Specific binding protein for free oestradiol and method for its production and use

Abstract

The application belongs to the technical field of immune detection, and particularly relates to a specific binding protein of free estradiol-3, and a preparation method and application thereof. The application provides a high-specificity binding protein for free estradiol-3 (uE3), which comprises specific complementarity determining regions. The specific binding protein is immunologically prepared based on a hapten designed by computer-aided simulation, a mouse is immunized after the hapten is coupled with a carrier protein, and a high-affinity and high-specificity anti-uE3 monoclonal antibody is obtained by screening through a hybridoma technology. The specific binding protein can also be applied to the development of an in-vitro diagnostic reagent for free estradiol-3, so as to realize accurate quantitative detection of uE3 in a clinical blood sample, significantly improve the accuracy and reliability of uE3 detection, and provide a more reliable basis for prenatal screening and placental function evaluation.

Description

Technical Field

[0001] This application belongs to the field of immunoassay technology, specifically relating to a specific binding protein of free estriol and its preparation method and application. Background Technology

[0002] Unconjugated estriol (uE3), as a core component of maternal serum biomarkers in mid-pregnancy, is of vital clinical value for prenatal screening of fetal chromosomal aneuploidy disorders such as Down syndrome and Edwards syndrome, due to the accuracy and reliability of its quantitative detection. However, the quality of the detection performance fundamentally depends on the quality of its core reagent—a highly specific and high-affinity antibody.

[0003] Currently, the preparation technology of monoclonal antibodies against uE3 mainly relies on two traditional routes, both of which have significant limitations:

[0004] Direct conjugation immunization: uE3 molecules are directly conjugated to carrier proteins (such as BSA, OVA) before immunizing animals. Although this method is simple to operate, the key antigenic epitopes of the small molecule hapten (uE3) are easily spatially masked or embedded by the large molecular carrier after conjugation, resulting in incomplete or unsatisfactory epitope recognition by the immune system, and the obtained antibody specificity is often poor.

[0005] Random introduction of a spacer arm: To avoid the masking effect of direct coupling, a "spacer arm" is often chemically synthesized and introduced into the uE3 molecule before being linked to a carrier protein. However, the selection of the spacer arm's connection site, chemical structure, and length largely relies on empirical trial-and-error screening. This strategy is highly arbitrary and random, resulting in a lengthy and resource-intensive development cycle. Furthermore, it is difficult to ensure at the molecular level that key characteristic epitopes (such as the C16α-hydroxyl group, which distinguishes it from estradiol and estrone) are optimally exposed to the solvent to achieve the most effective immunostimulation.

[0006] The common consequence of the aforementioned technical bottlenecks is that the quality of the prepared antibodies is unstable between batches, the cross-reactivity rate with structural analogs (such as other estrogens) is high, and the affinity is inconsistent. Ultimately, this restricts the sensitivity, specificity, and consistency of clinical test kits, making it difficult to meet the ever-increasing demand for precision medicine.

[0007] Therefore, it is necessary to conduct more in-depth research on the specific binding proteins of free estriol. Summary of the Invention

[0008] Based on this, one embodiment of this application provides a specific binding protein for free estriol, its preparation method, and its application.

[0009] This application provides a free estriol-specific binding protein, wherein the free estriol-specific binding protein has a free estriol-binding domain.

[0010] The free estriol binding domain has heavy chain CDR1 to heavy chain CDR3 as shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain CDR1 to light chain CDR3 as shown in SEQ ID NO.4 to SEQ ID NO.6.

[0011] In some embodiments, the amino acid sequence of the heavy chain variable region of the free estriol binding domain is shown in SEQ ID NO.7.

[0012] In some embodiments, the amino acid sequence of the light chain variable region of the free estriol binding domain is shown in SEQ ID NO.8.

[0013] In some embodiments, the specific binding protein of free estriol further includes a heavy chain constant region and a light chain constant region.

[0014] In some embodiments, the specific binding protein of the free estriol has a sequence of any one of the constant regions of IgG, IgA, IgM, IgE, and IgD.

[0015] In some of these embodiments, the species sources of the heavy chain constant region and the light chain constant region are each independently selected from any one of the following: human, mouse, rabbit, sheep, cattle, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck, and goose.

[0016] This application, in another respect, provides a nucleic acid molecule encoding a specific binding protein of the aforementioned free estriol.

[0017] This application also provides a carrier comprising the aforementioned nucleic acid molecule.

[0018] This application also provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned vector.

[0019] This application also provides a method for preparing the cells described above, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

[0020] This application also provides a method for preparing the specific binding protein of free estriol, the method comprising the following steps:

[0021] The cells were cultured; and a specific binding protein for free estriol was isolated from the resulting culture.

[0022] In another aspect, this application provides a free estriol detection kit, the detection kit comprising a specific binding protein for the free estriol.

[0023] This application also provides a method for detecting free estriol in a sample to be tested. The method uses a specific binding protein of free estriol as a detection antibody and determines the presence of free estriol in the sample to be tested through an immunobinding reaction.

[0024] This application also provides a medicament comprising the specific binding protein of the aforementioned free estriol.

[0025] This application provides a highly specific binding protein for unconjugated estriol (uE3) containing specific complementarity-determining regions (CDRs). This binding protein is prepared by immunization using a computer-aided design hapten. Mice are immunized after conjugation of the hapten with a carrier protein, and high-affinity, high-specificity anti-uE3 monoclonal antibodies are obtained through hybridoma technology screening. This specific binding protein can also be used to develop in vitro diagnostic reagents for unconjugated estriol, enabling accurate quantitative detection of uE3 in clinical blood samples, significantly improving the accuracy and reliability of uE3 detection, and providing a more reliable basis for prenatal screening and placental function assessment. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The hapten synthesis steps provided in one embodiment of this application;

[0028] Figure 2 The ultraviolet absorption spectra of the estriol hapten, carrier protein, and artificial antigen provided in an embodiment of this application are shown. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0030] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0032] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0033] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0034] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0035] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0036] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0037] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0038] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0039] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0040] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0041] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0042] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0043] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof) through at least one antigen recognition site located within the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins including antibodies, and any other modified immunoglobulin molecule, provided that the antibody exhibits the desired biological activity. Antibodies may be any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α, δ, ε, γ, and μ, respectively, based on the characteristic of their heavy chain constant structure. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies may be naked or conjugated to other molecules (such as toxins, radioisotopes, etc.).

[0044] The term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of a complete antibody that specifically binds to an antigen. An antigen-binding fragment may contain an antigen recognition site of the complete antibody (e.g., a CDR region (complementarity-determining region) sufficient to specifically bind to an antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

[0045] The terms "anti-free estriol antibody" and "antibody bound to free estriol" refer to antibodies that specifically bind to free estriol with sufficient affinity, enabling them to be used as diagnostic and / or therapeutic agents targeting free estriol. As used herein, the terms "specifically bind," "immunely specifically bind," "immunely specifically recognize," and "specifically recognize" are similar terms in the context of antibodies or their antigen-binding fragments. These terms indicate that the antibody or its antigen-binding fragment binds to the epitope via its antigen-binding domain, and that binding requires a degree of complementarity between the antigen-binding domain and the epitope.

[0046] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the amino terminus of the mature light chain, which differs in sequence between antibodies and is responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called CDRs, while more conserved regions within a variable domain are called FR regions (Framework regions). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region is a rodent or mouse variable region.

[0047] The term "heavy chain variable region" consists of 4 FR regions and 3 CDR regions. According to the IMGT database, the genes of the heavy chain variable region are defined as the V gene, D gene, and J gene.

[0048] The term "light chain variable region" consists of 4 FR regions and 3 CDR regions. According to the IMGT database, the genes of the light chain variable region are defined as the V gene and the J gene.

[0049] The term "heavy chain" generally includes one variable region and three constant regions (CH1 / CH2 / CH3).

[0050] The term "light chain" generally includes one variable region and one constant region (CL).

[0051] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen-binding fragments. In some respects, CDRs can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31 to 35 (which may optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically located at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In one specific embodiment, the CDRs of the antibody described herein have been determined according to the Kabat numbering scheme.

[0052] As used herein, the terms “constant region” and “constant domain” are interchangeable and have their common meanings in the art. A constant region is an antibody portion that does not directly participate in antibody-antigen binding but can exhibit a variety of effector functions, such as an antibody portion interacting with an Fc receptor, or, for example, the carboxyl-terminal portion of the light chain and / or heavy chain. Compared to the variable domains of immunoglobulins, the constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence. In some respects, antibody or antigen-binding fragments include constant regions or portions thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0053] As used herein, based on the amino acid sequence of constant structural domains, the term "heavy chain" in relation to antibody use can refer to any different type, for example, α, δ, ε, γ, and μ that produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4. The amino acid sequence of the heavy chain is well known in the art. In a specific embodiment, the heavy chain is a human heavy chain. In a specific embodiment, the heavy chain is a rodent or mouse heavy chain.

[0054] As used herein, based on the amino acid sequence of a constant domain, the term "light chain" can refer to any different type, such as κ or λ, in relation to antibody use. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain. In a specific embodiment, the light chain is a rodent or mouse light chain.

[0055] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequence is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies or antigen-binding fragments thereof derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions are homologous to sequences in antibodies or antigen-binding fragments thereof derived from another species (usually human) to avoid triggering an immune response in said species.

[0056] The term "human" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment thereof is prepared using any technique known in the art. This definition of human antibody or antigen-binding fragment thereof includes complete or full-length antibodies and fragments thereof.

[0057] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of molecule X for its partner Y can generally be represented by a dissociation constant (K). Affinity can be measured and / or represented in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant.

[0058] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (linear or continuous epitope), or an epitope can be, for example, derived from two or more discontinuous regions of one or more polypeptides (conformal, nonlinear, discontinuous, or non-continuous epitopes). In some embodiments, the epitope to which the antibody or its antigen-binding fragment specifically binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / atmosphere exchange in combination with mass spectrometry (e.g., liquid chromatography-electrospray ionization mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody / its antigen-binding fragment: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be improved using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymo1 (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt1): 37-60; Bricogne G (1997) Meth Enzymo276A: 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For descriptions of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al., (1995) JBiol Chem 270:1388-1394 and Cunningham BC and Wells JA (1989) Science 244:1081-108.

[0059] If an antibody binds to a given epitope or overlapping epitope of a reference antibody such that it partially blocks the binding of the reference antibody to the epitope, then it can be said that the antibody "competitively inhibits" the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. It can be said that the antibody competitively inhibits the binding of the reference antibody to the given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0060] In this application, the term "sample" refers to a human sample, but animal samples may also be used in the practice of this application. Non-limiting sources of samples used in this application include, for example, solid tissues, biopsies, ascites, aspirates, fluid leachates, blood (including circulating tumor cells), plasma, serum, cerebrospinal fluid, lymph, external sections of skin, respiratory tract, intestinal and genitourinary tract, tears, saliva, breast milk, tumors, organs, cell cultures, and / or cell culture components.

[0061] Terms such as “treatment” and “relief” refer to therapeutic measures that can cure, slow down, alleviate, reduce the symptoms of a pathological disease or condition and / or stop its progression. Therefore, those who require treatment include those who have been diagnosed with or are suspected of having the aforementioned condition.

[0062] This application uses computer-aided simulation to design a hapten-coupled protein to immunize mice, prepares a monoclonal antibody for detecting unconjugated estriol (uE3), and uses this paired antibody to develop an in vitro diagnostic kit for the quantitative detection of uE3 in clinical blood samples.

[0063] uE3 is a crucial "messenger" during pregnancy, and its unique synthetic mechanism makes it a highly sensitive indicator for assessing fetal health and placental function. This application uses computer-aided simulation to design and screen uE3 haptens, selecting the optimal hapten for immunizing Balb / c mice, and then utilizes hybridoma technology to prepare uE3 monoclonal antibodies.

[0064] This application provides a free estriol-specific binding protein, wherein the free estriol-specific binding protein has a free estriol-binding domain.

[0065] The free estriol binding domain has heavy chain CDR1 to heavy chain CDR3 as shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain CDR1 to light chain CDR3 as shown in SEQ ID NO.4 to SEQ ID NO.6.

[0066] The specific sequences are shown in Table 1:

[0067] Table 1

[0068]

[0069] The free estriol-specific binding protein of this application may have the aforementioned CDRs or derivative fragments having the aforementioned CDRs. The derivative fragments are formed by replacing amino acids at no more than six sites relative to their corresponding CDRs (“conservative modification” or “conservative substitution”), retaining the biological activity consistent with their corresponding complementarity-determining regions. For example, the derivative fragments may replace one amino acid with another, or one amino acid with multiple amino acids (e.g., two), at sites 1, 2, 3, 4, 5, or 6 of their corresponding complementarity-determining regions.

[0070] In the CDRs provided in this application, the derived fragments (conserved variants) refer to polypeptides formed by replacing one, two, or three amino acids with amino acids of similar or related properties compared to the amino acid sequence of the antibody in this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0071] Table A

[0072]

[0073] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will understand that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, the substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.

[0074] In some embodiments, the amino acid sequence of the heavy chain variable region of the free estriol binding domain is shown in SEQ ID NO.7.

[0075] The amino acid sequence of the variable region of the estriol antibody heavy chain (SEQ ID NO.7):

[0076] EVQLQQSGAELARPGASVKLSCKASGYIFSNYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKDKATLTADNSSSTAYMQLSTLASEDSAVYYCAKPYDHDEAWFAYWGQGTLVTVSA.

[0077] In some embodiments, the amino acid sequence of the light chain variable region of the free estriol binding domain is shown in SEQ ID NO.8.

[0078] The amino acid sequence of the variable region of the light chain of the estriol antibody (shown in SEQ ID NO.8):

[0079] DIVMSQSPSSLAVSVGEKVTMNCKSSQNLLFSSNQKNYLA WYQQKPGQSPKLLIYW

[0080] ASTRES GVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPWTTFGGGTKLEIK.

[0081] The specific binding protein for free estriol provided in this application can be an antibody, an antigen-binding fragment of an antibody, or a small modular immunodrug. Further, the binding protein can be a monoclonal antibody, an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, a ScFv fragment, a biclonal antibody, a multispecific antibody, a microantibody, a chelated recombinant antibody, an internal antibody, a nanobody, a binding domain immunoglobulin fusion protein, or a small modular immunodrug.

[0082] Alternatively, this application does not specifically limit the species origin of the heavy chain constant region and the light chain constant region, and may include, but is not limited to, any of the following species: human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck and goose.

[0083] This application does not specifically limit the types of heavy chain constant regions and light chain constant regions of the antibody, which may include, but are not limited to, at least one of IgG1, IgG4, IgA, IgM and IgE.

[0084] This application also provides a nucleic acid molecule containing a specific binding protein for encoding the aforementioned free estriol.

[0085] This application also provides a vector comprising the aforementioned nucleic acid molecule. This application does not specifically limit the type of recombinant expression vector, which may include, but is not limited to, antibody expression vectors. This application does not specifically limit the antibody expression vector, which may include, but is not limited to, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or combinations thereof.

[0086] This application also provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned vector. This application does not specifically limit the type of host cell, including but not limited to CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0087] This application also provides a method for preparing the cells described above, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

[0088] In one example, the import method uses transfection.

[0089] The term “transfection” refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chuetal, 1981, Gene 13:197.

[0090] This application also provides a method for preparing the specific binding protein of free estriol, the method comprising the following steps: culturing the cells, and isolating the specific binding protein of free estriol from the resulting culture.

[0091] In another aspect, this application provides a free estriol detection kit, the detection kit comprising a specific binding protein for the free estriol.

[0092] This application also provides a method for detecting free estriol in a sample to be tested. The method uses a specific binding protein of free estriol as a detection antibody and determines the presence of free estriol in the sample to be tested through an immunobinding reaction.

[0093] The term "immunobinding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen (for which the antibody is specific). The strength or affinity of the immunobinding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The properties of the immunobinding between two molecules can be quantified using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex.

[0094] This application relates to methods for immunodetection or determination of target antigens (e.g., free estriol), reagents for immunodetection or determination of target antigens (e.g., free estriol), methods for immunodetection or determination of cells expressing target antigens (e.g., free estriol), and diagnostic agents for diagnosing diseases associated with target antigen (e.g., free estriol) positive cells, comprising, as active ingredients, antibodies or antibody fragments that specifically recognize the target antigen (e.g., free estriol) and bind to the amino acid sequence or its three-dimensional structure in the extracellular region.

[0095] In this application, the method for detecting or determining the amount of a target antigen (e.g., free estriol) can be any known method. For example, it includes immunoassay or assay methods. Immunoassay or assay methods are methods that use labeled antigens or antibodies to detect or determine the amount of antibody or antigen. Examples of immunoassay or assay methods include radiolabeled antibody immunoassays (RIA), colloidal gold immunochromatography, enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0096] For example, a combination of Hapten C-OVA as the coating antigen and a complex of estriol antibody and colloidal gold as an immunoprobe can be used for the colloidal gold method of detecting free estriol in blood.

[0097] To detect cells expressing peptides, known immunoassay methods can be used, such as immunoprecipitation, fluorescent cell staining, and immunohistochemical staining. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0098] The application does not impose any particular restrictions on the test sample used to detect or determine the target antigen (e.g., free estriol), as long as it has the potential to include cells expressing the target antigen (e.g., free estriol), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0099] Depending on the required detection method, the detection product may also contain reagents for performing antigen-antibody reactions or for detecting the reaction. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragments, or conjugates, and substrates corresponding to the labeled antibodies.

[0100] This application also provides a medicament comprising the specific binding protein of the aforementioned free estriol.

[0101] This application also provides a treatment method comprising administering to a subject an effective dose of the specific binding protein of the free estriol or the drug.

[0102] The treatment methods described in this application may be for disease diagnosis and treatment purposes or for non-disease diagnosis and treatment purposes.

[0103] In this context, "application," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Application," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Application," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0104] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments of this application, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of the disease, inducing the regression of such symptoms or inhibiting their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Studentt test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0105] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this application or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of this disclosure in patients.

[0106] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0107] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0108] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0109] Example 1

[0110] This embodiment provides a method for preparing a specific binding protein of free estriol.

[0111] I. Computer-aided simulation of hapten design and hapten synthesis

[0112] The structures of free estriol and the designed hapten were constructed using Gauss View 6.0 software (Gaussian, Wallingford, CT, USA) to obtain the lowest energy conformations. Then, B3LYP density functional theory calculations and 6-31G(d) basis set density functional theory (DFT) calculations were performed using Gaussian 09 software (Gaussian, Wallingford, CT, USA) to obtain the lowest energy conformations of all molecules. The output files from the Gaussian calculations were imported into Multiwfn 3.7 (dev) software, and the structural parameters of acetaminophen and its designed hapten were extracted. The hydrophobic constant (Log P) and molecular weight (MW) of the molecular structures were extracted using Chemdraw software (PerkinElmer, Waltham, MA, USA). The specific surface area (SA) and molecular polarity index (MPI) of the molecular structure were extracted using VMD (Visual Molecular Dynamics) software and Multiwfn code. As shown in Figure 1, the physicochemical properties of Hapten B are quite similar to those of estriol, making it the preferred immunogen for immunization, which will be verified by subsequent immunization results. The physicochemical properties and structural parameters of free estriol and the hapten molecule are shown in Table 1.

[0113] Table 1

[0114]

[0115] Taking hapten A as an example, 4.8 mmol / L (1.4 g) of estriol was added to a 100 mL round-bottom flask, along with 20 mL of DMSO (anhydrous dimethyl sulfoxide). The mixture was stirred at room temperature until the starting material dissolved, and then 12.5 mmol / L (0.7 g) of KOH was added. 9.6 mmol / L (1.3 g) of bromoacetic acid was added, and the mixture was reacted at room temperature for 2 h. 50 mL of ice water was added, and unreacted estriol was extracted with ethyl acetate (repeated three times). The pH of the aqueous phase was adjusted to approximately 3.0 with 2 mol / L HCl, resulting in a large amount of white precipitate. The reaction was monitored by thin-layer chromatography on a silica gel plate (developing solvent: methanol:chloroform = 1:5, estriol Rf = 0.7, product Rf = 0.1). The aqueous phase was filtered, and the residue was washed three times with acidic ice water to obtain a white solid powder. TLC revealed the presence of a small amount of raw material. The sample was passed through a 200-300 mesh silica gel column with a methanol:chloroform ratio of 1:10 as the developing solvent. The synthetic routes for hapten B and hapten C were the same as for hapten A, with the reaction arms replaced by bromobutyric acid and bromohexanoic acid, respectively. Subsequently, the synthesized hapten and coating antigen were directly coupled to BSA OVA.

[0116] II. Immunogen Preparation

[0117] Substances with a molecular weight below 5 kDa lack immunogenicity and cannot elicit an immune response. Therefore, in this embodiment, the prepared hapten needs to be coupled with a carrier protein to stimulate an immune response. The artificial antigen is prepared by coupling the carboxyl-containing hapten with the carrier protein using the active ester method. Taking the Hapten A-BSA artificial antigen as an example, the specific operation is as follows:

[0118] (1) Accurately weigh 2 mg (0.01 mmol) Hapten A, 1.6 mg NHS (0.015 mmol) and 5.2 mg EDC (0.015 mmol) and dissolve them in 300 µL DMF. Stir at room temperature in the dark for 4 h to obtain Hapten A activation solution. Accurately weigh 10 mg BSA and add it to 1 mL PBS buffer (0.01 mol / L, pH 7.4) to obtain protein solution.

[0119] (2) Add Hapten A activation solution slowly dropwise to the protein solution and stir at 4°C in the dark for 12 h.

[0120] (3) After the reaction is complete, transfer the product to a dialysis bag and dialyze it with PBS buffer (0.01 mol / L, pH 7.4) for 3 days, changing the buffer 3 times a day;

[0121] (4) After dialysis, collect the solution and make up to 2 mL to obtain 5 mg / mL artificial antigen (Hapten A-BSA), which is stored at -20℃ for later use.

[0122] Artificial antigens Hapten A-OVA, Hapten B-BSA, Hapten B-OVA, Hapten C-BSA, and Hapten C-OVA were also prepared according to the above steps.

[0123] III. Mouse Immunization and Hybridoma Cell Screening

[0124] Balb / c mice aged 8-12 weeks were immunized with the prepared Hapten A / B / C-BSA. Mice tail blood coated with Hapten A / B / C-OVA was then detected by ELISA until the titer reached 10. 5 Immunization was stopped, and mouse spleens were removed. After processing, the spleens were fused with mouse myeloma cells Sp2 / 0, and positive hybridoma cell lines were screened using a limiting dilution method. Positive hybridomas were injected into the abdomen of mice to prepare ascites. The results of free estriol-containing mouse antiserum activity assays are shown in Table 2. The combination of Hapten B-BSA as the immunogen and Hapten C-OVA as the coating agent showed the best effect. The hybridoma supernatant activity was detected using indirect ELISA ("+" indicates that the OD value exceeds the measurement range of the microplate reader). The results are shown in Table 3. The E3-4 cell line was selected for subsequent monoclonal screening.

[0125] Table 2

[0126]

[0127] Table 3

[0128]

[0129] Example 2: uE3 antibody colloidal gold method for detecting clinical blood samples

[0130] 1. Preparation of NC membranes coated with Hapten C-OVA antigen:

[0131] The coating antigen was prepared into a 1.0 mg / mL NC coating solution using NC membrane coating diluent. The solution was then coated onto a pre-attached NC membrane PVC plate using a membrane coating machine, and finally dried in a 50°C drying oven for 24 hours.

[0132] 2. Preparation of colloidal gold:

[0133] This application uses the trisodium citrate reduction method to prepare colloidal gold nanoparticles. The main steps are as follows:

[0134] (1) Cleaning: In order to ensure the quality of the prepared gold nanoparticles, the glassware and rotors to be used are soaked in aqua regia in advance to remove metal ions and other impurities, then rinsed with ultrapure water and dried for later use.

[0135] (2) Preparation of solution: First, measure 192 mL of ultrapure water and 8 mL of 1% HAuCl4 into an Erlenmeyer flask to prepare a 0.04% (w / w) HAuCl4 aqueous solution.

[0136] (3) Burning gold: Heat the solution to reflux to boiling, and quickly add 10 mL of 1% trisodium citrate solution prepared in advance while stirring. The color of the solution can be observed to change from light yellow to transparent, and after a few seconds it changes from transparent to black. Then a bright wine red color appears. Continue to heat and stir at a constant speed for 10 min, then remove it, stop heating and cool to room temperature, and store it in a 4℃ refrigerator for later use.

[0137] 3. Preparation of colloidal gold immunoprobes:

[0138] The gold-labeled probes were prepared using an electrostatic adsorption method. The specific preparation steps are as follows:

[0139] (1) pH adjustment: Rinse the 1.5 mL centrifuge tube three times with ultrapure water, add 1 mL of colloidal gold solution to the tube, adjust the pH value of the solution with a certain amount of 0.2 mol / L K2CO3, and shake to mix evenly.

[0140] (2) Antibody conjugation: Take an appropriate amount of estriol antibody and dilute it to a certain concentration with a suitable antibody diluent. Add a certain amount of the diluted antibody solution to the pH-adjusted colloidal gold solution, shake and mix evenly, and let it stand at room temperature for 30 minutes.

[0141] (3) Blocking: After the above reaction is completed, add 50 μL of 10% BSA solution and place at room temperature for 30 min to block the sites on the colloidal gold that are not bound to antibodies.

[0142] (4) Centrifugation: After sealing, place it in a high-speed centrifuge and centrifuge at 10,000 r / min for 20 min at 4℃, then discard the supernatant.

[0143] (5) Redissolution: The precipitate after centrifugation is redissolved in 200 μL of redissolution solution, and the precipitate is sonicated to be evenly dispersed in the redissolution solution. It is then stored at 4℃ for later use.

[0144] 4. Blood sample processing:

[0145] First, the blood sample is centrifuged to obtain serum; then, it is diluted with PBS at a ratio of 1:9 mL pH 7.4 before testing.

[0146] 5. Clinically positive serum samples were collected and tested using the colloidal gold method. The results of visual observation of positive and negative results are shown in Table 4. The conclusion is that the test results for clinically positive serum samples were positive, while the test results for normal clinical serum samples were negative. This indicates that the developed test strip can be used to detect unconjugated estriol in clinical serum samples and provides a certain basis for clinical diagnosis.

[0147] Table 4: Results of Colloidal Gold Method for Detecting Tissue Samples

[0148]

[0149] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A specific binding protein for free estriol, characterized in that, The free estriol-specific binding protein has a free estriol-binding domain; The free estriol binding domain has heavy chain CDR1 to heavy chain CDR3 as shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain CDR1 to light chain CDR3 as shown in SEQ ID NO.4 to SEQ ID NO.

6.

2. The specific binding protein for free estriol according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the free estriol-binding domain is shown in SEQ ID NO. 7; and / or, The amino acid sequence of the light chain variable region of the free estriol binding domain is shown in SEQ ID NO.

8.

3. The specific binding protein for free estriol according to any one of claims 1 to 2, characterized in that, The specific binding protein for free estriol also includes a heavy chain constant region and a light chain constant region.

4. The specific binding protein for free estriol according to claim 3, characterized in that, The specific binding protein of the free estriol satisfies one or more of the conditions shown in (1) and (2) below: (1) The specific binding protein of the free estriol has a sequence having a constant region of any one of IgG, IgA, IgM, IgE, and IgD; and, (2) The species sources of the heavy chain constant region and the light chain constant region are each independently selected from any one of the following: human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck and goose.

5. A nucleic acid molecule, characterized in that, Encodes a specific binding protein for free estriol as described in any one of claims 1 to 4.

6. A carrier, characterized in that, Includes the nucleic acid molecule as described in claim 5.

7. A cell, characterized in that, It includes the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A method for preparing the cell according to claim 7, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

9. A method for preparing the specific binding protein of free estriol according to any one of claims 1 to 4, characterized in that, The preparation method includes the following steps: Cultivating the cells according to claim 7; and The specific binding protein of free estriol was isolated from the resulting culture.

10. A free estriol detection kit, characterized in that, The test kit includes a specific binding protein for free estriol as described in any one of claims 1 to 4.

11. A method for detecting free estriol in a sample, characterized in that, The detection method uses the specific binding protein of free estriol as described in any one of claims 1 to 4 as the detection antibody, and determines the presence of free estriol in the sample to be tested through an immunobinding reaction.

12. A drug, characterized in that, The drug comprises a specific binding protein of free estriol as described in any one of claims 1 to 4.

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