances.

In the present invention, the term "affinity constant" or "K _D (M)" or "K _D" is characterized by K _D (M) =kd (or koff, dissociation rate of a particular binding molecule-target molecule interaction)/Ka (or kon, association rate of a particular binding molecule-target molecule interaction). Smaller K _D (M) indicates stronger affinity of antigen-antibody. The K _D value can be determined by methods well known in the art, for example, using Surface Plasmon Resonance (SPR) in a BIACORE instrument or ELISA methods. SPR is based on optical principles, which cause changes in the refractive index of the chip surface when biomolecules bind or dissociate at the sensor chip surface, resulting in changes in the SPR angle, which are monitored to analyze interactions between molecules in real time. An exemplary SPR procedure mainly comprises the steps of fixing an antigen to be detected on the surface of a sensor chip, enabling antibodies to be detected with different concentration gradients to flow through the chip as flow dependence, monitoring dynamic changes of SPR angles in the processes of molecular binding and dissociation in real time and generating a binding/dissociation curve, and finally calculating a binding rate constant (kon), a dissociation rate constant (koff) and an affinity constant (K _D) by fitting curve data through software, so that the affinity of the antibodies is evaluated. The ELISA method for detecting the affinity of the antibody is characterized in that the antibody (or antigen) is subjected to gradient dilution under the condition that a small amount of antigen (or antibody) exists, the concentration of an antigen-antibody complex is detected, and when the concentration of the complex is half of the total antigen concentration, the corresponding antibody concentration is the affinity constant K _D. The ELISA flow mainly comprises the following steps of firstly coating an antigen to be detected on an ELISA plate and closing unbound sites, then adding an antibody to be detected into a hole for incubation after gradient dilution to wash unbound components, then adding an ELISA secondary antibody for incubation and cleaning, adding a chromogenic substrate for reaction, then measuring absorbance (OD value), and finally determining antibody concentration corresponding to a half saturation point by drawing an OD value and antibody concentration relation curve, thus calculating affinity constant for evaluating affinity.

The first aspect of the invention relates to an 18-OHB antibody or antigen binding fragment thereof, which comprises a heavy chain complementarity determining region with an amino acid sequence shown in SEQ ID NO. 1-3 and a light chain complementarity determining region with an amino acid sequence shown in SEQ ID NO. 4-6.

In some embodiments, the 18-OHB antibody or antigen binding fragment thereof comprises a heavy chain variable region having an amino acid sequence shown in SEQ ID NO.7 and a light chain variable region having an amino acid sequence shown in SEQ ID NO. 8.

In order to increase/alter the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications in the nucleotide sequences encoding the antibodies or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into 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. Variations of the above amino acid sequences are considered to be within the scope of the present invention, as would be conventionally understood by those skilled in the art. Variants of the 18-OHB antibodies or antigen binding fragments thereof of the invention, which comprise a mutation of up to 1,2 or 3 amino acids in the CDR region (if present) compared to any of the polypeptides of SEQ ID NO: 1-SEQ ID NO:6, respectively, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity relative to the overall sequence of SEQ ID NO:7 or SEQ ID NO: 8. Typically, the mutation is a conservative amino acid substitution.

"Conservative amino acid substitutions" are well known in the art and refer to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such similarities include, for example, similarities in polarity, chargeability, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. The exemplary amino acid substitution criteria are as follows:

1) Physicochemical property similarity priority substitution

The acidic amino acid is ASP (D) and GLU (E) are exchanged;

basic amino acids including ARG (R), LYS (K) and HIS (H);

Hydrophobic amino acids ILE (I), LEU (L), VAL (V), MET (M) are interchanged;

the aromatic amino acids PHE (F), TYR (Y) and TRP (W) are selectively interchanged;

Polar uncharged amino acids SER (S) and THR (T) are interchanged, ASN (N) and GLN (Q) are interchanged;

ALA (A) and GLY (G) are exchanged.

2) Evolutionarily conserved guided substitutions

High frequency substitution pair based on Dayhoff/PAM matrix:

SER(S)- THR(T);

LYS(K)- ARG(R);

ILE(I)- VAL(V);

ASP(D)- ASN(N)/GLU(E)。

3) Structural conservation constraint

Beta-sheet region, preferential VAL (V), ILE (I), THR (T) interchange;

Alpha-helical regions ALA (A), LEU (L) and GLU (E) are replaced;

the corner region PRO (P) can be replaced by ALA (A) or GLY (G).

4) Principle of avoidance of functional loci

Antigen binding critical sites (e.g., CDR regions) that avoid substitution of residues involved in hydrogen bonding/hydrophobic interactions such as TYR (Y), TRP (W);

disulfide-related CYS (C) allows only substitution to SER (S) or ALA (a).

5) Treatment of specific residues

Cysteine (CYS) unpaired CYS is preferentially mutated to SER (S) or ALA (a) to reduce aggregation;

Histidine (HIS) the pH sensitive region is replaced with LYS (K) or ARG (R);

Proline (PRO) rigid structural domain replacement requires binding molecule kinetic simulation verification.

Affinity maturation of antibody/antigen binding fragments is also possible in the art, and current affinity maturation methods mainly include directed evolution based on display technology, rational design, and computational simulation aided optimization. The method comprises the specific steps of (1) constructing an antibody mutation library through random mutation or focused mutation by using a phage display platform, enriching high-affinity clones through multiple rounds of antigen binding screening and amplification circulation, (2) adopting a site-directed saturation mutation technology to design mutation combinations aiming at key residues of a Complementarity Determining Region (CDR) or a Framework Region (FR), evaluating binding performance, (3) introducing random mutation in the amplification process by using error prone PCR to generate a diversity library and obtaining optimized variants through functional screening, and (4) guiding directed mutation by predicting key sites affecting affinity through molecular docking, free Energy Perturbation (FEP) or a machine learning model by means of computer aided design. In practical application, various strategies are often integrated, for example, potential hot spots are screened through calculation, and verification and optimization are performed by combining an in-vitro display technology, so that efficient and accurate affinity promotion is realized.

In the field of antibody applications, it is also desirable to produce cysteine engineered antibodies, artificially introducing highly reactive sulfhydryl groups (-SH). The directional coupling is achieved by labelling or coating with thiol groups and maleimides by selecting for replacement of one or more residues in the antibody with cysteine residues, the replaced residues typically being in the constant region of the antibody in order not to affect the specific binding of the antibody to the antigen. By replacing specific amino acid residues with cysteines, reactive thiol groups are generated that make the antibody available at specific sites for conjugation of the antibody to other moieties, e.g., antibody-labels, antibody-solid phase carriers, antibody-specific proteins, etc.

Isoelectric point (pI) is the pH at which the net charge of a protein in solution is zero, and is determined by the pKa of the ionizable groups in the amino acid sequence (e.g., carboxyl groups of ASP/D, GLU/E, amino groups of LYS/K, ARG/R). In antibody preparation, pI affects solubility (e.g., colloidal stability of high concentration formulations), purification efficiency (ion exchange chromatography binding properties). In the detection field, the stability of the reagent is generally improved by adjusting the pI of the antibody to be close to the pH of the reaction buffer to avoid aggregation. Strategies for regulating the isoelectric point of antibodies include substitution of surface charge residues by site-directed mutagenesis (e.g., LYS. Fwdarw. GLN decreases pI), chemical modification (PEGylation introduces negative charge), or glycosylation engineering (regulates the sialylation level of the Fc region). For example, substitution of basic residues in the CDR regions of an antibody with neutral residues can reduce pI from 8.5 to 7.2. In practical application, the pI is predicted by combining a calculation tool (such as ExPASy ProtParam) and verified by capillary isoelectric focusing electrophoresis.

The 18-OHB antibody or antigen binding fragment thereof of the invention may have a constant region, which may be a constant region of any of IgG (including any of IgG1, igG2, igG3, igG 4), igE, igD, igA, igM. The constant region may be derived from a species selected from the group consisting of cattle, horses, pigs, sheep, goats, rats, mice, guinea pigs, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, alpacas, and humans.

The antigen binding fragments of the antibodies provided by the invention can be designed as antibody fragments with specific functions or application advantages, which optimize physicochemical properties while retaining antigen binding capacity by structural simplification or engineering. Common antibody fragments include Fab fragments (which are disulfide-linked to the intact light chain by the heavy chain variable region VH and the first constant region CH1, and have a molecular weight of about 50 kDa), fab ' fragments (which retain a portion of the hinge region on the Fab basis and contain free sulfhydryl groups and which can form multivalent structures by chemical coupling), F (ab ') ₂ fragments (which are disulfide-linked by the hinge region to form bivalent binding forms by two Fab's), fv fragments (which contain only the heavy and light chain variable regions VH and VL, relying on non-covalent interactions to maintain structural stability), and scFv fragments (which form single chain structures by the flexible polypeptide linker tandem VH and VL, enhancing stability and facilitating genetic engineering operations). The preparation method is divided into two types, namely an enzymatic digestion method (such as papain hydrolysis of an IgG hinge region to generate a monovalent Fab fragment, or pepsin cleavage of the C-terminal end of an IgG heavy chain to generate a bivalent F (ab ') ₂ fragment, treatment with a reducing agent such as beta-mercaptoethanol to obtain a Fab' fragment) and a recombinant expression technology (such as direct synthesis of scFv fragments by an E.coli, yeast or mammalian cell expression system, screening of high affinity variants by combining with phage display technology, and improvement of thermodynamic stability by introducing additional disulfide bonds or optimizing a connecting peptide).

The functional fragments of the above antibodies can also be synthesized by commercially available automated peptide synthesizers.

A second aspect of the invention relates to an antibody conjugate comprising an 18-OHB antibody or antigen binding fragment thereof as described above.

The antibody conjugate refers to a complex formed by coupling an antibody with a functional molecule (such as a chemiluminescent label, a fluorescent dye, an enzyme, a radioisotope), a solid phase, a protein or the like. This technique is widely used in biomedical research, diagnosis and therapy. The thus obtained include antibody-solid phase conjugates, antibody-signal substance conjugates, antibody-polypeptide conjugates, and the like.

For antibody-solid phase conjugates, "solid phase" refers to a solid support for immobilization of an antibody, which functions to provide a stable solid-liquid reaction system to achieve specific capture, separation, and signal detection of the target. The use of solid phases is well known in the fields of chemistry, biochemistry, pharmacy and molecular biology.

The solid phase may be classified into various types according to its characteristics, including organic polymers such as polystyrene, polypropylene, glass, and silicone rubber, 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 nanocomposite materials.

Immobilization on a solid phase may be accomplished using a method in which the solid phase is modified or activated to contain functional groups capable of covalent coupling with an antibody or antibody complex (an antibody may form a complex with a specific molecule including carrier protein, biotin/avidin, FITC, etc.). The functional group is selected from one or more of carboxyl, amino, mercapto and tosyl. The antibody or antibody complex may also be non-covalently attached to the solid phase by, for example, ionic or hydrophobic mechanisms.

In one embodiment, the solid phase may be a sheet, a preformed tray, a cylinder, fibrous or granular, generally allowing for proper contact. The size of the solid phase suitable for use in the method of the invention may vary depending on the method chosen. Antibodies may be bound to only one solid phase (e.g., one multi-well plate), or may be bound to a number of solid phases (e.g., beads). In one embodiment, the solid phase may be fibrous or particulate to allow optimal contact. The size of the solid phase may vary and may be selected according to the method to be performed.

In the present invention, the shape of the term "particle" (sometimes referred to as a "bead" or "microbead" or "microsphere") may generally vary. For example, in one particular embodiment, the particles are spherical. However, it should be understood that other shapes are also contemplated by the present invention, such as plates, rods, discs, bars, tubes, irregular shapes, and the like. In addition, the particle size may also vary. For example, the average size (e.g., diameter) of the particles may range from about 0.1nm to 1mm, such as 1nm, 10nm, 100nm, 500nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 500 μm, for example, of the diameter of the particles.

In some embodiments, the particles are magnetic spheres, preferably nanomagnetic microspheres, such as, for example, ferroferric oxide magnetic nanoparticles. The magnetic microsphere is a colloidal composite material which can be uniformly dispersed in a certain base solution, and has the characteristics of superparamagnetism, higher specific surface area, modifiable functional groups and the like. Surface functional groups include, but are not limited to, reactive groups such as carboxyl, amino, hydroxyl, or sulfhydryl groups. The antibody coupled magnetic beads are mainly used for realizing directional combination through physical adsorption or chemical coupling. Physical adsorption relies on electrostatic action, hydrophobic action and van der Waals forces, chemical coupling utilizes amino, carboxyl, hydroxyl or sulfhydryl groups modified on the surface of magnetic beads to bind with charged groups of antibodies, usually incubated in a suitable buffer, and non-specific sites blocked with BSA.

For antibody-signal substance conjugates, wherein the signal substance refers to any substance capable of generating a signal for direct or indirect detection. Thus, the signal substance may be detected directly or indirectly. For direct detection, the signal material suitable for use in the present invention may be selected from any known set of detectable labels. In some embodiments, the signal species is selected from chromogens, fluorescent groups, chemiluminescent groups (e.g., isoluminol and derivatives thereof, acridinium esters or dioxetanes), electrochemiluminescent compounds, catalysts, enzymes, enzyme substrates, dyes, fluorescent dyes (e.g., fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine, and derivatives thereof). Other examples of such signal substances are luminescent metal complexes such as ruthenium or europium complexes (e.g. for ECLIA), enzymes (e.g. for ELISA) and radioisotopes (e.g. for RIA). In some embodiments, the signal material is selected from the group consisting of ABEI and its derivatives, acridinium esters, alkaline phosphatase, and horseradish peroxidase. Indirect detection systems include, for example, labeling a particular molecule with a first partner of a bioaffinity binding pair. Examples of suitable binding pairs are biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin/avidin or streptavidin, sugar/lectin, nucleic acid or nucleic acid analogue/complementary nucleic acid.

Preferred are chemiluminescent signal substances including, but not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, and peroxyoxalate and its derivatives. Antibodies form antibody-signal substance conjugates by direct and indirect coupling. The direct coupling is that the reactive group of the antibody is directly connected with the reactive group of the luminescent agent, and the indirect coupling is that the antibody is connected with the luminescent agent through a bridge, and the bridge can introduce new active groups and reduce space obstruction. Common coupling methods include carbodiimide, glutaraldehyde and N-hydroxysuccinimide active ester methods, such as the reaction of an antibody with an amino residue of lysine on the antibody to form an amide bond using a direct label ABEI, ABEI cyclic lactone. The main process of antibody labeling ABEI includes preparing labeling buffer solution, labeling antibody, stirring reaction at 25 deg.c to react with ABEI, and dialysis to eliminate small molecule.

The antibody-polypeptide conjugate can be a fusion protein formed by fusing an antibody and a polypeptide which does not influence the structure of the antibody, wherein the polypeptide can be one or more of 6 multiplied by His, GST, MBP, FLAG, HA, c-Myc, SUMO, strep, and the like, or can be a complex formed by coupling the antibody with other antibodies and specific molecules, and the specific molecules comprise one or more of carrier proteins, biotin/avidin, FITC, and the like.

A third aspect of the invention relates to a kit comprising an 18-OHB antibody or antigen binding fragment thereof as described above, or an antibody conjugate as described above.

The term "kit" in the present invention may refer to any article of manufacture (e.g., package or container) comprising at least one device comprising a detection agent according to the present invention. The kit may further comprise instructions, supplemental reagents and/or components or assemblies for use in the methods described herein or steps thereof.

If desired, the kit may further comprise any one or more of a standard substance, a sample pretreatment reagent (e.g., a sample purification enrichment reagent, a lysate, etc.), a buffer/diluent (e.g., a phosphate buffer), a substrate for a signal substance (usually a chromogenic reagent, such as ABEI, sodium hydroxide and hydrogen peroxide, if the signal substance is ABEI), and a preservative (any one or more of sodium azide, sodium nitrite, sodium benzoate, proclin series, potassium sorbate). These components may be packaged separately or may be mixed in advance in two or more kinds to facilitate use/preservation. One or more components of the kit may be liquid or lyophilized and present as a solid powder.

Kits of the invention include, but are not limited to, enzyme-linked immunosorbent assay kits, chemiluminescent kits, radioimmunoassay kits, and the like, fluorescent immunoassay kits, and the like.

In some specific embodiments, the kit is a chemiluminescent kit comprising a solid phase reagent comprising an antibody-solid phase conjugate, wherein the solid phase is a nanomagnetic microsphere, and a label reagent comprising a label-solid phase conjugate, wherein the label is ABEI and derivatives thereof. The detection step of the kit comprises the steps of simultaneously or sequentially contacting a sample to be detected with a solid-phase reagent and a marker reagent to form an antigen-antibody immune complex, and determining the amount of a target substance in the sample to be detected by detecting the amount of the immune complex. The sample to be tested may be blood, urine, cerebrospinal fluid, tissue samples or other fluids containing the test substance.

The fourth aspect of the invention relates to the use of an 18-OHB antibody or antigen-binding fragment thereof, or an antibody conjugate as described above, in the preparation of a kit or kit for diagnosis, differential diagnosis, typing, therapy monitoring or prognosis of a disease associated with a cortisol hormone disorder.

In some embodiments, the cortisol hormone disorder related disorder is selected from the group consisting of primary aldosteronism, pseudoaldosteronism, cushing syndrom, endocrine hypertension, hypoaldosteronism, aldosteronism and edison's disease.

The primary aldosteronism comprises five subtypes, namely, aldosteronism (aldosterone producing adenoma, APA), idiopathic aldosteronism (or called bilateral adrenal hyperplasia, idiopathic hyperaldosteronism, IHA), primary adrenal cortical hyperplasia (or called unilateral adrenal hyperplasia, PRIMARY ADRENALHYPERPLASIA, PAH), familial aldosteronism (familial hyperaldosteronism, FH, including FH-i, II, III, IV type), adrenal cortical carcinoma and the like.

The subject of the above method or use may refer to a patient or an animal suspected of suffering from a disease associated with a plasma hormone disorder, particularly a mammal, preferably a primate, more preferably a human.

The fifth aspect of the invention relates to an isolated nucleic acid capable of encoding an 18-OHB antibody or antigen binding fragment thereof as described above.

The term "isolated nucleic acid" in the present invention refers to a polymer of deoxyribonucleic acid or ribonucleic acid in single-stranded or double-stranded form. The isolated nucleic acids include RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and unless otherwise indicated, the polypeptides also include natural polynucleotides, sugars, or base-altered analogs. According to one aspect of the invention, the polynucleotide is a light chain polynucleotide.

The isolated nucleic acid includes a nucleotide sequence encoding an amino acid sequence of a protein complex, as well as a nucleotide sequence complementary thereto. The complementary sequences include fully complementary sequences and substantially complementary sequences, which refers to sequences that hybridize under stringent conditions known in the art to nucleotide sequences encoding amino acid sequences of protein complexes.

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. Polynucleotides encoding protein complex amino acid sequences may be construed to include nucleotide sequences that have substantial identity to the isolated nucleic acid. The substantial identity aligns the nucleotide sequences to additional random sequences in a manner that maximizes their correspondence, which sequences may exhibit greater than 80% homology, such as greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology when the aligned sequences are analyzed using algorithms common in the art.

Furthermore, the codons of the corresponding nucleic acid of the 18-OHB antibody or antigen binding fragment thereof may be optimized according to the species of the host cell.

A sixth aspect of the invention relates to a vector comprising a nucleic acid as described above.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby 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), phages, such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

A seventh aspect of the invention relates to a host cell comprising a nucleic acid as described above, or transformed with a vector as described above.

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

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, suitable host cells or cell lines for expressing the antigen binding proteins of the 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 may also be used. Human cells can be used, thus allowing the molecule to be modified with a human glycosylation pattern. Or 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 generation and purification, are known in the art.

Prokaryotic cells, such as bacterial cells, may prove useful as host cells suitable for expressing the proteins or other embodiments of the invention. However, since proteins expressed in bacterial cells tend to be in an unfolded or incorrectly folded form or in a non-glycosylated form, any protein produced in bacterial cells must be screened to preserve 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 alternative embodiments, the molecule may be expressed in a bacterial host followed by refolding. For example, various E.coli strains for expression are well known host cells in the biotechnology field. Various strains of Bacillus subtilis, streptomyces, other Bacillus, and the like may also be used in the method.

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

In some embodiments, the nucleic acid is inserted into the genome of the cell and is capable of stable expression. The manner of insertion may be by way of a vector as described above, or by direct transfer of the nucleic acid into the cell without attachment of the vector (e.g., liposome-mediated transfection techniques).

An eighth aspect of the invention relates to a method of producing an 18-OHB antibody or antigen binding fragment thereof as described above comprising culturing a host cell as described above under culture conditions suitable for expression of the antibody.

In addition, the method includes the optional step of isolating and recovering the expressed antibodies from the host cells or their culture medium.

Antibodies provided herein can be prepared by recombinant techniques, and reference can be made specifically to the relevant methods disclosed in U.S. Pat. No. 4,816,567. In one embodiment of the invention, it relates to isolating and obtaining a nucleic acid fragment encoding said antibody. These nucleic acid molecules comprise the amino acid sequence of the light chain variable region (VL) and/or the heavy chain variable region (VH) of the antibody. Further, the nucleic acid may be cloned into one or more vectors, particularly expression vectors, for expression within a host cell.

After the antibodies of the invention are produced, 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 ordinary skill in the art and do not limit the present invention. Culturing host cells is also understood to mean the expression of antibodies in animals, in particular transgenic animals or nude mice. This involves an expression system that utilizes animal casein promoters, which when transgenically incorporated into a mammal, allow a female to produce the desired recombinant protein in its milk. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present invention, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

The main reagents and instrument sources used in the following examples were as follows:

the detection enzyme label instrument is purchased from BioTek, model number ELX800;

protein isolation and purification systems were purchased from cytiva, model AKTA Pure;

18-OHB (Shanghai screening, accession No. IR-15291), 18-OXOF (Shanghai Bei Moda, cat No. TB-131081), 18-OHF (Shanghai screening, ZS-20214), deoxycortisone (Allatin, C302974), 11-deoxycorticosterone (Allatin, D133971), corticosterone (Allatin, C104537), progesterone (Allatin, P106427), aldosterone (Allatin, cat No. A299444), 17 a-hydroxyprogesterone (Allatin, H137675), hydrocortisone (Altin, H110523), prednisolone (Altin, P276607), triamterene (Altin, T303644), dexamethasone (Altin, D137736), spironolactone (Altin, S303875), eplerenone (Altin, E129934), amiloride (Altin, A131615), fludrocortisone (Altin, F340821), altin (Altin, D119445), altin (Altin, D3754).

Example 1 antibody preparation procedure

1) Gene transfer

After positive clone cells are collected by centrifugation, mRNA is extracted, cDNA products are obtained by reverse transcription, a target fragment is amplified by adopting a light/heavy chain FR1 and FR4 region universal primer through Taq enzyme, and then a tail is added, a pMD19-T vector is connected, the target fragment is transformed into JM109 competence, and 10 positive colonies are selected for sequencing after colony PCR verification.

2) Antibody gene sequence analysis and alignment

And (3) analyzing and comparing the gene sequences obtained by sequencing in an IMGT antibody database to determine that the antibody has complete structure. The sequences were selected for their expression by comparison with the repeat of the analysis sequences on SnapGene software.

3) Recombinant antibody expression plasmid construction

And designing specific upstream and downstream primers of the light and heavy chains according to the sequencing result, wherein the 5' -end of the primer carries a homology arm of about 15bp and the connecting tail end of the linearization vector. The light and heavy chain gene segment with homologous arm is obtained through PCR amplification, concretely comprising a front end homologous arm-VL/VH-rear end homologous arm;

Constructing a recombinant antibody expression vector by taking pEE 6.1 as a vector, carrying out HindIII/EcoRI double digestion on the pEE 6.1, recovering a linear vector by electrophoresis, connecting by a homologous recombination scheme, transforming JM109 competent, carrying out colony PCR positive verification and carrying out sequencing verification, and carrying out amplification culture to extract endotoxin plasmids to obtain a vector plasmid pL carrying a light chain and a vector plasmid pH carrying a heavy chain.

4) Expression purification

After resuscitating Chinese Hamster Ovary (CHO) cells, the flask was placed in a carbon dioxide shaker (100 rpm,37 ℃,5% CO ₂), the cells were cultured to a density of ≡3×10 ⁶ cells/mL, passaged, expanded, cell counts were collected and activity was calculated, plated at a density of 1×10 ⁶ cells/mL, cultured overnight, plasmid was mixed at a ratio pL: ph=1.5:1, and the mixed plasmid was transfected into CHO cells according to Gibco liposome transfection instructions.

And (3) purifying the expression supernatant according to the operation of the GE AKTA Pure protein separation and purification system instruction, so as to obtain the expression antibody.

Wherein, the heavy chain variable region sequence of the antibody A is shown as SEQ ID NO. 7:

EQLMESGGRLVTPGTPLTLTCTVSGFSLSSYTMSWVRQAPGKGLEWIGIISSSGSTYYATWAKGRFTISKASTTVDLKITSPTTEDTATYFCAREPRVWSYDDYDDINIWGPGTLVTISS Shown;

the sequence of the light chain variable region is shown as SEQ ID NO. 8:

ALVMTQTPSPVSATVGGTVSISCQSNKSIYNNNWLNWFQQKPGQPPKLLIYGASTLASGVPSRFKGSGSGTQFTLTISDVQCDDAATFYCAGGYSSTSDTYAFGGGTEVVVK As shown.

The heavy chain CDR1 of antibody A is shown in SEQ ID NO. 1: GFSLSSYT, CDR2 is shown in SEQ ID NO. 2: ISSSGST, and CDR3 is shown in SEQ ID NO. 3: AREPRVWSYDDYDDINI. Light chain CDR1 is shown in SEQ ID NO. 4:KSIYNNW, CDR2 is shown in SEQ ID NO. 5:IYGAT, and CDR3 is shown in SEQ ID NO. 6: AGGYSSTSDTYA.

Example 2 Performance validation of the antibodies themselves

1. Affinity for

The instrument and reagent are SPR instrument (Biacore 8K), chip selection of S series CM5/CM7 chip (covalent coupling chip), amino coupling kit (EDC, NHS, ethanolamine), buffer solution of 10 XPBS-P+ (200 mM phosphate, 27mM KCl,1.37M NaCl, 0.5% Tween 20), 10mM sodium acetate (coupling diluent, pH 4-5.5), 10mM Gly-HCl (chip regeneration solution, pH 1.5-3.0)

The experimental steps are as follows:

(1) Fixing antibody A, namely fixing the ligand by adopting an amino direct coupling method, firstly injecting EDC/NHS (for preparation at present, mixing 1:1) for 5-10min, activating carboxyl groups, then diluting the antibody A to 10ug/ml by using sodium acetate buffer solution with pH of 4.5, injecting a detection channel to a target coupling level (small molecule analyte, high coupling and 20000 RU), then injecting ethanolamine (with pH of 8.5) for 5-10min, closing unreacted sites, and finally flushing with 1 XPBS-P buffer solution until the base line is stable.

(2) Analyte detection 18-OHB was made up to 10mM stock solution, then diluted to 100uM, then diluted 2-fold for 6 gradients, plus 8 total 0 concentrations. The method adopts a multi-cycle dynamic detection mode, and sequentially samples from low concentration to high concentration, the flow rate is 10uL/min, the dissociation time is 600s, and the regeneration time is 120s by using 10mM Gly-HCl (pH 2.0).

Data fitting and analysis were performed using analysis software to calculate affinities.

Experimental results show that K _D (M) of the antibody A reaches 5.27+/-0.11 multiplied by 10 ^-9, and the requirements of immunodetection can be met.

2. Specificity and interference resistance

Sample preparation in order to verify the specificity and anti-interference capability of the antibody, the sample is set as a blank group (50 mMPBS), 18-OHB with 10ng/ml concentration is prepared by adopting PBS solution and analogue solutions with different concentrations are prepared for standby, and the determination standard of the concentration of the analogue is 10 times of the actual concentration in the reported clinical sample in order to approach the clinical detection environment.

OD values were measured using antibody a according to the following procedure. The OD values of the blank group were designated as B ₀, the OD values of the test substance 18-OHB as B ₁, and the analogues 18-OHF, 18-OXOF, dexamethasone, deoxycortisone, aldosterone, cortisol, prednisolone, corticosterone, aminopterine, spironolactone, amiloride, fludrocortisone, mitotane, 11 deoxy-18-hydroxycortidone were measured using antibody A according to the following procedure. The corresponding OD values were B ₂、B₃……B₂₁, respectively.

The experimental steps are as follows:

The 18-OHB-bovine IgG conjugate is coated on an ELISA plate, 1% OVA is added after plate washing for sealing at 37 ℃ for 0.5 h, 50 mu L of an object to be detected and 50 mu L of an antibody to be detected (the concentration is 1 mu g/ml) are added after plate washing, incubation is carried out for 30min at 37 ℃, goat anti-mouse secondary antibody marked with HRP is added after plate washing, incubation is carried out for 30min at 37 ℃, chromogenic solution TMB is added after plate washing, incubation is carried out for 10min at room temperature, and stopping solution 3M H ₂SO₄ is added for detecting OD value at 450 nm.

0.98< B _n/B₀ <1.02 is considered unbound, the result is marked as "-", 1.02≤B _n/B₀≤2.0 is considered bound, the result is marked as "+", B _n/B₀ >2.0 is considered to be strongly bound, the result is marked as "++", the experimental results are shown in Table 1, and the results show that the antibody of the scheme only specifically binds 18-OHB.

TABLE 1

Analogues	18-OHB	18-OHF(50ng/ml)	18-OXOF(50ng/ml)
				Detection result	++	-	-
Analogues	Deoxycortisone (100 ng/ml)	11-Deoxycorticosterone (10 ng/ml)	Corticosterone (100 ng/ml)
				Detection result	-	-	-
Analogues	Progesterone (100 ng/ml)	Aldosterone (5 ng/ml)	17 A-hydroxyprogesterone (50 ng/ml)
				Detection result	-	-	-
Analogues	Hydrocortisone (1000 ng/ml)	Prednisolone (800 ng/ml)	Ammonia benzene pteridine (200 ng/ml)
				Detection result	-	-	-
Analogues	Dexamethasone (30 ng/ml)	Spironolactone (200 ng/ml)	Eplerenone (1000 ng/ml)
				Detection result	-	-	-
Analogues	Amiloride (200 ng/ml)	Fluohydrocortisone (6 ng/ml)	Mitotane (100. Mu.g/ml)
				Detection result	-	-	-
Analogues	Cortisone (200 ng/ml)	Prednisone (200 ng/ml)
				Detection result	-	-

3. Sensitivity of

Sample preparation:

18-OHB solutions with concentration gradients of 0, 200, 400, 1000, 2000, 5000, 10000pg/ml were prepared for use.

The detection steps are as follows:

the competition method detection step comprises the following steps:

1) Reagent for detecting 18-OHB project

(1) Buffer 0.05M Tris Buffer.

(2) The magnetic ball reagent is an antibody which specifically recognizes 18-OHB small molecules, the feeding ratio of the magnetic ball to the antibody is 1mg to 10 mug, and the buffer solution is 0.05M PBS buffer solution.

(3) The ABEI labeling reagent comprises 18-OHB-carrier protein and ABEI, wherein the feeding ratio of the ABEI to the antigen is 0.18mg to 10.08 mug, and the buffer solution is 0.05M Tris buffer solution.

2) The detection process comprises the following steps:

Detection is carried out on a chemiluminescent immunoassay analyzer MAGLUMI X of new products in Shenzhen City, the technological parameters of instrument sample adding are that 40 mu L of sample is added with 50 mu L of Buffer and 20 mu L of magnetic ball, incubation is carried out for 9min at 37 ℃, and 50 mu L of ABEI is added at 37 ℃ for incubation 9 min. NaOH and H ₂O₂ are added for 3 times of cleaning, and a photomultiplier detects the optical signals.

According to the light intensity detection result, the detected distinguishing degree is calculated, the calculation formula is the ratio of 0-value light intensity to each gradient light intensity, and the 0-value light intensity/200 pg/ml sample light intensity (distinguishing degree) >2.0 is considered as the reliable detection result.

When 200pg/ml of the sample is detected by the antibody A, the sample is distinguished from a 0-value sample by 2.16, and the experimental result is shown in the table 2, so that the result shows that the antibody A can accurately detect the 200pg/ml of the sample and meets the detection requirement.

TABLE 2

(pg/ml)	0	200	400	1000	2000	5000	10000
								Ratio of	/	2.16	4.85	7.31	20.56	37.69	69.47

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is, therefore, indicated by the appended claims, and the description may be intended to interpret the contents of the claims.

Claims

The 18-OHB antibody or antigen binding fragment thereof is characterized by comprising a heavy chain complementarity determining region with an amino acid sequence shown in SEQ ID NO. 1-3 and a light chain complementarity determining region with an amino acid sequence shown in SEQ ID NO. 4-6.
2. The 18-OHB antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID No. 7, and a light chain variable region having the amino acid sequence shown in SEQ ID No. 8.
3. An antibody conjugate comprising the 18-OHB antibody or antigen-binding fragment thereof of claim 1 or 2.
4. A kit comprising the 18-OHB antibody or antigen-binding fragment thereof of claim 1 or 2, or the antibody conjugate of claim 3.
5. The kit of claim 4, wherein the kit is a chemiluminescent kit comprising a solid phase reagent and a label reagent, the solid phase reagent or label reagent comprising the 18-OHB antibody or antigen binding fragment thereof of claim 1 or 2, or the antibody conjugate of claim 3.
6. Use of an 18-OHB antibody or antigen-binding fragment thereof according to claim 1 or 2, or an antibody conjugate according to claim 3, for the preparation of a reagent or kit for diagnosis, differential diagnosis, typing, therapeutic monitoring or prognostic assessment of a disease associated with a cortisol hormone disorder.
7. The use according to claim 6, wherein the disorder associated with cortisol hormone dysfunction is selected from the group consisting of primary aldosteronism, pseudoaldosteronism, cushing syndrom, endocrine hypertension, hypoaldosteronism, aldosteronism deficiency and edison's disease.
8. An isolated nucleic acid capable of encoding the 18-OHB antibody or antigen-binding fragment thereof of claim 1 or 2.
9. A vector comprising the nucleic acid of claim 8.
10. A host cell comprising the nucleic acid of claim 8 or transformed with the vector of claim 9.