Specific binding protein of MHBs, nucleic acid molecule, carrier, cell, detection kit as well as preparation method and application of specific binding protein

Abstract

The invention belongs to the technical field of immunodetection, and particularly relates to a specific binding protein of MHBs, a nucleic acid molecule, a carrier, a cell, a detection kit and a preparation method and application thereof. The invention provides a specific binding protein of MHBs, which has specific CDRs and relatively high affinity and specificity, solves the problem that large protein, medium protein and small protein of hepatitis B virus cannot be distinguished in the prior art, ensures that an antibody is not subjected to cross reaction with the large protein and the small protein, improves the detection specificity, and reduces the detection cost. And a false positive result is avoided. The specific binding protein of the MHBs can be combined with a chemiluminescence detection system, so that high-sensitivity detection of the antigen level of the MHBs in a clinical sample is realized, the specific binding protein has high consistency with a nucleic acid detection result, and an important tool is provided for hepatitis B infection state and disease progress monitoring.

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 a heavy chain CDR1 to a heavy chain CDR3 with 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 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 as set forth 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 as set forth in SEQ ID NO. 8.

[0010] In some embodiments, the specific binding protein of MHBs further comprises a heavy chain constant region and a light chain constant region.

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

[0012] In some embodiments, the heavy chain constant region and the light chain constant region are independently selected from any one of human, murine, rabbit, sheep, bovine, equine, porcine, canine, feline, camel, donkey, deer, mink, chicken, duck and goose species origin.

[0013] Another aspect of the present application provides a nucleic acid molecule encoding the specific binding protein of MHBs.

[0014] Another aspect of the present application provides a vector comprising the nucleic acid molecule.

[0015] Another aspect of the present application provides a cell comprising the nucleic acid molecule or the vector.

[0016] Another aspect of the present application provides a method for preparing the cell, the method comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

[0017] Another aspect of the present application provides a method for preparing the specific binding protein of MHBs, the method comprising the steps of:

[0018] culturing the cell, and isolating the specific binding protein of MHBs from the resulting culture.

[0019] Another aspect of the present application provides a MHBs detection kit, the kit comprising the specific binding protein of MHBs.

[0020] Another aspect of the present application provides a method for detecting MHBs in a sample to be tested, the method using the specific binding protein of MHBs as a detection antibody to determine the presence of MHBs in the sample to be tested by an immunological binding reaction.

[0021] Another aspect of the present application provides a medicament comprising the specific binding protein of MHBs.

[0022] The application provides a specific binding protein of MHBs, which has specific CDRs, high affinity and specificity, solves the problem that a large protein, a medium protein and a small protein of hepatitis B virus cannot be distinguished in the prior art, and ensures that the antibody does not cross-react with S protein, improves the specificity of detection, and avoids false positive results. The specific binding protein of MHBs of the application can be combined with a chemiluminescence detection system, thereby realizing high-sensitivity detection of the MHBs antigen level in a clinical sample, and having high consistency with nucleic acid detection results, thereby providing an important tool for hepatitis B infection state and disease progression monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application, more completely understand the application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0024] Figure 1 MHBsAg-Ab purification results;

[0025] Figure 2 MHBsAg-Ab antibody affinity detection results. DETAILED DESCRIPTION

[0026] The application will be further described in detail below in combination with the embodiments and examples. It should be understood that the embodiments and examples are only used to illustrate the application and not to limit the scope of the application, and the purpose of providing the embodiments and examples is to make the understanding of the disclosed content of the application more thorough and comprehensive. It should also be understood that the application can be realized in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the application, and the equivalent forms obtained thereby also fall within the protection scope of the application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs.

[0028] Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings:

[0029] The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0030] In the present application, "multiple", "various", "multiple times", "multiple" and the like are used without specific limitation, which means greater than or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0031] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof" and the like include all suitable combination manners of any two or more listed items.

[0032] In the present application, "suitable", "suitable", "any suitable manner" and the like are described in the context of being able to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0033] In the present application, "further", "more further", "particularly" and the like are used for description purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present application.

[0034] In the present application, "optionally", "optional", "optional" means optional, i.e. selected from two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, and there is no specific description, and no contradictory relationship or mutual restriction, each "optional" is independent.

[0035] In the present application, the technical features described in an open manner include both the closed technical solution consisting of the listed features and the open technical solution of the listed features.

[0036] In the present application, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered to be continuous, and includes both numerical endpoints (i.e. the minimum and maximum values) of the numerical range, and every numerical value between the two numerical endpoints. When a numerical interval refers to integers within the numerical interval, unless otherwise specified, the two endpoints and every integer between the two endpoints are included, and in this context, are considered to be directly enumerated. For example, if t is an integer selected from 1-10, then t is any one of the integers selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe a characteristic or property, the ranges can be combined. In other words, unless otherwise specified, ranges disclosed herein are to be understood to include any and all sub-ranges subsumed therein.

[0037] In the present application, unless otherwise specified, the temperature parameter allows for constant temperature treatment, and also allows for variation within a certain temperature interval. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy of the instrument control. Fluctuations within a range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0038] In the present application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0039] All documents mentioned in the present application are incorporated by reference into the present application as if each document were individually incorporated. Unless and to the extent that the disclosure of the present application conflicts with the disclosure of the incorporated documents, the present application incorporates by reference the incorporated documents in their entirety. When the present application refers to the incorporated documents, the definitions of the relevant technical features, terms, names, phrases, etc. in the incorporated documents are also incorporated. When the present application refers to the incorporated documents, the examples of the relevant technical features that are incorporated by reference are also incorporated into the present application, subject to the ability to implement the present application. It should be understood that when the incorporated content conflicts with the description in the present application, the present application is given priority or is modified adaptively according to the description in the present application.

[0040] The term "MHBs" refers to a middle hepatitis B virus surface protein. MHBs is one of the hepatitis B virus envelope proteins and is a component of the hepatitis B virus particles (Dane particles) and subviral particles (tubular particles and spherical particles). It is one of the middle molecular weight proteins of the hepatitis B virus surface antigen (HBsAg). It includes the entire sequence of the PreS2 region and the S region. In contrast, the large protein includes the PreS1 region, PreS2, and the S region, and the small protein includes only the S region.

[0041] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins including an antibody, and any other modified configuration of the immunoglobulin molecule that exhibits the desired biological activity, so long as the antibody exhibits the desired biological activity. Antibodies can have any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), as well as derivatives thereof, which are based on their heavy chain constant region. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0042] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen binding fragment," "antigen binding domain," or "antigen binding region" refers to a portion of an intact antibody that specifically binds to an antigen. An antigen binding fragment can contain the antigen recognition site of the intact antibody (e.g., the CDR regions 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. An antigen binding fragment of an antibody can be derived from any animal species, such as a rodent (e.g., mouse, rat, or hamster) and human, or can be artificially produced.

[0043] The terms "anti-MHBs antibody" and "antibody that binds to MHBs" refer to an antibody that specifically binds to MHBs with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting MHBs. As used herein, the terms "specifically binds to," "immunospecifically binds to," "immunospecifically recognizes," and "specifically recognizes" in the context of an antibody or antigen binding fragment thereof are analogous terms. These terms indicate that the antibody or antigen binding fragment thereof binds to an epitope via its antigen binding domain, and that binding requires some 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. The variable region generally refers to the portion of an antibody, generally a portion of a light or heavy chain, typically about the first 110 to 120 amino acids or 110 to 125 amino acids in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, that varies in sequence among antibodies and is used in the binding and specificity of a particular antibody to its particular antigen. The variability of sequence is concentrated in those regions known as CDRs, while the more conserved regions of the variable domain are called FR regions. Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of both light and heavy chains are primarily responsible for the interaction of the antibody with the antigen and specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region is a rodent or murine variable region.

[0045] The term "heavy chain variable region" is composed of 4 FR regions and 3 CDR regions, the genes defining the heavy chain variable region according to the IMGT database are V, D and J genes.

[0046] The term "light chain variable region" is composed of 4 FR regions and 3 CDR regions, the genes defining the light chain variable region according to the IMGT database are V and J genes.

[0047] The term "heavy chain" generally includes 1 variable region and 3 constant regions (CH1 / CH2 / CH3).

[0048] The term "light chain" generally includes 1 variable region and 1 constant region (CL).

[0049] The terms "Kabat numbering" and like terms are art-recognized and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody or antigen-binding fragment thereof. In certain aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., 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, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are generally found at amino acid positions 31 to 35 (which optionally can include one or two additional amino acids after 35 (referred to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are generally found 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 particular embodiment, the CDRs of the antibodies 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 meaning in the art. Constant regions are portions of an antibody molecule, e.g., carboxy-terminal portions of the light and / or heavy chains, that are not directly involved in binding of the antibody to an antigen, but which can exhibit various effector functions, such as interaction with Fc receptors. Constant regions of immunoglobulin molecules generally have a more conserved amino acid sequence relative to the variable domains of the immunoglobulin. In certain aspects, an antibody or antigen-binding fragment includes a constant region or portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0051] As used herein, the term "heavy chain" when used in reference to an antibody, based on the amino acid sequence of the constant domain, can refer to any of several distinct types, e.g., a, d, e, g, and m, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGl, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In particular embodiments, the heavy chain is a human heavy chain. In particular embodiments, the heavy chain is a rodent or murine heavy chain.

[0052] As used herein, the term "light chain" when used in reference to an antibody, based on the constant domain amino acid sequence, can refer to any of the different types, e.g., kappa or lambda. Light chain amino acid sequences are well known in the art. In particular embodiments, the light chain is a human light chain. In particular embodiments, the light chain is a rodent or murine light chain.

[0053] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof in which the 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 an antibody or antigen-binding fragment thereof having the desired specificity, affinity, and capability derived from one mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous to sequences in an antibody or antigen-binding fragment thereof derived from another species (typically human) to avoid eliciting an immune response in that 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 which is derived from a human immunoglobulin locus, where such antibody or antigen-binding fragment thereof is produced using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0055] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y can be generally represented by the dissociation constant (Kd). 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, an "epitope" is a term of art in the field and refers to a local region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, a contiguous amino acid of a polypeptide (linear or contiguous epitope), or the epitope can be, for example, from two or more noncontiguous regions of one or more polypeptides (conformational, nonlinear, discontinuous, or noncontiguous epitope). In certain embodiments, the epitope to which an antibody or antigen-binding fragment thereof specifically binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA assays, hydrogen / deuterium exchange by mass spectrometry (e.g., liquid chromatography electrospray 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 known method 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 / antigen-binding fragment thereof:antigen crystals can be studied using well-known X-ray diffraction techniques, and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) Vols. 114 and 115, Wyckoff HW, et al., eds.; U.S. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276 A: 361-423, Carter CW, ed.; Roversi P, et al. (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323). Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, e.g., Champe M, et al. (1995) J Biol Chem 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-108.

[0057] An antibody can be said to "compete for binding" to a given epitope if it binds to the same epitope as a reference antibody, or to an overlapping epitope, such that it blocks binding of the reference antibody to the epitope to some extent. Competition can be determined by any method known in the art, for example, a competitive ELISA assay. An antibody can be said to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0058] A "sample" of the present application is, in embodiments, a human sample, but animal samples can also be used in the practice of the present application. Non-limiting sources of samples for use in the present application include, for example, solid tissue, biopsies, ascites, aspirates, fluid lavage, blood (including circulating tumor cells), plasma, serum, spinal fluid, lymph fluid, external sections of skin, respiratory, intestinal, and urogenital tracts, tears, saliva, milk, tumors, organs, cell cultures, and / or cell culture constituents.

[0059] The term "chemiluminescence" refers to the phenomenon in which an excited state intermediate is produced during a chemical reaction, which releases a photon when it returns to the ground state. In immunoassays, chemiluminescent substances are labeled on antibodies, after the formation of immune complexes, the luminescent substrate is added, and a chemical reaction produces a light signal, the intensity of the signal is proportional to the concentration of the antigen to be measured. In the present application, chemiluminescence is a technical platform to achieve high sensitivity detection. Its function is to amplify the small antigen-antibody binding event and convert it into a light signal that can be accurately measured, thereby achieving the detection of trace MHBs in clinical samples. The lower concept of chemiluminescent substances used in the present application is acridinium ester, which is a highly efficient chemiluminescent label with high luminescent efficiency, low background signal, rapid reaction, etc. Combined with high affinity antibodies, it constitutes the technical basis for the excellent sensitivity of the kit of the present application.

[0060] The word "label" when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody so as to generate a "labeled" antibody. The label can itself be detectable (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, can catalyze chemical alteration of a substrate compound or composition to generate a detectable product.

[0061] Terms such as "treat," "treatment," "treating," "alleviate," and "alleviating" refer to therapeutic measures that can cure, slow down, lessen symptoms of, and / or halt progression of a pathological condition or disorder. Those in need of treatment include, therefore, those already diagnosed with or suspected of having the disorder. In certain embodiments, a subject's cancer is successfully treated according to the methods of the present application if the patient shows one or more of the following.

[0062] In order to solve the problem of insufficient sensitivity and specificity of protein detection in hepatitis B in the prior art, the application provides a preparation of an antibody against middle hepatitis B protein (MHBs) and application thereof in chemiluminescence detection.

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

[0064] The specific binding protein of MHBs provided by the application has extremely high specificity and sensitivity, can specifically bind to recombinant MHBs antigen, and does not cross-react with large and small proteins, thereby solving the problem that the prior art cannot distinguish between large protein, middle protein and small protein of hepatitis B virus. Through Biacore detection, the monoclonal antibody provided by the application has high affinity, and the dissociation constant KD≤10 -10 M, which is significantly better than the antibody in the prior art.

[0065] The MHBs binding domain has heavy chain CDR1 to heavy chain CDR3 shown in SEQ ID NO. 1 to SEQ ID NO. 3, and light chain CDR1 to light chain CDR3 shown in SEQ ID NO. 4 to SEQ ID NO. 5.

[0066] The specific binding protein of MHBs provided by the application can have the above-mentioned CDRs, or a derivative fragment of the above-mentioned CDRs. The derivative fragment is formed by no more than 6 site amino acid substitutions (“conservative modification” or “conservative substitution or replacement”) of the corresponding CDR, and retains the biological activity consistent with the corresponding complementarity determining region. For example, the derivative fragment has 1, 2, 3, 4, 5 or 6 site substitutions relative to the corresponding complementarity determining region, which can be one amino acid replaced by another amino acid, or one amino acid replaced by multiple (for example, 2) amino acids.

[0067] Table 1

[0068]

[0069] The specific binding protein of MHBs of the application can have the above-mentioned CDRs, or a derivative fragment of the above-mentioned CDRs. The derivative fragment is formed by no more than 6 site amino acid substitutions (“conservative modification” or “conservative substitution or replacement”) of the corresponding CDR, and retains the biological activity consistent with the corresponding complementarity determining region. For example, the derivative fragment has 1, 2, 3, 4, 5 or 6 site substitutions relative to the corresponding complementarity determining region, which can be one amino acid replaced by another amino acid, or one amino acid replaced by multiple (for example, 2) amino acids.

[0070] In the CDRs provided by the application, the derivative fragment (conservative variant) refers to a polypeptide formed by replacing 1, 2, 3 amino acids with amino acids of similar or similar properties compared with the amino acid sequence of the antibody of the application. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 2.

[0071] Table 2

[0072]

[0073] "Conservative modifications" or "conservative substitutions or replacements" refer 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, etc.) such that the change can be made frequently without altering the biological activity of the protein. As is known to those skilled in the art, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). In addition, substitutions of amino acids of similar structure or function are not likely to disrupt biological activity.

[0074] The specific binding protein of MHBs provided by the present application can be an antibody, an antigen binding fragment of an antibody, or a small modular immunopharmaceutical. Further, the binding protein is a monoclonal antibody, a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, a ScFv fragment, a diabody, a multispecific antibody, a minibody, a chelating recombinant antibody, an intrabody, a nanobody, a binding domain immunoglobulin fusion protein, or a small modular immunopharmaceutical.

[0075] Further alternatively, the present application does not particularly limit the species origin of the heavy chain constant region and the light chain constant region, which can be, including but not limited to, any one of the species origin of human, mouse, rabbit, sheep, bovine, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck, and goose.

[0076] The present application does not particularly limit the species of the heavy chain constant region and the light chain constant region of the antibody, which can be, including but not limited to, IgG1, IgG4, IgA, IgM, and IgE.

[0077] Another aspect of the present application provides a nucleic acid molecule containing a nucleotide sequence encoding the specific binding protein of MHBs.

[0078] Another aspect of the present application provides a vector comprising the nucleic acid molecule. The present application does not particularly limit the species of the recombinant expression vector, which can be, including but not limited to, an antibody expression vector. The present application does not particularly limit the antibody expression vector, which can be, including but not limited to, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or a combination thereof.

[0079] Another aspect of the present application provides a cell comprising the nucleic acid molecule or the vector. The present application does not particularly limit the species of the host cell, including but not limited to, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, or HEK293 cell.

[0080] Another aspect of the present application provides a method for preparing the cell, comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

[0081] In one example, the introduction is by transfection.

[0082] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0083] Another aspect of the present application provides a method for preparing the specific binding protein of MHBs, comprising the steps of culturing the cell, and isolating the specific binding protein of MHBs from the resulting culture.

[0084] Specifically, the method comprises the following steps: Step 1: constructing a recombinant antigen comprising an epitope specific to the protein (MHBs) in hepatitis B virus; Step 2: immunizing an animal 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 a hybridoma cell strain that specifically binds to MHBs but not to large and small proteins; and Step 5: isolating and purifying the monoclonal antibody or antigen-binding fragment thereof from the hybridoma cell strain. The method provided by the present application successfully obtains a monoclonal antibody with high affinity and specificity through specific immunization with a recombinant antigen and strict screening steps, and solves the problem of insufficient antibody specificity and affinity in the prior art.

[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 design of recombinant antigen includes the specific epitope of MHBs and can induce the production of specific antibodies. Specifically, the PreS2 region can be selected from the amino acid sequence of aa 121-174, the S region can be selected from the amino acid sequence of aa 175-449, and the purification tag can be selected from any one of 6xHis tag, GST tag, and MBP tag.

[0086] The application fuses the PreS2 region, the S region and the purification tag for expression, and simultaneously covers the neutralizing antibody target (S region) and the high immunogenicity target (PreS2 region) for hepatitis B virus, wherein the PreS2 region contains important antigen epitopes of the virus surface and can induce the body to produce antibodies against the PreS2 region. These antibodies have a synergistic neutralizing effect on the antibodies against the S region, and can more effectively prevent the virus from invading liver cells. The design (PreS2-S) also better mimics the natural antigen structure: in the middle protein (MHBs) of the virus, the PreS2 region is naturally located at the N-terminal of the S region. This design (PreS2-S) faithfully reproduces the spatial conformation of the natural virus middle protein, and helps to induce correct antibodies that can recognize the natural virus.

[0087] In some embodiments, the animal in step 2 is a BALB / c mouse, the immunization method is subcutaneous injection, the number of immunization times is 3, and Freund's adjuvant is used to promote immunization. BALB / c mice are commonly used animal models 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 in 50 μg, 100 μg, 150 μg, 200 μg; the immunization interval can be selected from any value in 2 weeks, 3 weeks, 4 weeks.

[0088] In some embodiments, the myeloma cells in step 3 are SP2 / 0 cells, and HAT medium is used to screen hybridoma cells after fusion. SP2 / 0 cells are commonly used myeloma cell lines, which can produce hybridoma cells after fusion with spleen cells, and HAT medium can screen successful hybridoma cells. Specifically, the fusion ratio of spleen cells to myeloma cells can be selected from any value in 5:1, 10:1, 15:1; the HAT screening time can be selected from any value in 7 days, 10 days, 14 days.

[0089] In some embodiments, the screening method in step 4 includes ELISA, Western blot and immunofluorescence. These screening methods can verify the specificity and affinity of the antibody from multiple angles to ensure that high-quality antibodies are obtained. Specifically, ELISA screening can detect the binding ability of the antibody to the antigen, Western blot can verify the specificity of the antibody, and immunofluorescence can confirm the binding of the antibody to the natural antigen.

[0090] The embodiments of the application do not particularly limit the type of detection product, for example, diagnostic reagents, test strips, detection plates or kits. In the detection product, the specific binding protein of MHBs competes for the binding of MHBs in the test product.

[0091] In some embodiments, the MHBs detection kit provided by the present application comprises the specific binding protein of MHBs and the goat anti-human HBsAg antibody labeled with acridinium ester.

[0092] The detection kit provided by the present application has high sensitivity and specificity, can be used for detecting the level of MHBs antigen in a clinical sample, has high consistency with the nucleic acid detection result, and can accurately reflect the hepatitis B infection state and disease progression.

[0093] In some embodiments, the antibody purification method can be selected from any one or a combination of Protein A affinity chromatography, Protein G affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and molecular sieve.

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

[0095] In some embodiments, the antibody solid-phase carrier can be selected from any one of magnetic beads, a microplate, colloidal gold, and latex microspheres.

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

[0097] Another aspect of the present application provides a method for detecting MHBs in a sample to be tested, wherein the specific binding protein of MHBs is used as a detection antibody, and the presence of MHBs in the sample to be tested is determined by an immunological binding reaction.

[0098] The detection method of the present application can be a method for diagnostic purposes or a method for non-diagnostic purposes.

[0099] The term “immunological binding” 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 an immunological binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction, wherein a smaller KD value indicates a higher affinity. The immunological binding properties between two molecules can be quantified using methods known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation.

[0100] The present application relates to a method for immunoassay or measurement of a target antigen (e.g., MHBs), a reagent for immunoassay or measurement of a target antigen (e.g., MHBs), a method for immunoassay or measurement of a cell expressing a target antigen (e.g., MHBs), and a diagnostic agent for diagnosing a disease associated with a cell positive for a target antigen (e.g., MHBs), which comprises an antibody or antibody fragment of the present application that specifically recognizes a target antigen (e.g., MHBs) and binds to an amino acid sequence or a three-dimensional structure of an extracellular region as an active ingredient.

[0101] In the present application, the method for detecting or measuring the amount of a target antigen (e.g., MHBs) can be any known method. For example, it includes an immunoassay or measurement method. The immunoassay or measurement method is a method for detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunoassay or measurement method include a radioisotope-labeled immunological antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a fluorescent immunoassay (FIA), a luminescent immunoassay, a Western blotting method, a physical-chemical method, and the like.

[0102] In order to detect a cell expressing a polypeptide, a known immunoassay method such as an immunoprecipitation method, a fluorescent cell staining method, an immunohistological staining method, and the like can be used. In addition, a fluorescent antibody staining method using an FMAT8100 HTS system (Applied Biosystem) or the like can be used.

[0103] In the present application, there is no particular limitation on the sample to be measured for detecting or measuring a target antigen (e.g., MHBs) as long as it has a possibility of including a cell expressing a target antigen (e.g., MHBs), such as a tissue cell, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or a culture solution.

[0104] Depending on the detection method required, the detection product can further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction. The reagent for performing an antigen-antibody reaction includes a buffer, a salt, and the like. The reagent for detection includes a reagent generally used in an immunoassay or measurement method, such as a labeled second antibody recognizing the monoclonal antibody, an antibody fragment thereof, or a conjugate thereof, and a substrate corresponding to the label, and the like.

[0105] Another aspect of the present application provides a medicament comprising the specific binding protein of MHBs.

[0106] Another aspect of the present application provides a therapeutic method comprising administering an effective dose of the specific binding protein of MHBs or the medicament to a subject.

[0107] "administering," "giving," and "treating" when applied to an animal, human, test subject, cell, tissue, organ, or biological fluid, means the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, test subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of the reagent with the cell, as well as contact of the reagent with a fluid that is in contact with the cell. "Administering," "giving," and "treating" also mean by a reagent, diagnostic, binding composition, or by another cell in vitro and ex vivo treatment of, for example, a cell. "Treating" when applied to a human, veterinary, or research subject means therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0108] "Treating" means administering to a patient in need of a therapeutic agent, for example, a composition comprising any of the antibodies or antigen-binding fragments of the present application, that is known to have a therapeutic effect on the symptoms of the disease. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or to inhibit progression of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular symptom of the disease (also referred to as "therapeutically effective amount") can vary depending on factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical measurement method typically used by a physician or other professional health care provider to assess the severity or progression of the symptom. While embodiments of the present disclosure (e.g., therapeutic methods or articles of manufacture) can not be effective in alleviating each target disease symptom, it is determined that the target disease symptom should be alleviated in a statistically significant number of patients according to any statistical test known in the art, such as Student's t-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" refers to the amount of a drug, compound, or pharmaceutical composition that is necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include eliminating or reducing risk, lessening severity, or delaying onset of a disease or disorder, including biochemical, histological, and / or behavioral symptoms of the disease or disorder, and intermediate pathological phenotypes presenting during development of the disease or disorder. For therapeutic use, beneficial or desired results include clinical results, such as decreasing incidence of various disorders associated with the target antigen of the present application, or ameliorating one or more symptoms of the disorder, reducing the dose of other medications required to treat the disorder, enhancing effect of another medication, and / or delaying progression of the target antigen of the present disclosure related disorder in a patient.

[0110] The embodiments of the present application will be described in detail below with examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. The experimental methods in the following examples, if no specific conditions are indicated, the priority is given to the guidance given in the present application, but also can be in accordance with the experimental manual or conventional conditions in the art, but also in accordance with the conditions suggested by the manufacturer, or reference to the known experimental methods in the art.

[0111] In the following specific examples, the amount of raw material components involved in the parameter, such as no special mention, there may be a slight deviation in the range of weighing accuracy. Involving temperature and time parameters, the acceptable deviation caused by the accuracy of the instrument or the operation accuracy is allowed.

[0112] It should be understood that the size of the sequence number of the above-mentioned processes in various embodiments of the present application does not mean the order of execution, and the execution order of the processes should be determined by its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0113] Example 1

[0114] The present example provides a monoclonal antibody specifically binding to the protein (MHBs) in hepatitis B virus and a method for preparing the same.

[0115] Raw materials for preparing MHBsAg-Ab monoclonal antibody:

[0116] The recombinant antigen MHBsAg: MHBs additional region (aa 121-174, PreS2) is spliced to the S region (aa 175-449), and the C-terminal fusion 6×His tag sequence is shown in SEQ ID NO. 9. It is expressed by HEK293F cells, purified by Ni-NTA affinity chromatography, and the purity is >95%.

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

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

[0119] MQWNSTTFHQALLDPKVRGLYFPAGGSSSGTVNPVPTTASISSIFSRTGDPAPNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPSQHQSPSNLSPFHQPNLLDKTWMKFQIGLASLQVGYQFQTLKQDLKGLYQMLWACRCPTPIRPTPNRPISPIPQRSIPLHQVNLTRFQNLPISSYSYLQAPIRPGQPYSHLQRSGDSYGTHLYLVQTGLVQSHLPKSLMHQLQSLRSPLPSTPLPPSSRNLLSLLYPLLNQASLSKLQAMLSYTRFLGLDHYSFYASWMVQTLHQKDMMQQLGFHSQVNLKRQKRKRLKHHHHHH

[0120] BALB / c mice: 6-8 weeks old female, purchased from Beijing VitoLiuHua Co., Ltd.

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

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

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

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

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

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

[0127] Fetal bovine serum: purchased from Hyclone Company.

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

[0129] Ion exchange chromatography column: purchased from Cytiva Company, 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 after 5 days of culture, the cell supernatant was collected, and the recombinant MHBsAg antigen was purified by Ni-NTA affinity chromatography, and the purity was >95% by SDS-PAGE.

[0132] Mouse immunization: 100 μg of recombinant MHBsAg antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously into BALB / c mice; 2 weeks later, 100 μg of recombinant MHBsAg antigen was emulsified with an equal volume of Freund's incomplete adjuvant and used for the second immunization; 2 weeks later, the same method was used for the third immunization. Ten days after the third immunization, blood was collected from the tail vein, and the serum titer was detected by ELISA. The titer was >10 5 Splenocytes were collected after immunization.

[0133] Hybridoma cell preparation: The spleen of the immunized mouse was taken to prepare a splenocyte suspension; the splenocytes were mixed with SP2 / 0 myeloma cells at a ratio of 10:1 and fused under the action of PEG; the fused cells were selected 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: ELISA method was used to screen positive clones: the recombinant MHBsAg antigen was coated on the enzyme-labeled plate, the 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 positive clones; at the same time, the recombinant SHBsAg antigen was coated on the enzyme-labeled plate to exclude clones that cross-reacted with SHBsAg; the specificity of the positive clones was further verified by Western blot and immunofluorescence.

[0135] Monoclonal antibody purification: the positive hybridoma cells selected were cultured in large quantities, the cell supernatant was collected, and the monoclonal antibody MHBsAg-Ab was purified by Protein A affinity chromatography and ion exchange chromatography. The purity was >95% by SDS-PAGE.

[0136] Antibody sequence determination: total RNA was extracted from the hybridoma cells, the antibody variable region gene was amplified by RACE technology, and the heavy chain 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] QIQLVQSGPELKKPGETVKISCKASGYTFTNYSMHWVKQAPGKGLKWVGWINTNTGEPIYADEFKGRFAFSLETSARTAYLQINNLKNEDTATYFCARSILRIGPMDYWGQGTSVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

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

[0141] Table 3

[0142]

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

[0144] >MHBsAg-Ab-L KAPPA

[0145] DIVLTQSPASLAVSLGQRATISCRASKSVSASAYSYMHWYQQKPGQPPKVLIYLASNLDSGVPARFSGSGSGTDFTLNIHPVQEEDAATYYCQQSNELPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

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

[0147] Table 4

[0148]

[0149] Example 2

[0150] The present example provides a method for detecting the affinity of MHBsAg-Ab monoclonal antibody.

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

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

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

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

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

[0156] Ethanolamine: purchased from Merck Company.

[0157] Biacore T200 instrument: purchased from Ctyvia Company. Method for detecting the affinity of MHBsAg-Ab monoclonal antibody:

[0158] Chip preparation: select CM5 chip, pre-activate carboxyl functional groups on the chip surface by amino coupling method. Dilute recombinant MHBsAg antigen (ligand) to 10 μg / mL with sodium acetate buffer (pH 5.0), inject it to the chip surface, and the reaction time is about 10 min, so that the antigen fixation amount reaches about 1000 RU. Use 1 M ethanolamine (pH 8.5) to block the non-specific binding sites to prevent non-specific binding.

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

[0160] Experimental method setting: set the experimental template through Biacore T200 software, select "kinetics / affinity" detection mode. Set the injection parameters: binding time 120 seconds, flow rate 30 μL / min, dissociation time 300 seconds, regeneration condition is 10 mM glycine-HCl (pH 1.5). Set multiple concentration sample repeated injection, multiple cycle detection.

[0161] Detection process: the running starts, the sample enters the chip flow path in turn and is combined with the fixed antigen. The combination and dissociation processes are monitored in real time, and the RU change curve is collected. The non-specific binding signal is deducted through the reference channel to ensure the accuracy of the data.

[0162] Data analysis: using Biacore Evaluation software, selecting 1:1 Langmuir binding model to fit the curve. Calculate the association rate constant (ka), dissociation rate constant (kd) and affinity constant (KD), the MHBsAg-Ab antibody affinity detection results are as shown in Figure 2 The detection data results are shown in Table 5:

[0163] Table 5: MHBsAg-Ab antibody affinity detection results

[0164]

[0165] Example 3

[0166] The present embodiment provides a detection kit and a preparation method thereof.

[0167] Preparation method of detection kit:

[0168] Preparation of antibody magnetic beads: wash the carboxyl magnetic beads with MES buffer (pH 5.0) for 3 times, add EDC and NHS to activate the carboxyl on the surface of the magnetic beads, and react at room temperature for 30 min; After washing, add MHBsAg-Ab monoclonal antibody, react at room temperature for 2 hours; Block with PBS buffer containing 1% BSA, and store in PBS buffer containing 0.1% BSA and 0.02% NaN3 after washing, store at 4°C.

[0169] Acridinium ester labeled antibody: dialyze the goat anti-human HBsAg antibody with carbonate buffer (pH 9.5), add acridinium ester (antibody: acridinium ester molar ratio = 1:10), and react at room temperature for 2 hours; Remove free acridinium ester with PD-10 desalting column, collect the labeled antibody, and store at 4°C in the dark.

[0170] Preparation of chemiluminescent substrate: mix chemiluminescent substrate A (0.1% H2O2) and chemiluminescent substrate B (0.1 mol / L NaOH) in a volume ratio of 1:1, and prepare them immediately before use.

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

[0172] Example 4:

[0173] The embodiment provides a method for detecting a clinical sample by using a detection kit.

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

[0175] Full-automatic chemiluminescence analyzer: YHLO iflash3000.

[0176] Method for detecting a clinical sample: sample processing: take 50 μL of a clinical serum sample, add 50 μL of a sample diluent, and mix well for standby.

[0177] Detection steps:

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

[0179] (b) wash 3 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 3 times to remove unbound substances;

[0182] (e) add 100 μL of chemiluminescence substrate, and immediately read the relative light intensity (RLU) on a chemiluminescence analyzer.

[0183] Result calculation: calculate the concentration of MHBsAg in the sample according to the calibration curve.

[0184] Detection results:

[0185] 100 clinical samples (50 HBsAg positive samples and 50 HBsAg negative samples) are collected, and the detection kit of the embodiment is used for detection. The results show that the sensitivity and specificity of the kit of the application are significantly better than those of the same kind of kit for scientific research only (see Table 6).

[0186] Table 6: Comparison of sensitivity and specificity of chemiluminescence method

[0187]

[0188] Embodiment 5

[0189] Verification of clinical significance of hepatitis B middle protein

[0190] The basic definition and confirmation standard of the enrollment of the embodiment "confirmation of chronic HBV infection" need to meet one of the following two conditions:

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

[0192] 2. HBsAg, HBeAg or HBV DNA any index is positive, and anti-HBc immunoglobulin M antibody negative (exclude acute hepatitis B).

[0193] Main exclusion criteria:

[0194] 1. Decompensated cirrhosis patients (Child-Pugh B or C); 2. Patients with significantly abnormal liver function; 3. Patients with HIV co-infection; 4. Patients with HCV, HDV co-infection; 5. Immunosuppressed patients; 6. Patients with orthotopic liver transplantation; 7. Patients with hepatocellular carcinoma; 8. Patients receiving other investigational drugs; 9. Other obvious clinical abnormalities.

[0195] Collect 46 cases of chronic hepatitis B patients treated with NUC serum, using total HBsAg, MHBs, LHBs kit for tracking detection, of which 10 cases of total HBsAg disappeared, detection of total HBsAg disappearance and non-disappearance group before and during treatment serum LHBs, MHBs and total HBsAg level. 10 cases of total HBsAg disappearance group, MHBs disappeared on average 6 months before the total HBsAg disappearance group, LHBs disappeared in the first 5 months. In this application, MHBs can be used as an indicator for predicting HBsAg disappearance in advance.

[0196] The above-described embodiments only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the scope of patent protection. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present application are within the scope of protection of the appended claims of the present application. Therefore, the scope of protection of the patent of the present application should be based on the content of the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A specific binding protein for MHBs, characterized in that, The specific binding protein of MHBs has an MHBs binding domain; 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.

6.

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

8.

3. The specific binding protein for MHBs according to any one of claims 1 to 2, characterized in that, The specific binding proteins of the MHBs also include heavy chain constant regions and light chain constant regions.

4. The specific binding protein for MHBs according to claim 3, characterized in that, The specific binding proteins of the MHBs satisfy 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 the constant regions of IgG1, IgG4, IgA, IgM, and IgE; and, (2) The heavy chain constant region and the light chain constant region are 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.

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

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

7. A cell of MHBs, 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 MHBs 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 Specific binding proteins of MHBs were isolated from the resulting cultures.

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

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

12. A drug for MHBs, characterized in that, The drug comprises a specific binding protein of MHBs according to any one of claims 1 to 4.

Images