Antibodies and kits for anti-estradiol immune complexes

Abstract

The present application belongs to the field of medicine and detection, and relates to an antibody and a kit for anti-estradiol immune complex. The antibody of the present application can specifically bind to the estradiol immune complex, but cannot bind to the estradiol alone, nor can bind to the estradiol-specific antibody alone. The kit prepared by the antibody of the present application can effectively determine the estradiol, and has extremely high sensitivity and specificity.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and testing, and relates to an antibody and kit for anti-estradiol immune complexes. The estradiol immune complex refers to a complex formed by the binding of estradiol and an anti-estradiol antibody. Specifically, the kit is used to detect or determine estradiol levels. Background Technology

[0002] Estradiol (E2) is a type of estrogen that regulates various functions in the human body. It controls ovulation and the development of female sexual characteristics and is generally considered a reproductive hormone. Estradiol levels range widely in clinical and research settings. Physiological estradiol levels range from approximately 1 to 20,000 pg / mL in prepubertal children, adolescents, adult women (premenopausal, pregnant, and postmenopausal), and men. This range also includes estradiol levels obtained from breast cancer patients treated with aromatase inhibitors, postmenopausal women treated with estrogen, and patients undergoing ovarian stimulation therapy. In patients treated with aromatase inhibitors, this range can extend to a lower limit of <1 pg / mL. Therefore, estradiol assays used in clinical and research laboratories require accuracy and precision across concentration ranges of several orders of magnitude. Thus, it is essential to develop methods capable of detecting estradiol concentrations as low as pg / mL.

[0003] One method for determining estradiol levels in the blood is immunoassay. However, most current estradiol immunoassays are based on the principle of competition, which inherently does not allow for the measurement of very low concentrations of analytes (Simon Rattle et al., Handbook of Immunoassays (4th Edition), 2023, p. 178). Furthermore, estradiol is a low molecular weight substance and cannot accommodate two different antibody epitopes for a classic sandwich-type immunoassay, further complicating detection. Summary of the Invention

[0004] Through in-depth research and creative work, the inventors have obtained an antibody against estradiol immune complexes, which specifically binds to estradiol immune complexes but does not specifically bind to estradiol itself, nor to the anti-estradiol antibody constituting the estradiol immune complexes. This provides the following invention:

[0005] One aspect of the present invention relates to an antibody against an anti-estradiol immune complex or an antigen-binding fragment thereof, said anti-estradiol complex antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and said light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein,

[0006] HCDR1, HCDR2, and HCDR3 contain amino acid sequences selected from those shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are not identical to each other; and

[0007] LCDR1, LCDR2, and LCDR3 contain amino acid sequences selected from those shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are not identical to each other.

[0008] In some embodiments of the present invention, the antibody or antigen-binding fragment of the anti-estradiol immune complex, wherein,

[0009] HCDR1 contains an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:13.

[0010] HCDR2 contains an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:8, and SEQ ID NO:14.

[0011] HCDR3 contains an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:9, and SEQ ID NO:15.

[0012] LCDR1 contains an amino acid sequence selected from those shown in SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:16.

[0013] LCDR2 contains an amino acid sequence selected from those shown in SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:17, and

[0014] LCDR3 contains an amino acid sequence selected from those shown in SEQ ID NO:6, SEQ ID NO:12 and SEQ ID NO:18.

[0015] In some embodiments of the present invention, the antibody or antigen-binding fragment of the anti-estradiol immune complex, wherein,

[0016] HCDR1 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, HCDR3 contains the amino acid sequence shown in SEQ ID NO:3, LCDR1 contains the amino acid sequence shown in SEQ ID NO:4, LCDR2 contains the amino acid sequence shown in SEQ ID NO:5, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:6.

[0017] HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, HCDR2 contains the amino acid sequence shown in SEQ ID NO:8, HCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR1 contains the amino acid sequence shown in SEQ ID NO:10, LCDR2 contains the amino acid sequence shown in SEQ ID NO:11, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:12; or

[0018] HCDR1 contains the amino acid sequence shown in SEQ ID NO:13, HCDR2 contains the amino acid sequence shown in SEQ ID NO:14, HCDR3 contains the amino acid sequence shown in SEQ ID NO:15, LCDR1 contains the amino acid sequence shown in SEQ ID NO:16, LCDR2 contains the amino acid sequence shown in SEQ ID NO:17, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:18.

[0019] In some embodiments of the present invention, the antibody or antigen-binding fragment of the anti-estradiol immune complex, wherein,

[0020] The antibody includes a non-CDR region, and the non-CDR region is derived from rat antibody, mouse antibody, rabbit antibody, or human antibody;

[0021] Preferably, the antibody against the estradiol immune complex is a monoclonal antibody;

[0022] Preferably, the antibody against the estradiol immune complex is a murine monoclonal antibody.

[0023] In some embodiments of the present invention, the antibody or antigen-binding fragment of the anti-estradiol immune complex, wherein,

[0024] The antibody or its antigen-binding fragment for the anti-estradiol immune complex is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, or chimeric antibody.

[0025] In some embodiments of the present invention, the antibody or antigen-binding fragment of the anti-estradiol immune complex, wherein,

[0026] The antibody or antigen-binding fragment of the anti-estradiol immune complex binds to the K+ of the estradiol immune complex. D Less than or equal to 10 -8 M, less than or equal to 10 -9 M or less than or equal to 5x10 -10 M.

[0027] In some embodiments of the present invention, the antibody or antigen-binding fragment of the anti-estradiol immune complex, wherein,

[0028] The estradiol immune complex is a complex formed by the binding of estradiol and anti-estradiol antibody.

[0029] Preferably, the anti-estradiol antibody is an E2 monoclonal antibody from Wuhan Panoramic, with the product number AB-E2-01.

[0030] In some embodiments of the present invention, the antibody or antigen-binding fragment of the anti-estradiol immune complex, wherein,

[0031] The antibody or antigen-binding fragment of the anti-estradiol immune complex does not specifically bind to estradiol alone, nor does it specifically bind to an anti-estradiol antibody alone.

[0032] Preferably, the antibody against the estradiol immune complex is an antibody produced by a hybridoma cell line with accession numbers H-224, H-225, or H-226.

[0033] The hybridoma cell line is deposited in the All-Russian National Collection of Industrial Microorganisms (VKPM) NRC (Kurchatov Institute) on September 17, 2024.

[0034] Another aspect of the invention relates to isolated nucleic acid molecules that encode an antibody or antigen-binding fragment of the anti-estradiol immune complex described in any one of the invention.

[0035] Another aspect of the invention relates to a recombinant vector comprising the isolated nucleic acid molecules of the invention.

[0036] Another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecules of the invention, or comprising the recombinant vector of the invention.

[0037] Another aspect of the present invention relates to a hybridoma cell line with accession numbers H-224, H-225 or H-226, and said hybridoma cell line is deposited in the All-Russian National Collection of Industrial Microorganisms (VKPM) NRC.<Kurchatov Institute> The deposit date is September 17, 2024.

[0038] Another aspect of the present invention relates to a method for preparing an antibody or antigen-binding fragment thereof of the anti-estradiol immune complex as described in any one of the present invention, comprising culturing the host cells or hybridoma cells of the present invention under suitable conditions, and recovering the antibody or antigen-binding fragment thereof of the anti-estradiol immune complex from the cell culture.

[0039] Another aspect of the present invention relates to a conjugate comprising an antibody portion and a conjugation portion, wherein the antibody portion is an antibody or antigen-binding fragment of the anti-estradiol immune complex described in any one of the present invention, and the conjugation portion is a detectable label; preferably, the conjugation portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0040] Preferably, the enzyme is selected from one or more of alkaline phosphatase, horseradish peroxidase, luciferase, glucose oxidase, and β-galactosidase.

[0041] Another aspect of the present invention relates to a kit for detecting estradiol, comprising:

[0042] The antibody or antigen-binding fragment thereof of the anti-estradiol immune complex described in any one of the present invention, or the conjugate of the present invention,

[0043] Antibodies that specifically bind to estradiol, and

[0044] Optional estradiol standard.

[0045] In some embodiments of the present invention, the kit contains an antibody that specifically binds to estradiol; preferably, the anti-estradiol antibody is an E2 monoclonal antibody from Wuhan Panoramic Technology Co., Ltd. with catalog number AB-E2-01.

[0046] In some embodiments of the present invention, the kit is provided in which the antibody that specifically binds to estradiol is linked to a solid support.

[0047] Preferably, the solid support is a spherical or planar solid support;

[0048] Preferably, the solid support is selected from glass slides, polymer beads, magnetic beads, ceramics, silicon wafers, or plastic materials;

[0049] Preferably, the linker is connected to the solid support via biotin-streptomycin avidin, SpyTag / SpyCatcher, receptor-ligand, or OsASI1-BAH-OsSUVH6.

[0050] The kit described in some embodiments of the present invention, wherein...

[0051] The concentration of the antibody or its antigen-binding fragment, or the conjugate, of the anti-estradiol immune complex is 0.05-5 μg / ml, 0.1-3 μg / ml, 0.2-2 μg / ml, 0.3-1 μg / ml, 0.4-0.8 μg / ml, or 0.5 μg / ml;

[0052] The concentration of the antibody that specifically binds to estradiol is 0.05-2 μg / ml, 0.1-1 μg / ml, 0.1-0.8 μg / ml, 0.15-0.6 μg / ml, 0.2-0.4 μg / ml, or 0.25 mg / ml;

[0053] Preferably, the antibody or antigen-binding fragment of the anti-estradiol immune complex or the conjugate thereof is dissolved in a buffer solution (e.g., Tris-HCl buffer or MES buffer);

[0054] Preferably, the antibody that specifically binds to estradiol is dissolved in a buffer solution (e.g., Tris-HCl buffer or MES buffer);

[0055] Preferably, the buffer solution independently contains NaCl, BSA, Tween-20 and ProClin 300, and one or more selected from NaN3, MgCl2 and ZnCl2.

[0056] In some embodiments of the present invention, the kit contains NaCl, BSA, Tween-20, ProClin 300, and NaN3; preferably, the Tris-HCl buffer contains 150 mM NaCl, 2% BSA, 0.2% Tween-20, 0.05% ProClin 300, and 0.09% NaN3.

[0057] In some embodiments of the present invention, the kit contains NaCl, BSA, Tween-20, ProClin 300, MgCl2, and ZnCl2; preferably, the MES buffer contains 0.9% NaCl, 1% BSA, 0.05% Tween-20, 0.048% ProClin 300, 2mM MgCl2, and 0.1mM ZnCl2.

[0058] In some embodiments of the present invention, the kit has a LoQ for detecting estradiol of less than 10 pg / mL, less than 9 pg / mL, less than 8 pg / mL, less than 7 pg / mL, less than 6 pg / mL, less than 5 pg / mL, less than 4 pg / mL, less than 3 pg / mL, less than 2 pg / mL, less than 1 pg / mL, 1-10 pg / mL, 1-9 pg / mL, 1-8 pg / mL, 1-7 pg / mL, 1-6 pg / mL, 1-5 pg / mL, 1-4 pg / mL, or 1-3 pg / mL.

[0059] Another aspect of the present invention relates to the use of any antibody or antigen-binding fragment thereof of the anti-estradiol immune complex described in any one of the present invention, or the conjugate thereof, in the preparation of reagents or kits for detecting estradiol.

[0060] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0061] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and heavy chains also contain "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H1 C H2 and C H3 It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (CL It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L These regions form antigen-binding sites, respectively. The CDR sequences of the heavy chain variable region and the light chain variable region are defined by the Kabat numbering system, which refers to the EU index of Kabat et al. (see, for example, Kabat, Elvin A., et al. Sequences of Proteins of Immunological Interest. 5th ed., USDept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991.).

[0062] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0063] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but it can also be obtained using recombinant DNA technology (see US Patent 4,816,567).

[0064] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, Jones et al., Nature 1986; 321:522-525; Reichmann et al., Nature 1988; 332:323-329; Presta, Curr. Op. Struct. Biol., 1992; 2:593-596; and Clark M. Antibody humanization: a case of the 'Emperor's new clothes'? [J]. Immunol. Today, 2000; 21(8):397-402.

[0065] As used herein, the term "light chain" includes full-length light chains and segments thereof with sufficient variable region sequences to confer binding specificity. Full-length light chains include a variable region domain (VL) and a constant region domain (CL). The variable region domain of a light chain is located at the amino terminus of the polypeptide. Light chains include κ chains and λ chains.

[0066] As used herein, the term "heavy chain" includes the full-length heavy chain and its segments having sufficient variable region sequences to confer binding specificity. The full-length heavy chain includes a variable region domain (VH) and three constant region domains (CH1, CH2, and CH3). The VH domain is located at the amino terminus of the polypeptide, and the CH domain is located at the carboxyl terminus, with CH3 being closest to the carboxyl terminus of the polypeptide. The heavy chain can have any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 isotypes), IgA (including IgA1 and IgA2 isotypes), IgM, and IgE.

[0067] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0068] As used in this invention, the term "Fab fragment" consists of a light chain and a CH1 group, as well as a variable region of a heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0069] As used in this invention, the term "Fab' fragment" comprises a portion of a light chain and a portion of a heavy chain (containing a VH domain and a CH1 domain, as well as a portion of the region between the CH1 and CH2 domains) so that interchain disulfide bonds can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0070] As used in this invention, the term "F(ab')2 fragment" comprises two light chains and two heavy chains containing portions of a constant region between CH1 and CH2 domains, so as to form interchain disulfide bonds between the two heavy chains. The F(ab')2 fragment thus consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0071] As used in this invention, the term "Fv region" includes variable regions from both the heavy and light chains, but lacks constant regions.

[0072] As used in this invention, the term “Fd” fragment refers to an antibody fragment consisting of VH and CH1 domains (Ward et al., Nature 341:544-546 (1989)).

[0073] As used in this invention, the term “dAb” fragment (Ward et al., Nature 341:544-546 (1989)) consists of a VH domain.

[0074] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0075] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0076] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells, or human cells.

[0077] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0078] As used in this article, the term "K" D "Affinity" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the dissociation equilibrium constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have a dissociation equilibrium constant of less than approximately 10-1. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to antigens (e.g., estradiol immune complexes). K can be measured using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0079] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0080] In this invention, the term LoB (Limit of Blank) refers to the blank detection limit, which represents the highest measurement result that can be obtained using a blank sample under specific probability conditions. LoB indicates that even if the sample does not contain the analyte, the test result may not be zero due to the influence of reagent components, instruments, and other factors. Concentrations below the LoB value can be considered as zero concentrations.

[0081] In this invention, the term LoD (Limit of Detection) refers to the detection limit, which represents the lowest concentration that can be detected under specific probability conditions. LoD is used to determine whether a analyte is present in a sample, but it cannot determine the specific concentration. For example, if the LoD is 8 ng / mL and the detection result is 6 ng / mL, it means that the analyte is indeed present in the sample, but the concentration is too low to be accurately measured.

[0082] In this invention, the term LoQ (Limit of Quantitation) refers to the limit of quantitation, which indicates the lowest concentration that can be accurately measured. LoQ requires not only the detection of the analyte but also the assurance of accurate results. For example, if the LoQ is 10 ng / mL and the detection result is 12 ng / mL, it means that the concentration of the analyte in the sample is 12 ng / mL.

[0083] Beneficial effects of the invention

[0084] This invention achieves one or more of the following technical effects:

[0085] (1) The antibody of the present invention can specifically bind to estradiol immune complexes.

[0086] (2) The antibody of the present invention cannot bind to estradiol alone.

[0087] (3) The antibodies of the present invention cannot bind to individual estradiol-specific antibodies.

[0088] (4) The antibodies of the present invention have no cross-reactivity or very low cross-reactivity with estradiol analogues (estrone, estriol, testosterone, progesterone, aldosterone, etc.), which is significantly superior to reagents or kits for competitive detection of estradiol.

[0089] (5) The reagent kit prepared by the antibody of the present invention can effectively measure estradiol and has extremely high sensitivity and specificity. Attached Figure Description

[0090] Figure 1 The absorbance bars of antibodies E2_112, E2_129, and E2_130 of the present invention after incubation with estradiol-primary antibody immune complex, primary antibody, E2-BSA, and BSA, respectively, are shown. The results indicate that the antibodies of the present invention are only reactive with estradiol-primary antibody immune complex and are not reactive with primary antibody alone or with E2.

[0091] Figures 2A to 2L : Affinity assay curves for antibodies. The antibody samples are shown below:

[0092] Figure 2A E2_112, 5nM;

[0093] Figure 2B E2_112, 10nM;

[0094] Figure 2C E2_112, 50nM;

[0095] Figure 2D E2_112-BSA coupling;

[0096] Figure 2E ,E2_129, 5 nM;

[0097] Figure 2F ,E2_129, 10 nM;

[0098] Figure 2G ,E2_129, 50 nM;

[0099] Figure 2H ,E2_129-BSA conjugate;

[0100] Figure 2I ,E2_130, 5 nM;

[0101] Figure 2J ,E2_130, 10 nM;

[0102] Figure 2K ,E2_130, 50 nM;

[0103] Figure 2L ,E2_130-BSA conjugate.

[0104] Figure 3 : Total error of samples with different concentrations in the establishment of LoQ for Kit 1.

[0105] Figure 4 : Total error of samples with different concentrations in the establishment of LoQ for Kit 2.

[0106] Figure 5 : Total error of samples with different concentrations in the establishment of LoQ for Kit 3.

[0107] Figure 6 : Comparison of Kit 1 with mass spectrometry methodology.

[0108] Figure 7 : Comparison of Kit 2 with mass spectrometry methodology.

[0109] Figure 8 : Comparison of Kit 3 with mass spectrometry methodology.

[0110] Biological materials involved in preservation:

[0111] Hybridoma cell line the hybridoma of E2_112, which was deposited on September 17, 2024 at All-Russian National Collection of Industrial Microorganisms (VKPM) NRC <<Kurchatov Institute>>, with the deposit number H-224 and the deposit address at Russia 117545 Moscow 1 st Dorozhny proezd1.

[0112] The hybridoma cell line E2_129 was deposited on September 17, 2024, at the All-Russian National Collection of Industrial Microorganisms (VKPM) NRC. <kurchatovinstitute>>,with the deposit number of H-225 and the deposit address of Russia 117545 Moscow 1 st Dorozhny proezd1。

[0113] The hybridoma cell line, the hybridoma of E2_130, was deposited on September 17, 2024 with the All-Russian National Collection of Industrial Microorganisms (VKPM) NRC <<Kurchatov Institute>>, with the deposit number of H-226 and the deposit address of Russia 117545 Moscow 1 st Dorozhny proezd1。

[0114] Part of the sequence information related to this invention is shown in Table A below

[0115] Table A

[0116]

[0117] Detailed implementation manners

[0118] The embodiments of this invention will be described in detail below in conjunction with the examples. However, those skilled in the art will understand that the following examples are only for illustrating this invention and should not be regarded as limiting the scope of this invention. For those conditions not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0119] Estradiol (E2) was purchased from Sigma Aldrich (Cat# E4389).

[0120] The specific antibody against estradiol was purchased from Wuhan Panorama (product number: AB-E2-01, antibody name: E2 monoclonal antibody). The E2 monoclonal antibody is also referred to as the primary antibody in this invention.

[0121] The HRP-conjugated mouse IgG polyclonal antibody was purchased from Sigma Chemicals, St. Louis, Mo. The HRP-conjugated mouse IgG polyclonal antibody is also referred to as the secondary antibody in this invention.

[0122] Example 1: Preparation of hybridoma cell lines that secrete monoclonal antibodies against anti-estradiol immune complexes

[0123] E2 monoclonal antibody was incubated with E2 solution dissolved in PBS at room temperature for 1 hour. The E2 concentration was 100 times the molar concentration of the antibody. Excess estradiol was removed by desalting using a Zeba spin desalting column (Thermo Fisher Scientific) to obtain estradiol-primary antibody immune complexes.

[0124] Using the obtained estradiol-antibody immune complex, BALB / c mice were immunized according to the conventional method for immunizing mice to obtain hybridomas.

[0125] Using estradiol-anti-immune complex as a pre-adsorbed antigen, hybridoma cells producing estradiol-anti-immune complex-specific antibodies were screened using enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

[0126] Add 20 ng / 0.05 ml PBS of estradiol-primary antibody immune complex to each well and incubate with gentle shaking at room temperature;

[0127] After the antigen was adsorbed for 30 min, it was washed twice with PBS (PBST) containing 0.1% Tween-20 detergent.

[0128] Then incubate with 0.05 ml of conditioned medium collected from the growing hybridoma for 30 min, and wash twice with PBST;

[0129] Incubate with HRP-conjugated mouse IgG polyclonal antibody (secondary antibody) for 30 minutes to show mouse antibodies binding to pre-adsorbed antigen, with 0.05 ml of secondary antibody diluted 1:1000 in PBST added to each well;

[0130] After incubation with the secondary antibody, wash 6 times with PBST, and add 0.05 ml of 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate containing 0.03% hydrogen peroxide;

[0131] After incubation for 10 minutes, 0.05 ml of 1 M phosphate was added to stop the reaction, and the absorbance of each well was measured at 450 nm. The absorbance was measured using a Labsystems Multiscan microplate reader (Labsystems, Finland).

[0132] Hybridoma cells that produce estradiol-anti-immune complex-specific antibodies (under the above conditions, the absorbance at 450 nm is 0.5 times higher than the background absorbance) were selected for subsequent experiments.

[0133] These hybridoma cells were cloned using restricted dilution. Hybridoma cell clones secreting the target monoclonal antibody were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (HyClone Laboratories, Logan, UT).

[0134] Three hybridoma cell lines were obtained, and the antibodies secreted by them were named E2_112, E2_129, and E2_130, respectively. These three hybridoma cell lines are deposited at the All-Russian National Collection of Industrial Microorganisms (VKPM) NRC.<Kurchatov Institute> The deposit date is September 17, 2024; their accession numbers are H-224, H-225 and H-226 respectively.

[0135] Example 2: Antibody Sequence Analysis

[0136] Total RNA was extracted from the mouse hybridoma cells obtained in Example 1 using a Quick-RNA Microprep Kit (Quick-RNA Microprep Kit #1050-51, Zymo Research, USA). cDNA was synthesized via reverse transcription and then amplified by PCR. The PCR amplification products were sequenced and analyzed to obtain the heavy chain variable region and light chain variable region sequences of the antibody. The CDRs were labeled using the Kabat numbering system, as shown in Table A above.

[0137] Example 3: Preparation of Antibodies

[0138] Hybridoma cells producing monoclonal antibodies E2_112, E2_129, and E2_130 were grown in mice, and mouse ascites fluid was used to isolate E2_112, E2_129, and E2_130.

[0139] Antibodies (E2_112, E2_129, E2_130) were purified from ascites fluid using protein A affinity chromatography. The resin was from GE Healthcare Life Sciences (Piscataway, NJ), and purification was performed according to the manufacturer's instructions. The purified monoclonal antibodies were stored at 4°C in 50% ammonium sulfate solution.

[0140] Example 4: Plate-based indirect ELISA for antibody specificity determination

[0141] To determine the specificity of antibodies E2_112, E2_129, and E2_130, empty primary antibody, estradiol-primary antibody immune complex, E2-BSA conjugate (β-estradiol 6-(O-carboxy-methyl)oxime:BSA, Sigma, E5630), or BSA were adsorbed onto the surface of a plate at a concentration of 100 ng per well.

[0142] After incubation for 30 minutes, wash three times with PBST.

[0143] A series of diluted antibodies were prepared in PBST and incubated in plate wells.

[0144] After incubation for 30 minutes, wash three times with PBST.

[0145] HRP-conjugated anti-mouse antibody (Jackson ImmunoResearch, 115-035-003) was added to the wells of the plate, with the antibody diluted 1:1000 with PBST.

[0146] After incubation for 30 minutes, wash 6 times with PBST and add 0.05 ml of 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate containing 0.03% hydrogen peroxide.

[0147] After incubation for another 30 minutes, 0.05 ml of 1 M phosphate was added to stop the reaction, and the absorbance of each well was measured at 450 nm. The absorbance was measured using an EnVision microplate reader (Perkin Elmer, USA).

[0148] The results are as follows Figure 1 As shown in the figure. The results showed that antibodies E2_112, E2_129, and E2_130 reacted only with the estradiol-primary antibody immune complex, and not with the empty primary antibody or E2 alone.

[0149] Example 5: Measurement of antibody affinity

[0150] Biological layer interferometry was performed according to the following protocol to determine the affinity of the antibody for the E2 immune complex.

[0151] The antibody (10 μg / mL) in PBS solution was bound to the sensor to block it. Then, antigens (antibody or estradiol-anti-immune complex) at concentrations of 5 nM, 10 nM and 50 nM reacted with the antibody on the sensor.

[0152] The complete experimental protocol is as follows: sensor hydration → antibody binding to sensor → sensor cleaning → sensor blocking → binding (for each concentration of test antibody) → dissociation → sensor regeneration.

[0153] The reaction curve was obtained by subtracting the reaction of the single antibody from the reaction of the estradiol-antibody immune complex, and further quantitative analysis was performed. To determine the affinity for E2 separately, the E2-BSA conjugate (Sigma E5630) was reacted with the antibody on the sensor. The result was a flat line, which could not be quantitatively analyzed; therefore, it can be concluded that the antibody does not interact with the E2 antigen alone.

[0154] The results are as follows Figures 2A to 2L As shown in Table 1.

[0155] Table 1: Antibody Affinity Data

[0156] Antibody Ka(1 / Ms) Kd(1 / s) KD(M) E2_112 <![CDATA[9.84*10 5 ]]> <![CDATA[4.24*10 -4 ]]> <![CDATA[4.23*10 -10 ]]> E2_129 <![CDATA[8.64*10 4 ]]> <![CDATA[9.51*10 -6 ]]> <![CDATA[1.10*10 -10 ]]> E2_130 <![CDATA[4.78*10 5 ]]> <![CDATA[4.08*10 -4 ]]> <![CDATA[8.80*10 -10 ]]>

[0157] The results showed that the antibody of the present invention has good affinity for the estradiol-anti-immune complex.

[0158] Example 6: Preparation of a kit for estradiol immunoassay

[0159] Monoclonal antibodies E2_112, E2_129, and E2_130 were labeled with alkaline phosphatase (Cat#03137031103, Roche Custom Biotech) as detection antibodies; E2 monoclonal antibody was labeled with biotin (Cat#PG82075, Thermo) as capture antibody.

[0160] The capture antibody was incubated with streptavidin-labeled particles (MyOne T1 Dynabeads Streptavidin PMPHydrophobic particles 100mkm, Cat#35604D, Thermo) to obtain capture antibody-labeled magnetic beads. The conditions for using the capture antibody-labeled magnetic beads in the assay were as follows: the capture antibody-labeled magnetic beads were dissolved in 50mM pH 7.4 Tris-HCl buffer with a capture antibody concentration of 0.25 mg / ml, which also contained 150mM NaCl, 2% BSA, 0.2% Tween-20, 0.05% ProClin 300, and 0.09% NaN3.

[0161] The detection antibody was dissolved at a concentration of 0.5 μg / ml in 50 mM pH 6.0 MES buffer, which also contained 0.9% NaCl, 1% BSA, 0.05% Tween-20, 0.048% ProClin 300, 2 mM MgCl2 and 0.1 mM ZnCl2.

[0162] Estradiol (Sigma Aldrich, E4389) was used as the calibrator in the immunoassay, with estradiol concentrations of 0 pg / mL for calibrator 1, 500 pg / mL for calibrator 2, and 4800 pg / mL for calibrator 3.

[0163] Example 7: Sensitivity Testing and Methodological Comparison

[0164] Prepare three kits using the method described above. The antibodies for the magnetic bead reagent and enzyme-labeled reagent are shown in Table 2 below:

[0165] Table 2

[0166] Antibodies used in magnetic bead reagents Antibodies used in enzyme-labeled reagents Reagent Kit 1 AB-E2-01 E2_129 Reagent Kit 2 AB-E2-01 E2_112 Reagent Kit 3 AB-E2-01 E2_130

[0167] Estradiol at different dilution ratios was prepared under the conditions of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20 (the concentration of estradiol was the same as in Example 6).

[0168] Mix 30 μL of estradiol with 50 μL of magnetic bead suspension and 50 μL of monoclonal antibody-phosphatase complex solution. Perform a one-step immunoassay on a Mindray CL-6000i fully automated analyzer according to the manufacturer's instructions.

[0169] Chemiluminescence was detected using the substrate solution provided by the Mindray fully automated immunoassay system supplied with the analyzer. The active ingredient in the substrate solution was 3-(2-spirodaradinane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane (AMPPD).

[0170] Chemiluminescence is expressed in relative light units (RLU).

[0171] 7-1 Sensitivity Test Results

[0172] A series of samples with low estradiol concentrations (1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, 6 pg / mL, 7 pg / mL, 8 pg / mL, 9 pg / mL) and a blank sample without estradiol were prepared by serial dilution. These samples were tested continuously for 4 days, using 2 reagent batches and 1 instrument, with each instrument performing 2 replicates per day, resulting in a total of 32 test results. Following the classic method in the EP17-A2 guideline, the mean and standard deviation of each concentration sample, as well as the total standard deviation (SD) and coefficients, were calculated to obtain LoB and LoD. Based on the mean and standard deviation of the low-concentration samples, the deviation of the test value from the reference value and the total error were calculated. The lowest concentration that meets the total error index is defined as LoQ.

[0173] The results of calculating LoB and LoD are as follows:

[0174] Kit 1: LoB = 0.56 pg / mL; LoD = 1.89 pg / mL.

[0175] Kit 2: LoB = 1.33 pg / mL; LoD = 3.78 pg / mL.

[0176] Kit 3: LoB = 1.54 pg / mL; LoD = 3.89 pg / mL.

[0177] The results of calculating LoQ are as follows ( Figures 3 to 5 The LoQ results were all less than 10 pg / mL, especially for Kit 1, where the LoQ was less than 5 pg / mL. The commercially available E2 kit claims a minimum sensitivity of 10 pg / mL, indicating that the kit prepared using the antibody in this protocol has very high sensitivity.

[0178] 7-2 Methodological Comparison

[0179] 120 samples were randomly collected, including samples of different genders, ages and gestational ages. Each sample was divided into two parts. One part was sent to Mindray's traceability laboratory for mass spectrometry detection, and the other part was used for luminescence detection using reagent kits 1, 2 and 3 on Mindray's immunoluminescence instrument.

[0180] The results are as follows Figures 6 to 8 As shown in the figure. The results show that the current E2 detection kit and the LC-MS detection method have good methodological consistency, with correlation coefficients R2 greater than 0.95.

[0181] 7-3 Cross-reactivity experiment of analogues

[0182] Cross-reactivity of analogs was detected using competitive assay kits and sandwich assay kits (kits 1-3 above) prepared with E2 monoclonal antibody (primary antibody). Five analogs (estrone, estriol, testosterone, progesterone, and aldosterone) at corresponding concentrations were added to blank serum matrix samples without E2, resulting in five samples. The concentrations of the samples were detected using both the competitive assay and sandwich assay kits. The cross-reactivity rate was calculated as: sample value / analog concentration * 100%. The obtained cross-reactivity rates are shown in Table 3 below.

[0183] Table 3

[0184]

[0185]

[0186] The results showed that in the cross-reaction of four analogues—estriol, testosterone, and aldosterone—the E2 sandwich method kit of this invention was superior to the competitive method kit; in the cross-reaction of progesterone, the E2 sandwich method kit was on par with the competitive method. These results indicate that the sandwich method kit of this invention has a significant advantage over the competitive method in terms of analogue interference.

[0187] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.< / kurchatovinstitute>

Claims

1. An antibody or antigen-binding fragment thereof of an anti-estradiol immune complex, wherein the anti-estradiol complex antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein, HCDR1, HCDR2, and HCDR3 contain amino acid sequences selected from those shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are not identical to each other; and LCDR1, LCDR2, and LCDR3 contain amino acid sequences selected from those shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are not identical to each other.

2. The antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to claim 1, wherein, HCDR1 contains an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:

13. HCDR2 contains an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:8, and SEQ ID NO:

14. HCDR3 contains an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:9, and SEQ ID NO:

15. LCDR1 contains an amino acid sequence selected from those shown in SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:

16. LCDR2 contains an amino acid sequence selected from those shown in SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:17, and LCDR3 contains an amino acid sequence selected from those shown in SEQ ID NO:6, SEQ ID NO:12 and SEQ ID NO:

18.

3. The antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to any one of claims 1 to 2, wherein, HCDR1 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, HCDR3 contains the amino acid sequence shown in SEQ ID NO:3, LCDR1 contains the amino acid sequence shown in SEQ ID NO:4, LCDR2 contains the amino acid sequence shown in SEQ ID NO:5, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:

6. HCDR1 contains the amino acid sequence shown in SEQ ID NO:7, HCDR2 contains the amino acid sequence shown in SEQ ID NO:8, HCDR3 contains the amino acid sequence shown in SEQ ID NO:9, LCDR1 contains the amino acid sequence shown in SEQ ID NO:10, LCDR2 contains the amino acid sequence shown in SEQ ID NO:11, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:12; or HCDR1 contains the amino acid sequence shown in SEQ ID NO:13, HCDR2 contains the amino acid sequence shown in SEQ ID NO:14, HCDR3 contains the amino acid sequence shown in SEQ ID NO:15, LCDR1 contains the amino acid sequence shown in SEQ ID NO:16, LCDR2 contains the amino acid sequence shown in SEQ ID NO:17, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:

18.

4. The antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to any one of claims 1 to 3, wherein, The antibody includes a non-CDR region, and the non-CDR region is derived from rat antibody, mouse antibody, rabbit antibody, or human antibody; Preferably, the antibody against the estradiol immune complex is a monoclonal antibody; Preferably, the antibody against the estradiol immune complex is a murine monoclonal antibody.

5. The antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to any one of claims 1 to 4, wherein, in, The antibody or antigen-binding fragment of the anti-estradiol immune complex is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, or chimeric antibody.

6. The antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to any one of claims 1 to 5, wherein, The antibody or antigen-binding fragment of the anti-estradiol immune complex binds to the K+ of the estradiol immune complex. D Less than or equal to 10 -8 M, less than or equal to 10 -9 M or less than or equal to 5x10 -10 M.

7. The antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to any one of claims 1 to 6, wherein, The estradiol immune complex is a complex formed by the binding of estradiol and anti-estradiol antibody. Preferably, the anti-estradiol antibody is an E2 monoclonal antibody from Wuhan Panoramic, with the product number AB-E2-01.

8. The antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to any one of claims 1 to 7, wherein, The antibody or antigen-binding fragment of the anti-estradiol immune complex does not specifically bind to estradiol alone, nor does it specifically bind to an anti-estradiol antibody alone. Preferably, the antibody against the estradiol immune complex is an antibody produced by a hybridoma cell line with accession numbers H-224, H-225, or H-226. The hybridoma cell line is deposited at the All-Russian National Collection of Industrial Microorganisms (VKPM) NRC, Kurchatov Institute, on September 17, 2024.

9. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the antiestradiol immune complex as described in any one of claims 1 to 8.

10. A recombinant vector comprising the isolated nucleic acid molecule of claim 9.

11. A host cell comprising the isolated nucleic acid molecule of claim 9, or comprising the recombinant vector of claim 10.

12. A hybridoma cell line with accession number H-224, H-225 or H-226, and said hybridoma cell line is deposited in the All-Russian National Collection of Industrial Microorganisms (VKPM) NRC "Kurchatov Institute" on September 17, 2024.

13. A method for preparing an antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to any one of claims 1 to 8, comprising culturing the host cell of claim 11 or the hybridoma cell line of claim 12 under suitable conditions, and recovering the antibody or antigen-binding fragment thereof of the anti-estradiol immune complex from the cell culture.

14. A conjugate comprising an antibody portion and a conjugation portion, wherein, The antibody portion is an antibody or antigen-binding fragment of the anti-estradiol immune complex as described in any one of claims 1 to 8, and the conjugated portion is a detectable label; preferably, the conjugated portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. Preferably, the enzyme is selected from one or more of alkaline phosphatase, horseradish peroxidase, luciferase, glucose oxidase, and β-galactosidase.

15. A kit for detecting estradiol, comprising: The antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to any one of claims 1 to 8, or the conjugate according to claim 14. Antibodies that specifically bind to estradiol, and Optional estradiol standard; Preferably, the kit is a sandwich method kit for detecting estradiol; Preferably, the anti-estradiol antibody is an E2 monoclonal antibody from Wuhan Panoramic, with the product number AB-E2-01.

16. The reagent kit according to claim 15, wherein, The antibody that specifically binds to estradiol is linked to a solid support. Preferably, the solid support is a spherical or planar solid support; Preferably, the solid support is selected from glass slides, polymer beads, magnetic beads, ceramics, silicon wafers, or plastic materials; Preferably, the linker is connected to the solid support via biotin-streptomycin avidin, SpyTag / SpyCatcher, receptor-ligand, or OsASI1-BAH-OsSUVH6.

17. The kit according to any one of claims 15 to 16, wherein, The concentration of the antibody or its antigen-binding fragment, or the conjugate of claim 15, of the anti-estradiol immune complex is 0.05-5 μg / ml, 0.1-3 μg / ml, 0.2-2 μg / ml, 0.3-1 μg / ml, 0.4-0.8 μg / ml, or 0.5 μg / ml; The concentration of the antibody that specifically binds to estradiol is 0.05-2 μg / ml, 0.1-1 μg / ml, 0.1-0.8 μg / ml, 0.15-0.6 μg / ml, 0.2-0.4 μg / ml, or 0.25 mg / ml; Preferably, the antibody or antigen-binding fragment of the anti-estradiol immune complex or the conjugate thereof is dissolved in a buffer solution (e.g., Tris-HCl buffer or MES buffer); Preferably, the antibody that specifically binds to estradiol is dissolved in a buffer solution (e.g., Tris-HCl buffer or MES buffer); Preferably, the buffer solution independently contains NaCl, BSA, Tween-20 and ProClin 300, and one or more selected from NaN3, MgCl2 and ZnCl2.

18. Use of the antibody or antigen-binding fragment thereof of the anti-estradiol immune complex according to any one of claims 1 to 8, or the conjugate according to claim 14, in the preparation of a reagent or kit for detecting estradiol; preferably, the kit is a sandwich assay kit for detecting estradiol.

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