Specific binding protein of LHBs, 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 LHBs, 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 LHBs, 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 medium 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 LHBs can be combined with a chemiluminescence detection system, so that high-sensitivity detection of the antigen level of the LHBs 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 monitoring the infection state and disease progress of hepatitis B.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunodetection technology, and particularly relates to a specific binding protein of LHBs, 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 proteins (S proteins), medium proteins (MHBs) and large proteins (LHBs). Among them, the large protein contains unique PreS1 and PreS2 leader sequences, is mainly distributed on the surface of the virus shell, participates in the process of virus entering liver cells, and plays a role in virus adsorption and fusion function. These proteins play an important role in virus infection and immune evasion, and their expression level and antibody level can reflect the virus replication state and infection activity, and are closely related to liver cell damage, disease progression and treatment response, and are important molecular markers for monitoring the state of hepatitis B infection and disease progression.

[0003] The existing HBsAg chemiluminescence / ELISA detection kit cannot distinguish the content of large protein, medium protein and small protein of hepatitis B virus, and there is a lack of indicators for predicting functional cure of hepatitis B and observing curative effect in clinic. Due to the complex structure and various antigen epitopes of different proteins, they are easy to interfere with S proteins, resulting in insufficient sensitivity and specificity of traditional detection methods. In addition, the large protein in the PreS1 region includes a hepatocyte receptor binding site, which is an important target for the development of antiviral drugs and vaccines, so the development of large and medium protein detection technology with high sensitivity and specificity has important clinical value for the prevention and treatment of hepatitis B.

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

[0005] Based on this, an embodiment of the present application provides a specific binding protein of LHBs, 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 LHBs, wherein the specific binding protein of LHBs has a LHBs binding domain.

[0007] The LHBs 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 heavy chain variable region amino acid sequence of the LHBs binding domain is as set forth in SEQ ID NO. 7.

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

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

[0011] In some embodiments, the specific binding protein of LHBs has the sequence of any one of IgGl, 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 LHBs.

[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] In some embodiments, the method for preparing comprises the steps of:

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

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

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

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

[0022] The application provides a specific binding protein of LHBs, which has specific CDRs, high 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 LHBs of the application can be combined with a chemiluminescence detection system, thereby realizing high-sensitivity detection of the LHBs 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 LHBsAg-Ab purification results;

[0025] Figure 2 LHBsAg-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 closed technical solutions consisting of listed features and open technical solutions including 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 number 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, each integer is considered to be directly listed, e.g. t is an integer selected from 1-10 means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, 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 in the present application as if each document was individually incorporated by reference. Unless and to the extent that the disclosure of the present application conflicts with the disclosure of the incorporated documents, the present application incorporates the incorporated documents by reference 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 by reference. When the present application refers to the incorporated documents, the examples of the relevant technical features incorporated by reference can also be incorporated into the present application as reference, but only to the extent that the present application can be implemented. 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 according to the description in the present application.

[0040] The term "LHBs" refers to the large hepatitis B virus surface protein, which is one of the envelope proteins of the hepatitis B virus and is a component of the Dane particles and subviral particles (tubular and spherical particles). It is the largest protein in the hepatitis B virus surface antigen (HBsAg). It includes the entire sequence of the PreS1 region, the PreS2 region and the S region. In contrast, the middle protein only includes the PreS2 and S regions, and the small protein only includes 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 incorporating 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 class (IgG, IgM, IgA, IgD, IgE) and any subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) of immunoglobulin molecule based on the identity of their heavy chain constant structure. 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-LHBs antibody" and "antibody that binds to LHBs" refer to an antibody that specifically binds to LHBs with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting LHBs. The terms "specifically binds," "immunospecifically binds," "immunospecifically recognizes," and "specifically recognizes," as used herein 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 the amino-terminal, about 110 to 120 amino acids or 110 to 125 amino acids in mature heavy chains and about 90 to 115 amino acids in mature light chains, 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 LHBs 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] To address the shortcomings in sensitivity and specificity of existing hepatitis B large protein (LHB) detection technologies, this application provides a method for preparing an antibody against LHBs and its application in chemiluminescence detection.

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

[0064] The LHBs-specific binding protein provided in this application exhibits extremely high specificity and sensitivity, specifically binding to recombinant LHBs antigens without cross-reactivity with medium and small proteins, thus solving the problem in existing technologies that cannot distinguish between large, medium, and small hepatitis B virus 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 LHBS 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 LHBS-specific binding protein of this application may have the aforementioned CDRs or derivative fragments having the aforementioned CDRs. The derivative fragments are formed by replacing amino acids at no more than six sites relative to their corresponding CDRs (“conservative modification” or “conservative substitution”), retaining the biological activity consistent with their corresponding complementarity-determining regions. For example, the derivative fragments may replace one amino acid with another, or one amino acid with multiple amino acids (e.g., two), at sites 1, 2, 3, 4, 5, or 6 of their corresponding complementarity-determining regions.

[0070] In the CDRs provided in this application, the derived fragments (conserved variants) refer to polypeptides formed by replacing one, two, or three amino acids with amino acids of similar or related properties compared to the amino acid sequence of the antibody in this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 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 LHBS 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 optionally, the species origin of the heavy chain constant region and the light chain constant region is not particularly limited, and 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 species of the heavy chain constant region and the light chain constant region of the antibody is not particularly limited, and can be, including but not limited to, at least one of 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 LHBS.

[0078] Another aspect of the present application provides a vector comprising the nucleic acid molecule. The species of the recombinant expression vector is not particularly limited, and can be, including but not limited to, an antibody expression vector. The antibody expression vector is not particularly limited, and 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 species of the host cell is not particularly limited, and includes but is not limited to CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[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 the LHBS, comprising the steps of culturing the cell, and isolating the specific binding protein of the LHBS from the resulting culture.

[0084] Specifically, the method comprises the following steps: Step 1: constructing a recombinant antigen comprising an epitope specific to the large hepatitis B virus protein (LHBs); 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 LHBs but not to S protein; and Step 5: isolating and purifying the monoclonal antibody or antigen-binding fragment thereof from the hybridoma cell strain. The preparation 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, thereby solving 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 PreS1 region and all or part of the amino acid sequence of the S region, and carries a purification tag. This recombinant antigen design includes the specific epitope of LHBs and can induce the production of specific antibodies. Specifically, the PreS1 region can be selected from the amino acid sequence of aa 1-120, 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 a 6×His tag, a GST tag, and an MBP tag.

[0086] The present application constructs a recombinant antigen including an epitope specific to large hepatitis B virus protein (LHBs) as an immunogen, by directly splicing the extracellular region (PreS1) of LHBs with the core part of S region (aa175-449), eliminating the intermediate PreS2 region and the N-terminal part of S region, and the transmembrane region of LHBs. The solubility of expression can be effectively improved: the complete LHBs protein contains a transmembrane domain, which is prone to form inclusion bodies or be retained on the cell membrane in a recombinant expression system (such as E. coli, yeast, mammalian cells), resulting in low expression and difficult purification. The truncation designed in the present application only contains the extracellular region, greatly improving the solubility of the protein in the cytoplasm or culture medium, facilitating the subsequent purification process.

[0087] In some embodiments, the animal in step 2 is a BALB / c mouse, the immunization method is subcutaneous injection, the number of immunization is 3 times, and the Freund's adjuvant is used to promote immunization. BALB / c mouse is 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 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 the hybridoma cells are screened using HAT medium 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 capacity 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] Another aspect of the present application provides a detection kit, which comprises the specific binding protein of LHBs.

[0091] The embodiments of the present application do not particularly limit the type of detection products, for example, diagnostic reagents, test strips, detection plates or kits. In the detection product, the specific binding protein of LHBs competes with LHBs in the product to be tested.

[0092] In some embodiments, the LHBs detection kit provided by the present application comprises the specific binding protein of LHBs and the goat anti-human HBsAg antibody labeled with acridinium ester.

[0093] The detection kit provided by the present application has high sensitivity and specificity, can be used for detecting the level of LHBs 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.

[0094] 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.

[0095] In some embodiments, the chemiluminescent substance can be selected from any one of acridinium ester, luminol, isoluminol, alkaline phosphatase, and horseradish peroxidase.

[0096] In some embodiments, the antibody solid-phase carrier can be selected from any one of magnetic beads, a microplate, colloidal gold, and latex microspheres.

[0097] 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.

[0098] Another aspect of the present application provides a method for detecting LHBs in a sample to be tested, wherein the specific binding protein of LHBs is used as a detection antibody, and the presence of LHBs in the sample to be tested is determined by an immunological binding reaction.

[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 of a target antigen (e.g., LHBs), a reagent for immunoassay of a target antigen (e.g., LHBs), a method for immunoassay of a cell expressing a target antigen (e.g., LHBs), and a diagnostic agent for diagnosing a disease associated with a cell positive for a target antigen (e.g., LHBs), which comprises an antibody or antibody fragment of the present application that specifically recognizes a target antigen (e.g., LHBs) 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 assaying the amount of a target antigen (e.g., LHBs) can be any known method. For example, it includes an immunoassay method. The immunoassay method is a method for detecting or assaying the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunoassay method include a radioisotope-labeled immunological antibody method (RIA), an enzyme immunoassay method (EIA or ELISA), a fluorescent immunoassay method (FIA), a luminescent immunoassay method, a Western blotting method, a physical-chemical method, and the like.

[0102] 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 tested for detecting or assaying a target antigen (e.g., LHBs) as long as it has a possibility of including a cell expressing a target antigen (e.g., LHBs), such as a tissue cell, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or a culture solution.

[0104] Depending on the desired detection method, 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 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 LHBs.

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

[0107] The therapeutic method of the present application can be for a disease diagnosis and treatment purpose or a non-disease diagnosis and treatment purpose.

[0108] "administering," "administering" 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," "administering" and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contact of a reagent with a cell, as well as contact of a reagent with a fluid that is in contact with a cell. "Administering," "administering" and "treating" also mean treatment by a reagent, diagnostic, binding composition or by another cell in vitro and ex vivo, for example. "Treating" when applied to a human, veterinary or research subject means therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0109] "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., a method of treatment or article 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 method 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.

[0110] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to effect any one or more of the beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing risk, lessening severity, or delaying onset of a condition, including biochemical, histological and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes presenting during development of the condition. For therapeutic use, beneficial or desired results include clinical results, such as decreasing incidence or improving one or more symptoms of various target antigen-related disorders of the present application, reducing the dose of other medications required to treat a disorder, enhancing the effect of another medication, and / or delaying the progression of a target antigen-related disorder of the present disclosure in a patient.

[0111] 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.

[0112] In the following specific examples, the amount of raw material components involved in the parameter, such as no particular 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 accuracy of the operation is allowed.

[0113] It should be understood that the size of the sequence number of each process described above in various embodiments of the present application does not mean the order of execution, the execution order of each process 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.

[0114] Example 1

[0115] The present example provides a monoclonal antibody specifically binding to large hepatitis B virus protein (LHBs) and a preparation method thereof.

[0116] Preparation materials of LHBsAg-Ab monoclonal antibody:

[0117] Recombinant LHBsAg antigen: including PreS1 region (aa 1-120) and S region (aa 175-449), C-terminal fusion 6×His tag, sequence as shown in SEQ ID NO. 9, expressed by HEK293F cells, purified by Ni-NTA affinity chromatography, purity >95%.

[0118] The complete amino acid sequence of LHBsAg (PreS1-S+C-terminal His6) recombinant antigen is as follows:

[0119] LHBsAg, (PreS1-S+C-terminal His6) SEQ ID NO. 9:

[0120] MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEAHQVGAGAFGPGFTPPHGGLLGWSPQAQGILTTVPVAPPPASTVNRQSGRQPTPISPPLRDSHPQAMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPSQHQSPSNLSPFHQPNLLDKTWMKFQIGLASLQVGYQFQTLKQDLKGLYQMLWACRCPTPIRPTPNRPISPIPQRSIPLHQVNLTRFQNLPISSYSYLQAPIRPGQPYSHLQRSGDSYGTHLYLVQTGLVQSHLPKSLMHQLQSLRSPLPSTPLPPSSRNLLSLLYPLLNQASLSKLQAMLSYTRFLGLDHYSFYASWMVQTLHQKDMMQQLGFHSQVNLKRQKRKRLKHHHHHH

[0121] BALB / c mice: 6-8 weeks old female, purchased from Beijing VitoLiuHua Co., Ltd.

[0122] Freund's complete adjuvant: purchased from Sigma Company, F5881.

[0123] Freund's incomplete adjuvant: purchased from Sigma Company, F5506.

[0124] SP2 / 0 myeloma cells: purchased from Wuhan University Preservation Center.

[0125] Hybridoma-SFM medium: purchased from Gbico Company.

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

[0127] HT medium supplement (50x): purchased from Gbico Company.

[0128] Fetal bovine serum: purchased from Hyclone Company.

[0129] Protein A affinity chromatography column: purchased from Cytiva Company, HiTrap Protein A HP.

[0130] Ion exchange chromatography column: purchased from Cytiva Company, HiTrap DEAE Sepharose FF.

[0131] Preparation method of LHBsAg-Ab monoclonal antibody:

[0132] Recombinant antigen expression and purification: The LHBsAg gene shown in SEQ ID NO. 9 was constructed into a pcDNA3.1 expression vector, transfected into HEK293F cells, and after 5 days of culture, the cell supernatant was collected, and the recombinant LHBsAg antigen was purified by Ni-NTA affinity chromatography, and the purity was detected by SDS-PAGE to be >95%.

[0133] Mouse immunization: 100 μg of recombinant LHBsAg 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 LHBsAg 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.

[0134] 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 screened with HAT medium, and the medium was changed every 3 days. After 10 days, the growth of hybridoma clones was observed.

[0135] Hybridoma cell screening: ELISA method was used to screen positive clones: the recombinant LHBsAg antigen was coated on the enzyme-labeled plate, the hybridoma cell supernatant was added, the 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 cross-reacting with SHBsAg; the specificity of the positive clones was further verified by Western blot and immunofluorescence.

[0136] Monoclonal antibody purification: the positive hybridoma cells screened were cultured in large quantities, the cell supernatant was collected, and the purified LHBsAg-Ab monoclonal antibody was obtained by preliminary purification through Protein A affinity chromatography and further purification through ion exchange chromatography. The purity was detected by SDS-PAGE to be >95%.

[0137] 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.

[0138] SEQ ID NO. 7: LHBsAg-Ab murine monoclonal antibody heavy chain sequence

[0139] >LHBsAg-Ab-H mouse IgG1

[0140] DVQLQESGPGLVKPSQSLSLTCTVTGDSITTDFAWNWIRQFPGNKLEWMGYIYYSGTINYNPSLTSRISITRDTSKNQFFLELNSVTTEDTATYYCARGGTGFTYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0141] The sequence of the heavy chain variable region is shown in Table 3 below:

[0142] Table 3

[0143]

[0144] SEQ ID NO. 8: LHBsAg-Ab murine monoclonal antibody light chain sequence

[0145] >LHBsAg-Ab-L KAPPA

[0146] DIVLTQSPKSMSTSVGERVTLSCKASQNVDTAVSWYQQKPEQSPKLLIYGASSRYTGVPDRFTGSGSGTDFTLTISSVQAEDAATYYCGQSYSYPRTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0147] The sequence of the light chain variable region is shown in Table 4 below:

[0148] Table 4

[0149]

[0150] Example 2

[0151] The present example provides a method for detecting the affinity of LHBsAg-Ab monoclonal antibody.

[0152] Raw materials for detecting the affinity of LHBsAg-Ab monoclonal antibody:

[0153] Recombinant LHBsAg antigen: same as Example 1.

[0154] LHBsAg-Ab monoclonal antibody: same as Example 1.

[0155] CM5 chip: purchased from Cytiva. Company, BR100012.

[0156] 10x HBS-EP+ buffer: purchased from Cytiva Company, BR100669.

[0157] Amino coupling kit: purchased from Cytiva. Company, BR100050.

[0158] Ethanolamine: purchased from Merck Company.

[0159] Biacore T200 instrument: purchased from Ctyvia Company.

[0160] Method for detecting the affinity of LHBsAg-Ab monoclonal antibody:

[0161] Chip preparation: select CM5 chip, pre-activate carboxyl functional groups on the chip surface by amino coupling method. Dilute recombinant LHBsAg 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.

[0162] Sample preparation: dilute LHBsAg-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.

[0163] Experimental method setting: set the experiment 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.

[0164] Detection process: The running starts, and the sample enters the chip flow path in turn and binds with the fixed antigen. The binding 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.

[0165] Data analysis: Using Biacore Evaluation software, select 1:1 Langmuir binding model to fit the curve. Calculate the association rate constant (ka), dissociation rate constant (kd) and affinity constant (KD), the test chart is shown in Table 2, and the detection results are shown in Table 5:

[0166] Table 5: LHBsAg-Ab antibody affinity detection results

[0167]

[0168] Example 3

[0169] The present embodiment provides a detection kit and a preparation method thereof.

[0170] Preparation method of detection kit:

[0171] 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 LHBsAg-Ab monoclonal antibody, and 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, and store at 4°C.

[0172] 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.

[0173] Chemiluminescent substrate preparation: Mix chemiluminescent substrate A (0.1% H2O2) and chemiluminescent substrate B (0.1 mol / L NaOH) at a volume ratio of 1:1, and prepare them immediately before use.

[0174] 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 (series concentration of recombinant LHBsAg antigen) into a kit.

[0175] Example 4: The present embodiment provides a method for detecting clinical samples using a detection kit.

[0176] Clinical serum samples: collected from hospital laboratory, including HBsAg positive samples and HBsAg negative samples.

[0177] Automatic chemiluminescence analyzer: YHLO iflash3000.

[0178] Method for detecting clinical samples:

[0179] Sample processing: take 50 μL of clinical serum sample, add 50 μL of sample diluent, mix well for standby.

[0180] Detection steps:

[0181] (a) Take 50 μL of processed sample, add 50 μL of antibody magnetic beads, incubate at 37°C for 30 min;

[0182] (b) Wash 3 times to remove unbound substances;

[0183] (c) Add 50 μL of acridinium ester labeled antibody, incubate at 37°C for 30 min;

[0184] (d) Wash 3 times to remove unbound substances;

[0185] (e) Add 100 μL of chemiluminescence substrate, immediately read the relative luminescence intensity (RLU) on the chemiluminescence analyzer.

[0186] Result calculation: calculate the concentration of LHBsAg in the sample according to the calibration curve.

[0187] Detection results:

[0188] Collect 100 clinical samples (50 HBsAg positive, 50 HBsAg negative), use the detection kit of the present embodiment for detection, and compare with the commercially available kit. The results show that the sensitivity and specificity of the kit of the present application are significantly better than the commercially available kit (see Table 6).

[0189] Table 6: Comparison of sensitivity and specificity of chemiluminescence method

[0190]

[0191] Example 5

[0192] Verification of clinical significance of hepatitis B large protein

[0193] The basic definition and confirmation criteria of the enrollment of the present embodiment "confirmation of chronic HBV infection" need to meet one of the following two conditions:

[0194] 1. HBsAg or HBV DNA positive for at least 6 months.

[0195] 2. HBsAg, HBeAg or HBV DNA any index is positive, and anti-HBc immunoglobulin M antibody negative (exclude acute hepatitis B).

[0196] Main exclusion criteria:

[0197] 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.

[0198] Collect 46 cases of chronic hepatitis B patients after NUC treatment of patients with serum, using total HBsAg, LHBs, MHBs kit for tracking detection, in 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 levels. 10 cases of total HBsAg disappearance group, the average in total HBsAg disappearance group before 6 months MHBs disappeared, LHBs disappeared in the first 5 months. In this application, LHBs can be used as an indicator to predict HBsAg disappearance in advance.

[0199] 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 provided by the present application, through logical analysis, reasoning or limited experiments, are within the scope of protection of the appended claims. Therefore, the scope of protection of the patent of the present application should be based on the contents of the appended claims, and the description can be used to explain the contents of the claims.

Claims

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

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

8.

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

4. The specific binding protein of LHBs according to claim 3, characterized in that, The specific binding protein of LHBs satisfies one or more of the following conditions (1) and (2): (1) The specific binding protein of LHBs 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 of LHBs, characterized in that, A nucleic acid molecule encoding the specific binding protein of LHBs as claimed in any one of claims 1 to 4.

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

7. A cell producing LHBs, 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 producing the specific binding protein of LHBs 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 LHBs from the resulting culture.

10. An LHBs detection kit, characterized in that, The detection kit includes the specific binding protein of LHBs as claimed in any one of claims 1 to 4.

11. A method for detecting LHBs in a sample to be tested, characterized by, The detection method uses the specific binding protein of LHBs as claimed in any one of claims 1 to 4 as a detection antibody to determine the presence of LHBs in a sample to be tested by an immunological binding reaction.

12. A medicament of LHBs, characterized by, The drug includes the specific binding protein of LHBs as claimed in any one of claims 1 to 4.

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