MHBs-specific binding proteins, nucleic acid molecules, vectors, cells, detection kits, their preparation methods and applications

Abstract

This application belongs to the field of immunoassay technology, specifically relating to specific binding proteins of hepatitis B viruses (MHBs), nucleic acid molecules, vectors, cells, detection kits, and their preparation methods and applications. This application provides a specific binding protein of MHBs with specific CDRs, exhibiting high affinity and specificity. It solves the problem in existing technologies of being unable to distinguish between large, medium, and small hepatitis B virus proteins, and ensures that the antibody does not cross-react with large and small proteins, thus improving detection specificity and avoiding false positive results. The specific binding protein of MHBs in this application can bind to a chemiluminescent detection system, thereby achieving highly sensitive detection of MHB antigen levels in clinical samples, with high consistency with nucleic acid detection results, providing an important tool for monitoring hepatitis B infection status and disease progression.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunoassay, and particularly relates to a specific binding protein of MHBs, a nucleic acid molecule, a vector, a cell, a detection kit and a preparation method and application thereof. BACKGROUND

[0002] Hepatitis B virus surface antigen (HBsAg) is composed of small protein (S protein), medium protein (MHBs) and large protein (MHBs). Among them, the medium protein (MHBs) is encoded by Pre-S2 and S genes, and the N terminal contains the Pre-S2 domain (containing the essential N4 N-glycosylation site), and the C terminal is the S antigen main protein. Pre-S2 mediates the attachment and entry of the virus to the liver cells; MHBs deletion / variation indicates active replication of the virus. MHBs quantification has become a new biomarker for identifying asymptomatic carriers and predicting functional cure, which is superior to the total HBsAg concentration; its specific detection is of great significance for the stratified diagnosis of hepatitis B and the evaluation of immunotherapy.

[0003] The structures of hepatitis B virus large protein, medium protein and small protein are different and the antigen epitopes are diverse, which are easy to interfere with each other in detection, resulting in insufficient sensitivity and specificity of traditional detection methods such as ELISA. The existing HBSAg chemiluminescence detection reagent cannot distinguish the contents of hepatitis B virus large protein, medium protein and small protein. Thus, there is a lack of indicators for predicting functional cure and observing curative effect of hepatitis B in clinic. The presence of medium protein is a marker of active virus replication, which is closely related to the infectivity of HBV, disease progression and immune response state.

[0004] Therefore, it is of great significance to develop a specific binding antibody with high affinity and high specificity for hepatitis B virus outer membrane medium protein and establish a corresponding chemiluminescence detection method for improving the accuracy of hepatitis B diagnosis, predicting the treatment effect and evaluating the disease progression. SUMMARY

[0005] Based on this, an embodiment of the present application provides a specific binding protein of MHBs, a nucleic acid molecule, a vector, a cell, a detection kit and a preparation method and application thereof.

[0006] In one aspect, the present application provides a specific binding protein of MHBs, wherein the specific binding protein of MHBs has a MHBs binding domain.

[0007] The MHBs binding domain has heavy chain CDR1 to heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO. 1 to SEQ ID NO. 3, and light chain CDR1 to light chain CDR3 with an amino acid sequence as shown in SEQ ID NO. 4 to SEQ ID NO. 6.

[0008] In some embodiments, the amino acid sequence of the heavy chain variable region of the MHBs binding domain is shown in SEQ ID NO. 7.

[0009] In some embodiments, the amino acid sequence of the light chain variable region of the MHBs binding domain is shown in SEQ ID NO. 8.

[0010] In some embodiments, the specific binding proteins of the MHBs also include heavy chain constant regions and light chain constant regions.

[0011] In some embodiments, the specific binding protein of the MHBs has a sequence of any one of the constant regions of IgG1, IgG4, IgA, IgM, and IgE; and,

[0012] In some embodiments, the heavy chain constant region and the light chain constant region are independently selected from any one of the following species: human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck and goose.

[0013] This application also provides a nucleic acid molecule that encodes a specific binding protein of the aforementioned MHBs.

[0014] This application also provides a carrier comprising the aforementioned nucleic acid molecule.

[0015] This application also provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned vector.

[0016] 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.

[0017] This application also provides a method for preparing the specific binding protein of MHBs, the method comprising the following steps:

[0018] The cells were cultured, and the specific binding proteins of MHBs were isolated from the resulting cultures.

[0019] This application also provides an MHBs detection kit, the detection kit comprising the specific binding protein of the MHBs.

[0020] This application also provides a method for detecting MHBs in a sample to be tested, wherein the method uses the specific binding protein of MHBs as the detection antibody and determines the presence of MHBs in the sample to be tested through an immune binding reaction.

[0021] This application also provides a medicament comprising a specific binding protein of the aforementioned MHBs.

[0022] This application provides a specific binding protein for hepatitis B viruses (MHBs) with specific cross-reactivity densities (CDRs), exhibiting high affinity and specificity. This solves the problem of traditional techniques being unable to distinguish between large, medium, and small hepatitis B virus proteins, and ensures that the antibody does not cross-react with the S protein, thus improving detection specificity and avoiding false positive results. The MHB-specific binding protein of this application can bind to a chemiluminescent detection system, thereby achieving highly sensitive detection of MHB antigen levels in clinical samples. The results show high consistency with nucleic acid detection, providing an important tool for monitoring hepatitis B infection status and disease progression. Attached Figure Description

[0023] 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.

[0024] Figure 1 The results of MHBsAg-Ab purification;

[0025] Figure 2 The results are for the MHBsAg-Ab antibody affinity assay. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0029] 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").

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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℃.

[0038] 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.

[0039] 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. Examples of relevant technical features cited in this application 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.

[0040] The term "MHBs" refers to proteins on the surface of the hepatitis B virus. MHBs are envelope proteins of the hepatitis B virus and are components of both the hepatitis B virus particle (Dane particle) and subviral particles (tubular and spherical particles). It is a medium-molecular-weight protein in the hepatitis B surface antigen (HBsAg). It includes the complete sequences of the PreS2 and S regions. In contrast, larger proteins include the PreS1, PreS2, and S regions, while smaller proteins only include the S region.

[0041] 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.).

[0042] 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.

[0043] The terms "anti-MHB antibody" and "antibody bound to MHBs" refer to antibodies that specifically bind to MHBs with sufficient affinity, enabling them to be used as diagnostic and / or therapeutic agents targeting MHBs. 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.

[0044] 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 mature light chain, which differ in sequence between antibodies and are 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.

[0045] 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.

[0046] 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.

[0047] The term "heavy chain" generally includes one variable region and three constant regions (CH1 / CH2 / CH3).

[0048] The term "light chain" generally includes one variable region and one constant region (CL).

[0049] 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, the CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31 to 35 (optionally including 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.

[0050] 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).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] "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.

[0056] 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.

[0057] 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%.

[0058] 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.

[0059] The term "chemiluminescence" refers to the phenomenon where an excited-state intermediate is generated during a chemical reaction, and when it returns to its ground state, it releases photons. In immunoassay, chemiluminescent substances are labeled onto antibodies. After the formation of immune complexes, a luminescent substrate is added, and a chemical reaction generates a light signal. The intensity of the signal is proportional to the concentration of the analyte antigen. In this application, chemiluminescence is a technological platform for achieving high-sensitivity detection. Its function is to amplify minute antigen-antibody binding events and convert them into a precisely measurable light signal, thereby enabling the detection of trace amounts of MHBs in clinical samples. The sub-concept of the chemiluminescent substance used in this application is acridine ester, which is a highly efficient chemiluminescent label with advantages such as high luminescence efficiency, low background signal, and rapid reaction. Combined with high-affinity antibodies, it forms the technical basis for the excellent sensitivity of the reagent kit in this application.

[0060] The term “label” as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody in order to produce a “labeled” antibody. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzyme labeling, may catalyze a chemical change in a detectable substrate compound or composition.

[0061] Terms such as “treatment,” “curative,” “treatment,” “relief,” and “relief” refer to therapeutic measures that can cure, slow down, alleviate, and / or stop the progression of a pathological disease or condition. Therefore, those requiring treatment include those already diagnosed with or suspected of having the stated condition. In some embodiments, a subject’s cancer is successfully diagnosed according to the method of this application if the patient exhibits one or more of the following conditions.

[0062] To address the shortcomings in sensitivity and specificity of hepatitis B protein detection in existing technologies, this application provides a method for preparing an antibody against hepatitis B proteins (MHBs) and its application in chemiluminescence detection.

[0063] Based on this, this application provides a specific binding protein for MHBs, wherein the specific binding protein for MHBs has an MHBs binding domain.

[0064] The specific binding protein for hepatitis B virus (HBV) provided in this application exhibits extremely high specificity and sensitivity, specifically binding to recombinant HBV antigens without cross-reactivity with large or small proteins. This solves the problem in existing technologies that cannot distinguish between large, medium, and small HBV proteins. Biacore testing shows that the monoclonal antibody provided in this application has high affinity, with a dissociation constant KD ≤ 10. -10 M is significantly superior to antibodies in existing technologies.

[0065] The MHBs 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.5.

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

[0067] Table 1

[0068]

[0069] The specific binding protein for MHBs 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 2.

[0071] Table 2

[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] The specific binding protein for MHBs 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.

[0075] 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.

[0076] 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, IgG1, IgG4, IgA, IgM and IgE.

[0077] This application also provides a nucleic acid molecule containing a specific binding protein for encoding the aforementioned MHBs.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] In one example, the import method uses transfection.

[0082] 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.

[0083] This application also provides a method for preparing the specific binding protein of MHBs, the method comprising the following steps: culturing the cells and isolating the specific binding protein of MHBs from the resulting culture.

[0084] Specifically, the method includes the following steps: Step 1: Constructing a recombinant antigen containing specific epitopes of hepatitis B virus (MHB) proteins; Step 2: Immunizing animals with the recombinant antigen to obtain immune spleen cells; Step 3: Fusing the immune spleen cells with myeloma cells to obtain hybridoma cells; Step 4: Screening for hybridoma cell lines that specifically bind to MHBs but not to large or small proteins; Step 5: Isolating and purifying the monoclonal antibody or its antigen-binding fragment from the hybridoma cell line. The preparation method provided in this application, through specific recombinant antigen immunization and rigorous screening steps, successfully obtains monoclonal antibodies with high affinity and high specificity, solving the problems of insufficient antibody specificity and affinity in existing technologies.

[0085] In some embodiments, the recombinant antigen in step 1 comprises all or part of the amino acid sequence of the PreS2 region and all or part of the amino acid sequence of the S region, and carries a purification tag. This recombinant antigen design incorporates specific epitopes of MHBs, enabling the induction of specific antibodies. Specifically, the PreS2 region may be selected from the amino acid sequence aa 121-174, the S region may be selected from the amino acid sequence aa 175-449, and the purification tag may be selected from any one of a 6×His tag, a GST tag, or an MBP tag.

[0086] This application presents a design that fuses the PreS2 region, S region, and purified tag for expression, simultaneously encompassing both neutralizing antibody targets (S region) and highly immunogenic targets (PreS2 region) against hepatitis B virus. The PreS2 region contains important viral surface epitopes that can induce the body to produce antibodies against the PreS2 region. These antibodies synergistically neutralize antibodies against the S region, more effectively preventing viral invasion of hepatocytes. Furthermore, it better mimics the natural antigen structure: in the viral middle proteins (MHBs), the PreS2 region is naturally located at the N-terminus of the S region. This design (PreS2-S) faithfully reproduces the spatial conformation of proteins in the natural virus, facilitating the induction of the correct antibodies that recognize the natural virus.

[0087] In some embodiments, the animal in step 2 is a BALB / c mouse, immunized by subcutaneous injection three times, and Freund's adjuvant is used to enhance the immunization effect. BALB / c mice are a commonly used animal model for preparing monoclonal antibodies, subcutaneous injection is an effective immunization method, and Freund's adjuvant can enhance the immunization effect. Specifically, the immunization dose can be selected from any value among 50 μg, 100 μg, 150 μg, and 200 μg; the immunization interval can be selected from any value among 2 weeks, 3 weeks, and 4 weeks.

[0088] In some embodiments, the myeloma cells in step 3 are SP2 / 0 cells, which are fused and then screened for hybridoma cells using HAT medium. SP2 / 0 cells are a commonly used myeloma cell line, and hybridoma cells can be generated by fusing them with spleen cells. HAT medium can screen for successful hybridoma cells. Specifically, the fusion ratio of spleen cells to myeloma cells can be selected from any value among 5:1, 10:1, and 15:1; the HAT screening time can be selected from any value among 7 days, 10 days, and 14 days.

[0089] In some embodiments, the screening methods in step 4 include ELISA, Western blot, and immunofluorescence. These screening methods can verify the specificity and affinity of antibodies from multiple perspectives, ensuring the acquisition of high-quality antibodies. Specifically, ELISA screening can detect the binding ability of antibodies to antigens, Western blot can verify the specificity of antibodies, and immunofluorescence can confirm the binding of antibodies to natural antigens.

[0090] This application does not specifically limit the type of detection product, such as diagnostic reagents, test strips, test plates, or kits. In the detection product, the specific binding protein of MHBs competitively binds to the MHBs in the analyte.

[0091] In some embodiments, the MHBs detection kit provided in this application includes the specific binding protein of the above-mentioned MHBs and a goat anti-human HBsAg antibody labeled with acridinium ester.

[0092] The test kit provided in this application has high sensitivity and high specificity, and can be used to detect the level of MHBs antigen in clinical samples. It has a high degree of consistency with the results of nucleic acid testing and can accurately reflect the hepatitis B infection status and disease progression.

[0093] In some embodiments, the antibody purification method may be selected from any one or more combinations of Protein A affinity chromatography, Protein G affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and molecular sieves.

[0094] In some embodiments, the chemiluminescent substance may be selected from any one of acridine ester, luminol, isoluminol, alkaline phosphatase, and horseradish peroxidase.

[0095] In some embodiments, the antibody solid-phase support may be selected from any one of magnetic beads, microplates, colloidal gold, and latex microspheres.

[0096] Specifically, the monoclonal antibody or its antigen-binding fragment can be conjugated to any one of magnetic beads, microplates, or colloidal gold; the labeling concentration of the acridinium ester can be selected from any value among 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL.

[0097] This application also provides a method for detecting MHBs in a sample to be tested, wherein the method uses the specific binding protein of MHBs as the detection antibody and determines the presence of MHBs in the sample to be tested through an immune binding reaction.

[0098] The detection method described in this application can be a method for diagnostic purposes or a method for non-diagnostic purposes.

[0099] 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.

[0100] This application relates to methods for immunodetection or determination of target antigens (e.g., MHBs), reagents for immunodetection or determination of target antigens (e.g., MHBs), methods for immunodetection or determination of cells expressing target antigens (e.g., MHBs), and diagnostic agents for diagnosing diseases associated with target antigen (e.g., MHBs) positive cells, comprising, as active ingredients, antibodies or antibody fragments that specifically recognize target antigens (e.g., MHBs) and bind to the amino acid sequence or three-dimensional structure of the extracellular region thereof.

[0101] In this application, the method for detecting or determining the amount of a target antigen (e.g., MHBs) 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), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc.

[0102] 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.

[0103] The application does not impose any particular restrictions on the test sample used to detect or determine the target antigen (e.g., MHBs), as long as it has the potential to include cells expressing the target antigen (e.g., MHBs), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0104] 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.

[0105] This application also provides a medicament comprising a specific binding protein of the aforementioned MHBs.

[0106] This application also provides a treatment method comprising administering to a subject an effective dose of the specific binding protein of the MHBs or the drug.

[0107] 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.

[0108] "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.

[0109] "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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] Example 1

[0114] This embodiment provides a monoclonal antibody that specifically binds to proteins (MHBs) in hepatitis B virus and its preparation method.

[0115] Raw materials for the preparation of MHBsAg-Ab monoclonal antibody:

[0116] Recombinant antigen MHBsAg was constructed by splicing the MHBs additional region (aa 121–174, PreS2) to the S region (aa 175–449) and fusing a 6×His tag sequence at the C-terminus as shown in SEQ ID NO.9. It was expressed in HEK293F cells and purified by Ni-NTA affinity chromatography with a purity of >95%.

[0117] The complete amino acid sequence of the MHBsAg (PreS2–S+ C-terminal His6) recombinant antigen is as follows:

[0118] >MHBsAg, (PreS2-S+C end His6) SEQ ID NO.9:

[0119] MQWNSTTFHQALLDPKVRGLYFPAGGSSSGTVNPVPTTASISSIFSRTGDPAPNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPSQHQSPSNLSPFHQPNLLDKTWMKFQIGLASLQVGYQFQTLKQDLKGLYQMLWACRCPTPIRPTPNRP ISPIPQRSIPLHQVNLTRFQNLPISSYSYLQAPIRPGQPYSHLQRSGDSYGTHLYLVQTGLVQSHLPKSLMHQLQSLRSPLPSTPLPPSRNLLSLLYPLLNQASLSKLQAMLSYTRFLGLDHYSFYASWMVQTLHQKDMMQQLGFHSQVNLKRQKRKRLKHHHHHH*

[0120] BALB / c mice: 6-8 week old females, purchased from Beijing Vital River Co., Ltd.

[0121] Freund's complete adjuvant: purchased from Sigma, F5881.

[0122] Freund's incomplete adjuvant: purchased from Sigma, F5506.

[0123] SP2 / 0 myeloma cells: purchased from the Wuhan University Preservation Center.

[0124] Hybridoma-SFM medium: purchased from Gbico.

[0125] HAT medium supplement (50x) and HT medium: purchased from Gbico.

[0126] HT culture medium supplement (50x): purchased from Gbico.

[0127] Fetal bovine serum: purchased from Hyclone.

[0128] Protein A affinity chromatography column: purchased from Cytiva, HiTrap Protein A HP.

[0129] Ion exchange chromatography column: purchased from Cytiva, HiTrap DEAE Sepharose FF.

[0130] Preparation method of MHBsAg-Ab monoclonal antibody:

[0131] Recombinant antigen expression and purification: The MHBsAg gene shown in SEQ ID NO.9 was constructed into the pcDNA3.1 expression vector, transfected into HEK293F cells, and the cell supernatant was collected after 5 days of culture. The recombinant MHBsAg antigen was purified by Ni-NTA affinity chromatography, and the purity was >95% as detected by SDS-PAGE.

[0132] Mouse immunization: 100 μg of recombinant MHBsAg antigen was emulsified with an equal volume of Freund's complete adjuvant and subcutaneously injected into BALB / c mice. Two weeks later, 100 μg of recombinant MHBsAg antigen was emulsified with an equal volume of Freund's incomplete adjuvant for a second immunization. Two weeks after that, a third immunization was performed using the same method. Ten days after the third immunization, blood was collected from the tail vein, and serum titers were detected by ELISA. A titer ≥10 was required. 5 Spleen cells were then collected.

[0133] Hybridoma cell preparation: Spleens of immunized mice were collected and spleen cell suspensions were prepared; spleen cells were mixed with SP2 / 0 myeloma cells at a ratio of 10:1 and fused under the action of PEG; after fusion, cells were screened with HAT medium, and the medium was changed every 3 days. After 10 days, the growth of hybridoma clones was observed.

[0134] Hybridoma cell screening: Positive clones were screened using ELISA: Recombinant MHBsAg antigen was coated onto an ELISA plate, hybridoma cell supernatant was added, HRP-labeled goat anti-mouse IgG was used as the secondary antibody, and TMB was used as the substrate to screen for positive clones; simultaneously, recombinant SHBsAg antigen was coated onto the ELISA plate to exclude clones that cross-reacted with SHBsAg; the specificity of positive clones was further verified by Western blot and immunofluorescence.

[0135] Monoclonal antibody purification: The selected positive hybridoma cells were expanded and cultured, and the cell supernatant was collected. The supernatant was initially purified by Protein A affinity chromatography and then further purified by ion exchange chromatography to obtain purified MHBsAg-Ab monoclonal antibody. The purity was >95% as detected by SDS-PAGE.

[0136] Antibody sequencing: Total RNA was extracted from hybridoma cells, and the antibody variable region gene was amplified by RACE technology. The heavy and light chain variable region sequences were obtained by sequencing.

[0137] SEQ ID NO.7: MHBsAg-Ab murine monoclonal antibody heavy chain sequence

[0138] >MHBsAg-Ab-H mouse IgG1

[0139] QIQLVQSGPELKKPGETVKISCKASGYTFTNYSMHWVKQAPGKGLKWVGWINTNTGEPIYADEFKGRFAFSLETSARTAYLQINNLKNEDTATYFCARSILRIGPMDYWGQ GTSVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTIS KTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0140] The sequences of the variable region and constant region of the heavy chain are shown in Table 3 below:

[0141] Table 3

[0142]

[0143] SEQ ID NO.8: MHBsAg-Ab mouse monoclonal antibody light chain sequence

[0144] >MHBsAg-Ab-L KAPPA

[0145] DIVLTQSPASLAVSLGQRATISCRASKSVSASAYSYMHWYQQKPGQPPKVLIYLASNLDSGVPARFSGSGSGTDFTLNIHPVQEEDAATYYCQQSNELPPTFGGGTKLE IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0146] The sequences of the variable region and constant region of the light chain are shown in Table 4 below:

[0147] Table 4

[0148]

[0149] Example 2

[0150] This embodiment provides a method for detecting the affinity of MHBsAg-Ab monoclonal antibody.

[0151] Raw materials for MHBsAg-Ab monoclonal antibody affinity assay: recombinant MHBsAg antigen: same as in Example 1.

[0152] MHBsAg-Ab monoclonal antibody: Same as in Example 1.

[0153] CM5 chip: purchased from Cytiva, BR100012.

[0154] 10×HBS-EP+ buffer: purchased from Cytiva, BR100669.

[0155] Amino coupling kit: purchased from Cytiva, BR100050.

[0156] Ethanolamine: purchased from Merck.

[0157] Biacore T200 instrument: purchased from Ctyvia. Method for MHBsAg-Ab monoclonal antibody affinity detection:

[0158] Chip preparation: A CM5 chip was selected, and the carboxyl functional groups on the chip surface were pre-activated using an amino-coupled method. The recombinant MHBsAg antigen (ligand) was diluted to 10 μg / mL with sodium acetate buffer (pH 5.0) and injected onto the chip surface. The reaction time was approximately 10 min, resulting in an antigen immobilization volume of approximately 1000 RU. Non-specific binding sites were blocked with 1 M ethanolamine (pH 8.5) to prevent non-specific binding.

[0159] Sample preparation: The MHBsAg-Ab monoclonal antibody was diluted with HBS-EP buffer to different concentration gradients: 3.125 nM, 1.5625 nM, 0.781 nM, 0.391 nM, and 0.195 nM.

[0160] Experimental method setup: The experimental template was set using Biacore T200 software, and the "kinetics / affinity" detection mode was selected. Injection parameters were set as follows: binding time 120 seconds, flow rate 30 μL / min, dissociation time 300 seconds, and regeneration conditions of 10 mM glycine-HCl (pH 1.5). Multiple concentrations of samples were repeatedly injected, and multiple cycles of detection were performed.

[0161] Detection process: At the start of operation, samples sequentially enter the chip flow path and bind to the immobilized antigen. The binding and dissociation processes are monitored in real time, and the change curves of the sensing response unit (RU) are acquired. Non-specific binding signals are subtracted through the reference channel to ensure data accuracy.

[0162] Data Analysis: Using Biacore Evaluation software, a 1:1 Langmuir binding model was selected to fit the curves. The binding rate constant (ka), dissociation rate constant (kd), and affinity constant (KD) were calculated. The MHBsAg-Ab antibody affinity detection results are as follows: Figure 2 As shown in Table 5, the test data results are as follows:

[0163] Table 5: Results of MHBsAg-Ab antibody affinity assay

[0164]

[0165] Example 3

[0166] This embodiment provides a detection kit and its preparation method.

[0167] Preparation method of the test kit:

[0168] Antibody magnetic bead preparation: Carboxyl magnetic beads were washed three times with MES buffer (pH 5.0), and EDC and NHS were added to activate the carboxyl groups on the surface of the magnetic beads. The reaction was carried out at room temperature for 30 min. After washing, MHBsAg-Ab monoclonal antibody was added and the reaction was carried out at room temperature for 2 hours. The beads were blocked with PBS buffer containing 1% BSA, washed, and stored in PBS buffer containing 0.1% BSA and 0.02% NaN3 at 4°C.

[0169] Acridinium ester labeled antibody: Goat anti-human HBsAg antibody was dialyzed with carbonate buffer (pH 9.5), acridinium ester was added (antibody:cridinium ester molar ratio = 1:10), and the reaction was carried out at room temperature for 2 hours; free acridinium ester was removed using a PD-10 desalting column, the labeled antibody was collected, and stored at 4°C in the dark.

[0170] Preparation of chemiluminescent substrates: Chemiluminescent substrate A (0.1% H2O2) and chemiluminescent substrate B (0.1 mol / L NaOH) are mixed in a 1:1 volume ratio and prepared fresh for use.

[0171] Kit assembly: Assemble the prepared antibody magnetic beads, acrid ester labeled antibody, chemiluminescent substrate, washing buffer (PBS buffer containing 0.05% Tween-20) and calibrators (serial concentrations of recombinant MHBsAg antigen) into a kit.

[0172] Example 4:

[0173] This embodiment provides a method for testing clinical samples using a test kit.

[0174] Clinical serum samples: collected from the hospital laboratory, including HBsAg positive and HBsAg negative samples.

[0175] Fully automated chemiluminescence analyzer: YHLO iflash3000.

[0176] Clinical sample testing method: Sample processing: Take 50 μL of clinical serum sample, add 50 μL of sample diluent, mix well and set aside.

[0177] Testing steps:

[0178] (a) Take 50 μL of the treated sample, add 50 μL of antibody magnetic beads, and incubate at 37°C for 30 min;

[0179] (b) Wash three times to remove unbound substances;

[0180] (c) Add 50 μL of acridinium ester-labeled antibody and incubate at 37°C for 30 min;

[0181] (d) Wash three times to remove unbound substances;

[0182] (e) Add 100 μL of chemiluminescent substrate and immediately read the relative luminescence intensity (RLU) on the chemiluminescence analyzer.

[0183] Result calculation: The concentration of MHBsAg in the sample was calculated based on the calibration curve.

[0184] Test results:

[0185] One hundred clinical samples (50 HBsAg positive and 50 HBsAg negative) were collected and tested using the detection kit described in this embodiment. The results showed that the sensitivity and specificity of the kit were significantly better than those of similar kits intended only for research use (see Table 6).

[0186] Table 6: Comparison of Sensitivity and Specificity of Chemiluminescence Method

[0187]

[0188] Example 5

[0189] Clinical significance of protein in hepatitis B

[0190] The basic definition and confirmation criteria for inclusion in this embodiment, "confirmation of chronic HBV infection," requires meeting one of the following two conditions:

[0191] 1. HBsAg or HBV DNA positive for at least 6 months.

[0192] 2. Positive for any of the following indicators: HBsAg, HBeAg, or HBV DNA, and negative for anti-HBc immunoglobulin M antibody (excluding acute hepatitis B).

[0193] Main exclusion criteria:

[0194] 1. Patients with decompensated cirrhosis (Child-Pugh B or C); 2. Patients with significantly abnormal liver function; 3. Patients co-infected with HIV; 4. Patients co-infected with hepatitis C virus (HCV) or hepatitis D virus (HDV); 5. Immunosuppressed patients; 6. Patients who have undergone orthotopic liver transplantation; 7. Patients with hepatocellular carcinoma; 8. Patients currently receiving other investigational drug treatments; 9. Other significant clinical abnormalities.

[0195] Serum samples were collected from 46 patients with chronic hepatitis B who underwent NUC treatment. Total HBsAg, MHBs, and LHBs were monitored using kits. Ten patients showed complete disappearance of total HBsAg. Serum LHBs, MHBs, and total HBsAg levels were measured before and during treatment in both the total HBsAg disappearance and non-disappearance groups. In the 10 patients in the total HBsAg disappearance group, MHBs disappeared on average 6 months prior to the disappearance, and LHBs disappeared within the previous 5 months. In this application, MHBs can be used as an indicator for early prediction of HBsAg disappearance.

[0196] 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 MHBs, characterized in that, The specific binding protein of the MHBs has an MHBs binding domain; The MHBs binding domain has a heavy chain CDR1 to a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO. 1 to SEQ ID NO. 3, and a light chain CDR1 to a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO. 4 to SEQ ID NO.

6.

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

8.

3. The specific binding protein of MHBs according to any one of claims 1 to 2, characterized in that, The specific binding protein of the MHBs further includes a heavy chain constant region and a light chain constant region.

4. The specific binding protein of MHBs according to claim 3, characterized in that, The specific binding protein of the MHBs satisfies one or more of the following conditions (1) and (2): (1) The specific binding protein of the MHBs has a sequence of any one of IgG1, IgG4, IgA, IgM, and IgE constant regions; and, (2) The heavy chain constant region and the light chain constant region are selected from any one of human, mouse, rabbit, sheep, bovine, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck, and goose species.

5. A nucleic acid molecule, characterized in that, A nucleic acid molecule encoding the specific binding protein of the MHBs as claimed in any one of claims 1 to 4.

6. A vector, characterized in that, A nucleic acid molecule as claimed in claim 5.

7. A cell, characterized in that, A vector as claimed in claim 6.

8. A method of producing the cell as claimed in claim 7, the method comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

9. A method for the production of a specific binding protein of MHBs according to any one of claims 1 to 4, characterized in that, The method comprises the steps of: culturing the cell as claimed in claim 7; and isolating the specific binding protein of the MHBs from the resulting culture.

10. A test kit comprising, The detection kit includes the specific binding protein of the MHBs as claimed in any one of claims 1 to 4.

11. A medicament, characterized by comprising: The drug includes the specific binding protein of the MHBs as claimed in any one of claims 1 to 4.

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