Method for detecting HBcAg and antibody

Abstract

In the field of hepatitis B virus (HBV) detection, a method for detecting HBcAg by means of using a double antibody sandwich method, as well as antibodies and kits for detecting HBcAg, are disclosed, including monoclonal antibodies that can be used in the immunological detection of HBcAg in tissue or cell samples.

Description

[Technical Field]

[0001] This invention relates to the field of hepatitis B virus (HBV) detection. In particular, the invention provides a method for detecting HBcAg using a double antibody sandwich method, as well as antibodies and kits used for detection. The invention also provides monoclonal antibodies that can be used for the immunological detection of HBcAg in tissue or cell samples. [Background technology]

[0002] Hepatitis B virus infection, particularly chronic HBV infection, is one of the most important public health issues in the world (Non-Patent Document 1).

[0003] Currently, HBV serum markers (e.g., hepatitis B serological tests for HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb) are widely used as routine detection standards for ongoing and past HBV infections. However, due to the high mutation rate and the large number of HBV carriers, conventional hepatitis B serological tests for HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb have a high rate of false negatives. Furthermore, hepatitis B serological tests for HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb do not quantitatively reflect the degree of viral replication and infectivity, often producing questionable and difficult-to-explain results, and cannot directly determine whether the tested individual is infected with HBV.

[0004] HBV DNA is a direct indicator of HBV replication, and its dot blot test (or PCR test) is a criterion for determining infection and infectivity in hepatitis B patients and HBV carriers. While both PCR and dot blot tests can be used as direct indicators of HBV infection and infectivity, they are not suitable for large-scale screening and routine use.

[0005] Of all serum marker antigens (HBV pre-S1, HBcAg, HBxAg, DNAP, HBV pre-S2, etc.) that can be highly associated with HBV DNA, HBcAg has always been considered an antigen directly associated with HBV DNA, and the detection of HBcAg has particular importance in the quantification of replicating viruses and in the diagnosis of HBsAg-negative HBV-infected patients and HBV patients. Currently, no specific HBcAg detection reagents have been developed on the market. Reported or developed HBcAg immunodiagnostic reagents typically employ either pre-treatment of the sample before detection (viral lysis, membrane rupture, and HBcAb inactivation) or detection of the HBcAg-HBcAb immune complex. However, the former method is a complex procedure and not easily acceptable to clinical customers and is not suitable for large-scale screening and epidemiological surveys of blood donors, while the latter method is difficult to achieve ideal specificity and sensitivity due to the specificity of the detection method. In 2006, Patent Document 1 reported that after using an sAg antibody, the membrane was destroyed, the virus was lysed, and cAg in the core particles was detected for HBcAg detection, thereby capturing the virus particles. However, the sensitivity was not satisfactory. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Method and diagnostic kit for combined detection of hepatitis B virus pre-S1 antigen and core antigen [Non-patent literature]

[0007] [Non-Patent Document 1] Dienstag JL. Hepatitis B virus infection. N Engl J Med 2008 Oct 2;359(14):1486-1500 [Overview of the project] [Problems that the invention aims to solve]

[0008] Developing a simple, accurate, and highly sensitive HBcAg luminescence detection reagent is of urgent practical importance in evaluating antiviral efficacy and the prognosis of HBV patients. [Means for solving the problem]

[0009] After extensive experimental research, the inventors unexpectedly discovered a pair of antibodies that bind to a specific epitope, which proved particularly suitable for detecting HBcAg using a bi-antibody sandwich method. Based on this, the inventors developed a novel HBcAg quantitative detection kit and method. This detection method achieves a level of sensitivity comparable to DNA methods and enables rapid, high-throughput detection, thus possessing significant clinical value.

[0010] kit Therefore, in the first embodiment, the present invention is (i) A first antibody, an isolated nucleic acid molecule encoding the first antibody, a vector containing the isolated nucleic acid molecule, or a recombinant cell expressing the first antibody, wherein the first antibody is selected from an antibody or antigen-binding fragment thereof that can specifically bind to an epitope located at positions 150-183 of the HBcAg protein, (ii) A second antibody, an isolated nucleic acid molecule encoding the second antibody, a vector containing the isolated nucleic acid molecule, or a recombinant cell expressing the second antibody, wherein the second antibody is selected from an antibody or antigen-binding fragment thereof that can specifically bind to an epitope located at positions 141-154 of the HBcAg protein, We provide a kit that includes this.

[0011] As used herein, the expression "epitope contained in positions 150 to 183 of the HBcAg protein" or a similar expression means that the epitope is within or overlapping with amino acids 150 to 183 of the HBcAg protein. In other words, an antibody or its antigen-binding fragment that can specifically bind to the epitope contained in positions 150 to 183 of the HBcAg protein is an antibody or its antigen-binding fragment that can specifically bind to amino acids 150 to 183 of the HBcAg protein or its fragment.

[0012] In certain exemplary embodiments, the HBcAg protein has the sequence shown in SEQ ID NO: 17.

[0013] In certain embodiments, the second antibody is selected from an antibody or its antigen-binding fragment that can specifically bind to an epitope contained in positions 141 to 152 of the HBcAg protein.

[0014] In certain embodiments, the first antibody is the following antibody or its antigen-binding fragment: (i) a heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): HCDR1 having the sequence shown in SEQ ID NO: 3, HCDR2 having the sequence shown in SEQ ID NO: 4, and HCDR3 having the sequence shown in SEQ ID NO: 5, and / or a light chain variable region (VL) containing the following three complementarity-determining regions (CDRs): LCDR1 having the sequence shown in SEQ ID NO: 6, LCDR2 having the sequence shown in SEQ ID NO: 7, and LCDR3 having the sequence shown in SEQ ID NO: 8, an antibody or its antigen-binding fragment, or, (ii) a heavy chain variable region (VH) containing three CDRs contained in the heavy chain variable region shown in SEQ ID NO: 1, and / or a light chain variable region (VL) containing three CDRs contained in the light chain variable region shown in SEQ ID NO: 2, preferably the three CDRs contained in the heavy chain variable region, and / or the three CDRs contained in the light chain variable region are defined by the Kabat, Chothia or IMGT numbering system, an antibody or its antigen-binding fragment, or, (iii) Monoclonal antibodies produced by hybridoma cell line 18B2-2, which is deposited with the China Center for Type Culture Collection (CCTCC) and has deposit number C2019303, either as antibodies or antigen-binding fragments thereof. More likely to be selected.

[0015] In a particular embodiment, the first antibody is (a) A heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of (i) the sequence shown in SEQ ID NO: 1, (ii) a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) when compared to the sequence shown in SEQ ID NO: 1, or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared to the sequence shown in SEQ ID NO: 1. and / or, (b) A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of (iv) the sequence shown in SEQ ID NO: 2, (v) a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) when compared to the sequence shown in SEQ ID NO: 2, or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared to the sequence shown in SEQ ID NO: 2. Includes.

[0016] In a particular embodiment, the substitution described in (ii) or (v) is a conservative substitution.

[0017] In a particular embodiment, the first antibody comprises VH having the sequence shown in SEQ ID NO: 1 and VL having the sequence shown in SEQ ID NO: 2.

[0018] In a particular embodiment, the second antibody is the following antibody or its antigen-binding fragment: (i) an antibody or its antigen-binding fragment comprising the following three complementarity-determining regions (CDRs): a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 11, HCDR1 having the sequence shown in SEQ ID NO: 12, and HCDR3 having the sequence shown in SEQ ID NO: 13, and / or the following three complementarity-determining regions (CDRs): an antibody or its antigen-binding fragment comprising an antibody or its antigen-binding fragment comprising the following three complementarity-determining regions (CDRs): an LCDR1 having the sequence shown in SEQ ID NO: 14, an LCDR2 having the sequence shown in SEQ ID NO: 15, and an LCDR3 having the sequence shown in SEQ ID NO: 16, (ii) A heavy chain variable region (VH) containing three CDRs included in the heavy chain variable region shown in SEQ ID NO: 9, and / or a light chain variable region (VL) containing three CDRs included in the light chain variable region shown in SEQ ID NO: 10, preferably an antibody or its antigen-binding fragment, wherein the three CDRs included in the heavy chain variable region and / or the three CDRs included in the light chain variable region are defined by the Kabat, Chothia, or IMGT numbering system, (iii) Monoclonal antibodies produced by hybridoma cell line 2A7, which is deposited with the China Center for Type Culture Collection (CCTCC) and has deposit number CTCCC number C2019302, either as antibodies or antigen-binding fragments thereof. More likely to be selected.

[0019] In a particular embodiment, the second antibody is (a) A heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of (i) the sequence shown in SEQ ID NO: 9, (ii) a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) when compared to the sequence shown in SEQ ID NO: 9, or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared to the sequence shown in SEQ ID NO: 9. and / or, (b)(iv) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) when compared to the sequence shown in SEQ ID NO: 10, or (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared to the sequence shown in SEQ ID NO: 10, a light chain variable region (VL), Includes.

[0020] In a particular embodiment, the substitution described in (ii) or (v) is a conservative substitution.

[0021] In a particular embodiment, the second antibody comprises VH having the sequence shown in SEQ ID NO: 9 and VL having the sequence shown in SEQ ID NO: 10.

[0022] In a particular embodiment, the first antibody and / or the second antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL).

[0023] In a particular embodiment, the first antibody and / or the second antibody comprises a mouse heavy chain constant region and a mouse light chain constant region.

[0024] In a particular embodiment, the first antibody and / or the second antibody is an IgG, IgM, IgE, IgD, or IgA antibody. In a particular embodiment, the first antibody and / or the second antibody is an IgG antibody.

[0025] In a particular embodiment, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and single-domain antibody (sdAb).

[0026] In a particular embodiment, the antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.

[0027] In some embodiments, the second antibody has a detectable label.

[0028] In other embodiments, the kit further comprises a third antibody that is specifically capable of binding to a second antibody, the third antibody having a detectable label.

[0029] As used herein, a detectable label may be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrochemistry, optics, or chemical means. It is particularly preferable that such labels are suitable for immunological detection (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescence immunoassays, chemiluminescence immunoassays, etc.). Such labels are known in the art, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C, or 32The present invention comprises a biotin compound for binding to avidin modified by the above label (e.g., P), a fluorescent dye (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dot, or cyanine derivative (e.g., Cy7, Alexa 750)), a chemiluminescent substance (e.g., acridinium ester compound), and biotin for binding to avidin modified by the above label (e.g., streptavidin). The labels included in the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation calculators, and fluorescent labels can be detected using photodetectors that detect emitted light. Enzyme labels are generally detected by providing a substrate to an enzyme and detecting the reaction product produced by the enzyme's action on the substrate. Colorimetric labels are detected by simply visualizing the colored label. Chemiluminescent substances (e.g., acridinium ester compounds) are typically detected by light emitted by providing a trigger solution and / or catalyst to the luminescent substance. Biotin is typically detected by providing biotin with avidin (e.g., streptavidin) modified with the above-described label, and detecting the label possessed by the avidin linked to the biotin. In certain embodiments, the above-described detectable label may be attached to the antibody or its antigen-binding fragment of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0030] In certain embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium ester compounds), fluorescent dyes, or biotin.

[0031] In certain embodiments, the kit may further include reagents for enabling the detection of the corresponding detectable label. For example, if the detectable label is an enzyme, the kit may further include a chromogenic substance for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS, or a luminol compound for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, if the detectable label is a chemiluminescent reagent (e.g., an acridinium ester compound), the kit may further include a pre-trigger solution and / or trigger solution for chemiluminescence.

[0032] In certain embodiments, the kit further comprises a solid carrier. In certain embodiments, the solid carrier includes well plates, test tubes, beads (e.g., latex particles), or membranes (e.g., nitrocellulose membranes) made of or coated with polymer materials (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose), or magnetic beads pre-coated with functional groups (e.g., amino, carboxyl, biotin, or avidin). In certain embodiments, the solid carrier is selected from magnetic beads or microtiter plates (e.g., microwell plates or ELISA plates).

[0033] In a particular embodiment, the kit further comprises a coating reagent for coating a first antibody onto a solid carrier, such as a coating buffer (e.g., a carbonate buffer, a phosphate buffer, a Tris-HCl buffer, or a borate buffer). Methods for coating a protein or polypeptide onto a solid carrier are known in the art and include, for example, physical adsorption, covalent coupling via an aminated or carboxylated surface, or binding mediated by an avidin-biotin system, a polylysine pre-coated surface, or a protein A or protein G pre-coated surface.

[0034] In a particular embodiment, the first antibody is coated onto the surface of a solid carrier.

[0035] In a particular embodiment, the kit comprises at least a solid carrier and a first antibody in a separate container or in a separate compartment of a single container unit.

[0036] In certain exemplary embodiments, the kit comprises a first antibody and a second antibody having a detectable label. In certain exemplary embodiments, the kit comprises a first antibody coated on the surface of a solid carrier and a second antibody having a detectable label. In certain exemplary embodiments, the kit comprises one or more first antibodies and a secondary antibody having a detectable label. In certain exemplary embodiments, the kit comprises one or more first antibodies coated on the surface of a solid carrier and a secondary antibody having a detectable label. In certain embodiments, further types of the first antibody recognize different epitopes located at positions 150-183 of the HBcAg protein.

[0037] In certain embodiments, the kit further comprises a solvent for dissolving HBV virions. In this specification, the solvent for dissolving HBV virions refers to any active agent capable of dissolving Dane particles (i.e., destroying the viral envelope) to expose the HBcAg antigen. Such active agents are known to those skilled in the art and include, for example, surfactants such as NP40, LDS, or SDS.

[0038] In certain embodiments, the solvent comprises LDS or SDS. In certain embodiments, the kit further comprises a neutralizing agent, the neutralizing agent comprising CHAPS. In certain embodiments, the solvent comprises 20% LDS or 20% SDS. In certain embodiments, the solvent comprises 20% LDS or 20% SDS and balance water. In certain embodiments, the neutralizing agent comprises 10% CHAPS. In certain embodiments, the neutralizing agent comprises 10% CHAPS and 20 mM PBS. In certain embodiments, the neutralizing agent comprises 10% CHAPS, 20 mM PBS, and balance water.

[0039] In a particular embodiment, the kit further comprises one or more reagents or devices selected from the group consisting of standards (e.g., a series of samples containing different known amounts of HBcAg), positive control samples (e.g., samples containing a known amount of HBcAg), negative control samples (e.g., samples without HBcAg), solvents for lysing HBV virus (and optionally neutralizers), and devices for collecting and storing the samples to be tested (e.g., blood collection devices).

[0040] Antibody preparation The first and second antibodies described in the first embodiment can be prepared by a variety of methods known in the art, for example, by genetic engineering and recombination techniques. For example, the DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules may be inserted into an expression vector and then transfected into host cells. The transfected host cells may then be cultured under specific conditions to express the antibodies of the present invention.

[0041] The antigen-binding fragments described in the first embodiment may be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments may also be directly produced by recombinant host cells (as outlined by Hudson, Curr. Opin. Immunol. 11:548-557 (1999) and Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments may be obtained directly from host cells, and Fab' fragments may be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). Furthermore, Fv, Fab, or F(ab')2 fragments can also be isolated directly from the culture medium of recombinant host cells. Other techniques for preparing these antigen-binding fragments are known to those skilled in the art.

[0042] Therefore, in a second embodiment, the present invention is (i) a first antibody, an isolated nucleic acid molecule encoding the first antibody, a vector containing the isolated nucleic acid molecule, or a recombinant cell expressing the first antibody, wherein the first antibody is defined as in the first embodiment, (ii) A second antibody, an isolated nucleic acid molecule encoding the second antibody, a vector containing the isolated nucleic acid molecule, or a recombinant cell expressing the second antibody, wherein the second antibody is defined as in the first embodiment, We provide a kit that includes this.

[0043] In certain embodiments, the vector is a cloning vector or an expression vector. In certain embodiments, the vector is, for example, a plasmid, cosmid, phage, etc.

[0044] In certain embodiments, recombinant cells expressing a first antibody are host cells comprising an isolated nucleic acid molecule encoding the first antibody or a vector containing an isolated nucleic acid molecule, and recombinant cells expressing a second antibody are host cells comprising an isolated nucleic acid molecule encoding the second antibody or a vector containing an isolated nucleic acid molecule. Such host cells include, but are not limited to, prokaryotic cells, such as Escherichia coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DG44).

[0045] In a particular embodiment, the recombinant cells expressing the first antibody are hybridoma cell line 18B2-2, deposited with the China Center for Type Culture Collection (CCTCC) and having deposit number C2019303, and the recombinant cells expressing the second antibody are hybridoma cell line 2A7, deposited with the China Center for Type Culture Collection (CCTCC) and having deposit number C2019302.

[0046] Detection method and use In a third aspect, the present invention relates to a method for detecting the presence or level of HBcAg protein in a sample, (1) A step of contacting a first antibody with a sample to form an antibody-antigen complex, wherein the first antibody is defined as in the first embodiment; (2) A step of contacting a second antibody with an antibody-antigen complex to form an antibody-antigen-antibody complex, wherein the second antibody is defined as in the first embodiment; (3) A step to determine the amount of antibody-antigen-antibody complex, This provides a method that includes [something].

[0047] This method may be used for diagnostic or non-diagnostic purposes. In certain embodiments, the method of the present invention is used for non-diagnostic purposes. In such embodiments, the sample to be tested is known to contain HBcAg, i.e., the subject of the sample has been diagnosed before detection by the method of the present invention, and therefore the method of the present invention is not useful for diagnosing the sample. Thus, the direct object of the method of the present invention is not to obtain a diagnostic result for the subject of the sample, but to perform a more accurate and quantitative detection of a sample with known diagnostic information.

[0048] In some embodiments, the second antibody has a detectable label. In a particular embodiment, the determination described in step (3) includes the steps of (3a) detecting the amount of detectable label and (3b) comparing the amount of detectable label obtained in step (3a) with a standard curve of the relationship between a known amount of HBcAg and the amount of detectable label to obtain the HBcAg content. In a particular embodiment, the determination described in step (3) includes the steps of (3a) detecting the amount of detectable label (e.g., luminescence value) and (3b) comparing the amount of detectable label (e.g., luminescence value) obtained in step (3a) with a cutoff value, wherein if the ratio is less than 1, the sample is considered negative, and if the ratio is 1 or greater, the sample is considered HBcAg positive. In a particular embodiment, if the detectable label is an acridinium ester compound, the cutoff value is 9000.

[0049] In other embodiments, the second antibody does not have a detectable label. In such embodiments, the determination described in step (3) includes detecting the antibody-antigen-antibody complex using a third antibody having a detectable label. In certain embodiments, the third antibody is specifically bindable to the second antibody (e.g., specifically bindable to the constant region of the second antibody). In certain embodiments, the determination described in step (3) may include (3a) contacting the antibody-antigen-antibody complex with a third antibody having a detectable label; (3b) determining the amount of detectable label; and (3c) comparing the amount of detectable label obtained in step (3b) with a standard curve of the relationship between a known amount of HBcAg and the amount of detectable label to obtain the HBcAg content. In a particular embodiment, the determination described in step (3) includes the steps of (3a) contacting an antibody-antigen-antibody complex with a third antibody having a detectable label, (3b) detecting the amount of the detectable label (e.g., luminescence value), and (3c) comparing the amount of the detectable label (e.g., luminescence value) obtained in step (3b) with a cutoff value, wherein if the ratio is less than 1, the sample is considered negative, and if the ratio is 1 or greater, the sample is considered HBcAg positive. In a particular embodiment, if the detectable label is an acridinium ester compound, the cutoff value is 9000.

[0050] In a particular embodiment, the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium ester compounds), fluorescent dyes, or biotin.

[0051] In a particular embodiment, in step (3), the determination is selected from an enzyme immunoassay or a chemiluminescent immunoassay.

[0052] In a particular embodiment, prior to step (1), the method further includes a step of treating a sample, the treatment including mixing the sample with a solvent to lyse the virus. In a particular embodiment, the treatment further includes terminating the lysis reaction with a neutralizing agent.

[0053] In a particular embodiment, the solvent and neutralizing agent are defined as in the first embodiment.

[0054] In a particular embodiment, the first antibody is coated onto the surface of a solid carrier. In a particular embodiment, the solid carrier is selected from the group consisting of magnetic beads or microtiter plates (e.g., microwell plates or ELISA plates).

[0055] In a particular embodiment, a washing step is further included before step (2) and / or step (3). The washing step may remove unreacted substances.

[0056] In a particular embodiment, the sample is selected from whole blood, plasma, and serum.

[0057] In another embodiment, the present invention also relates to the use of a kit according to the first embodiment in the manufacture of a detection kit for detecting the presence or level of HBcAg protein in a sample.

[0058] In a particular embodiment, the kit is used to detect the presence or level of HBcAg protein in a sample by a method according to the third embodiment.

[0059] 2A7 mAb and its use The anti-HBcAg antibodies currently used for the immunological detection of HBcAg in tissue or cell samples (e.g., immunohistochemistry or immunofluorescence) are polyclonal antibodies. While polyclonal antibodies can improve detection sensitivity, they often have drawbacks such as high background and lower specificity, making it difficult to standardize immunohistochemical results. However, there are no reports on the use of anti-HBcAg monoclonal antibodies for the immunological detection of HBcAg in tissue or cell samples.

[0060] Unexpectedly, the inventors have discovered a monoclonal antibody suitable for the immunological detection of HBcAg in tissue or cell samples. The detection effect based on this monoclonal antibody can reach a level comparable to that of commercially available polyclonal antibodies, which is a surprising, unexpected, and highly desirable technical effect.

[0061] Therefore, in a fourth aspect, the present invention also relates to a monoclonal antibody or antigen-binding fragment thereof that can specifically bind to HBcAg, (i) A monoclonal antibody or its antigen-binding fragment contains a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): HCDR1 having the sequence shown in SEQ ID NO: 11, HCDR2 having the sequence shown in SEQ ID NO: 12, and HCDR3 having the sequence shown in SEQ ID NO: 13, and / or a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): LCDR1 having the sequence shown in SEQ ID NO: 14, LCDR2 having the sequence shown in SEQ ID NO: 15, and LCDR3 having the sequence shown in SEQ ID NO: 16, or (ii) A monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) containing three CDRs included in the heavy chain variable region shown in SEQ ID NO: 9, and / or a light chain variable region (VL) containing three CDRs included in the light chain variable region shown in SEQ ID NO: 10, preferably the three CDRs included in the heavy chain variable region and / or the three CDRs included in the light chain variable region are defined by the Kabat, Chothia, or IMGT numbering system, or (iii) The monoclonal antibody is a monoclonal antibody produced by hybridoma cell line 2A7, which is deposited with the China Center for Type Culture Collection (CCTCC) and has CCTCC deposit number C2019302. The present invention provides a monoclonal antibody or its antigen-binding fragment.

[0062] In a particular embodiment, a monoclonal antibody or its antigen-binding fragment is (a) A heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of (i) the sequence shown in SEQ ID NO: 9, (ii) a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) when compared to the sequence shown in SEQ ID NO: 9, or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared to the sequence shown in SEQ ID NO: 9. and / or, (b)(iv) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions) when compared to the sequence shown in SEQ ID NO: 10, or (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared to the sequence shown in SEQ ID NO: 10, a light chain variable region (VL), Includes.

[0063] In a particular embodiment, the substitution described in (ii) or (v) is a conservative substitution.

[0064] In a particular embodiment, the monoclonal antibody or its antigen-binding fragment comprises VH having the sequence shown in SEQ ID NO: 9 and VL having the sequence shown in SEQ ID NO: 10.

[0065] In certain embodiments, the monoclonal antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL). In certain embodiments, the monoclonal antibody is an IgG, IgM, IgE, IgD, or IgA antibody.

[0066] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and single-domain antibody (sdAb). In certain embodiments, the monoclonal antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.

[0067] In another embodiment, the present invention also relates to the use of a monoclonal antibody or its antigen-binding fragment according to the fourth embodiment in the preparation of a reagent for detecting HBcAg in a sample.

[0068] In a particular embodiment, the sample is a tissue sample (e.g., a tissue section) or a cell sample.

[0069] In certain embodiments, detection is immunological detection. In certain embodiments, immunological detection is selected from the group consisting of immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), and Western blotting.

[0070] In one embodiment, the monoclonal antibody or its antigen-binding fragment has a detectable label.

[0071] In another embodiment, the reagent for detecting HBcAg in the sample further comprises a secondary antibody having a detectable label.

[0072] In a particular embodiment, the secondary antibody is specific to the antibody of a species (e.g., mouse) from which the constant region contained in the monoclonal antibody or its antigen-binding fragment was obtained.

[0073] In a particular embodiment, the secondary antibody is an anti-immunoglobulin antibody, such as an anti-IgG antibody.

[0074] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a fluorescent dye, or biotin.

[0075] In certain exemplary embodiments, if immunological detection is selected from immunohistochemistry (IHC), immunocytochemistry (ICC), or Western blotting, the detectable marker is selected from enzymes.

[0076] In certain exemplary embodiments, when immunological detection is selected from immunofluorescence (IF), the detectable label is selected from a fluorescent dye.

[0077] Definition of Terms In this invention, unless otherwise specified, the scientific and technical terms used herein have meanings that are generally understood by those skilled in the art. Furthermore, all virological, biochemical, and immunological experimental procedures used herein are routine procedures widely used in their respective fields. On the other hand, for a better understanding of this invention, definitions and explanations of relevant terms are provided below.

[0078] As used herein, the term "HBcAg" refers to the core antigen of the hepatitis B virus (HBV), also known as the nucleocapsid protein, which is known to those skilled in the art (see, for example, NCBI GENBANK database deposit number GU357842.1). The HBcAg protein contains an assembly region involved in VLP assembly at its N-terminus and an arginine-rich domain (ARD) at its C-terminus.

[0079] In this specification, when referring to the amino acid sequence of HBcAg, the sequence shown in SEQ ID NO: 17 is used. For example, the expression "amino acid residues 150-183 of HBcAg" refers to the amino acid residues at positions 150-183 of the polypeptide shown in SEQ ID NO: 17. However, those skilled in the art will understand that mutations or alterations (including, but not limited to, substitutions, deletions and / or additions, e.g., HBcAg of different genotypes or subgenotypes) can occur naturally or be artificially introduced into the amino acid sequence of HBcAg without affecting its biological function. Therefore, in this invention, the term "HBcAg" includes all such sequences, including, for example, the sequence shown in SEQ ID NO: 17 and its natural or artificial variants. Furthermore, when describing a sequence fragment of HBcAg, not only the sequence fragment of SEQ ID NO: 17 but also the corresponding sequence fragments in its natural or artificial variants are included. For example, the expression "amino acid residues 150-183 of HBcAg" includes amino acid residues 150-183 of Sequence ID No. 17 and the corresponding fragments in its variants (natural or artificial). In accordance with the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment located at an equivalent position in the sequence being compared for optimal alignment, i.e., sequence alignment to obtain the highest identity percentage.

[0080] As used herein, the term “Dane particle,” also known as a megaspherical particle, refers to an intact infectious hepatitis B virus particle with a bilayer structure. HBcAg is typically present in the core of the Dane particle. Therefore, to detect HBcAg, it is typically necessary to first dissolve the outer shell of the Dane particle so that the HBcAg can be exposed and released.

[0081] As used herein, the term “specific binding” refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as a reaction between an antibody and an antigen to which the antibody is directed. The binding affinity between the two molecules is K DIt can be described by a value, K. D The value is the ratio of kd (the dissociation rate of the specific binding molecule-target molecule interaction, also known as koff) to ka (the association rate of the specific binding molecule-target molecule interaction, also known as kon), expressed as molar concentration (M), i.e., the dissociation constant obtained from kd / ka. K D The smaller the value, the tighter the binding between the two molecules and the higher the affinity. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) has an affinity (K -5 less than about 10 -6 M, for example, about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less) for binding to the antigen. K D The value can be determined by methods known in the art, for example, in a BIACORE device using surface plasmon resonance (SPR). D As used herein, the term "immunological assay" refers to an assay that utilizes the specific interaction / binding affinity between an antigen and an antibody, and such assays can generally be used to detect the presence or level of a specific antigen or antibody in a sample. Such immunological assays are known to those skilled in the art and include, but are not limited to, enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance method, etc. For a detailed description of immunological assays, see, for example, Fundamental Immunology, Ch. 7 Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989).

[0082]

[0083] ​As used herein, the term “antibody” generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains, each having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and therefore the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by “J” regions of about 12 or more amino acids, and the heavy chain also contains a “D” region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain does not directly participate in antibody binding to antigens, but rather mediates diverse effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including diverse cells of the immune system (e.g., effector cells) and classical complement system component 1 (C1q). The VH and VL regions can also be subdivided into highly diverse regions (referred to as complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino-terminus to the carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The assignment of amino acids to regions or domains may follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, or Chothia et al. (1989) Nature 342:878-883.

[0084] As used herein, the term “complementarity-determining region,” or “CDR,” refers to amino acid residues in the variable region of an antibody that is involved in antigen binding. The variable regions of the heavy chain and light chain each contain three CDRs, designated as CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al. al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, correspondences between different numbering systems are known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0085] In the present invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to a variety of numbering systems known in the art. In certain embodiments, the CDR contained in the antibody or its antigen-binding fragment is preferably determined by the Kabat, Chothia, or IMGT numbering system. In certain embodiments, the CDR contained in the antibody or its antigen-binding fragment is preferably identified by the Kabat numbering system.

[0086] As used herein, the term "framework region," or "FR" residue, refers to amino acid residues in the variable region of an antibody other than the CDR residues defined above.

[0087] The term "antibody" is not limited by any particular method of antibody production. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies may also be of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0088] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as the “antigen-binding moiety.” For general information, see Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989) (which, for all purposes, is incorporated herein by reference in its entirety)). Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Examples of antigen-binding fragments, not limited to these, include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabodies, single-domain antibodies, chimeric antibodies, linear antibodies, nanobodies (this technology originates from Domantis), and polypeptides containing at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide. Manipulated antibody variants are outlined in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0089] As used herein, the term "full-length antibody" refers to an antibody comprising two "full-length heavy chains" and two "full-length light chains." Here, a "full-length heavy chain" refers to a polypeptide chain comprising a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. If the full-length antibody is an IgE isotype, the antibody optionally includes a heavy chain constant region CH4 domain. Preferably, the "full-length heavy chain" is a polypeptide chain comprising VH, CH1, HR, CH2, and CH3 from the N-terminus to the C-terminus. The "full-length light chain" is a polypeptide chain comprising a light chain variable region (VL) and a light chain constant region (CL) from the N-terminus to the C-terminus. The two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be derived from a single species, such as human, or it may be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by VH and VL pairs that specifically recognize / specifically bind to the same antigen.

[0090] As used herein, the term "Fd" refers to an antibody fragment consisting of a VH domain and a CH1 domain; the term "dAb fragment" refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked via a disulfide bond in a hinge region; and the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond linking two heavy chain fragments in the F(ab')2 fragment, consisting of an intact light chain and a heavy chain Fd fragment (consisting of a VH domain and a CH1 domain).

[0091] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of one arm of the antibody. Fv fragments are generally considered the smallest antibody fragments capable of forming a complete antigen-binding site. Generally, six CDRs are thought to confer antigen-binding specificity to an antibody. However, a single variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can still recognize and bind to an antigen, albeit with potentially lower affinity than an intact binding site.

[0092] As used herein, the term "Fc" refers to an antibody fragment formed by linking the second and third constant regions of the first heavy chain of an antibody with the second and third constant regions of the second heavy chain of an antibody via disulfide bonds. Antibody Fc fragments have many different functions but are not involved in antigen binding.

[0093] As used herein, the term "scFv" refers to a single polypeptide chain containing a VL domain and a VH domain, with the VL and VH linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988), Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988), and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Appropriate prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, linkers containing amino acid sequence (GGGGS)4 or its variants may be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, disulfide bonds may also be present between VH and VL of scFv.

[0094] As used herein, the term “diabody” means a polypeptide in which its VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain, resulting in two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) and Poljak RJ et al, Structure 2:1121-1123 (1994)).

[0095] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy-chain variable region) that retains the ability to specifically bind to the same antigen to which a full-length antibody would bind. Single-domain antibodies are also known as nanobodies.

[0096] Each of the aforementioned antibody fragments possesses the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or the ability to compete with the full-length antibody in terms of specific binding to the antigen.

[0097] Antibody antigen-binding fragments (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., the antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as that used for intact antibodies.

[0098] In this specification, unless otherwise specified, when the term “antibody” is used, it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0099] As used herein, the term “chimeric antibody” refers to an antibody in which a portion of its light chain and / or heavy chain originates from one antibody (which may be of a specific species or belong to a specific antibody class or subclass), while another portion of its light chain and / or heavy chain originates from another antibody (which may be of the same or different species or belong to the same or different antibody class or subclass), but nevertheless retains binding activity to a target antigen (U.S. Patent No. 4,816,567 to Cabilly et al., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). For example, the term “chimeric antibody” may include an antibody in which the heavy chain and light chain variable region of the antibody originate from a first antibody (e.g., a mouse antibody) and the heavy chain and light chain variable region of the antibody originate from a second antibody (e.g., a human antibody) (e.g., a human-mouse chimeric antibody).

[0100] As used herein, the term “humanized antibody” refers to a genetically modified non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody. Generally, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, and reactivity. The donor antibody may be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody with the desired properties (e.g., antigen specificity, affinity, and reactivity).

[0101] The chimeric or humanized antibody of the present invention may be prepared based on the sequence of the mouse monoclonal antibody prepared above. The DNA encoding the heavy and light chains can be obtained from a target mouse hybridoma and can be manipulated using standard molecular biology techniques to include a non-mouse (e.g., human) immunoglobulin sequence.

[0102] To prepare chimeric antibodies, the murine immunoglobulin variable region may be ligated to the human immunoglobulin constant region using methods known in the art. For example, the DNA encoding VH may be functionally ligated to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be the IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but generally, the IgG1 or IgG4 constant region is preferred. For example, the DNA encoding VL may be functionally ligated to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a κ-constant region or a λ-constant region, but is generally preferred as a κ-constant region.

[0103] To prepare humanized antibodies, the mouse CDR region may be transplanted into a human framework sequence using methods known in the art (see, for example, U.S. Patent No. 5,225,539 for Winter; U.S. Patents No. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 for Queen et al., and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

[0104] As used herein, the term “vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it is capable of expressing a protein encoded by the inserted polynucleotide. A vector may be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements carried by the vector can be expressed in the host cell. Vectors are known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages, e.g., λ phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector may contain a variety of elements that regulate gene expression, including, but are not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may also contain an origin site for replication.

[0105] As used herein, the term “host cell” refers to a cell into which a vector can be introduced, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0106] As used herein, the term “identity” refers to the degree of similarity between two polypeptides or two nucleic acids. Two sequences being compared are identical at a particular site if they have the same monomer subunit of a base or amino acid at that site (for example, each of two DNA molecules has adenine at a particular site, or each of two polypeptides has lysine at a particular site). The percentage of identity between two sequences is a function of the number of identical sites common to the two sequences multiplied by 100, relative to the total number of sites being compared. For example, if six out of ten sites in two sequences are identical, these two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six sites are identical). In general, the comparison of two sequences is performed in a way that yields the greatest possible identity. Such alignment can be performed using a computer program such as the Align program (DNAstar, Inc.) based on the method of Needleman, et al. (J. Mol. Biol. 48:443-453, 1970). The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. In addition, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using Blossom The 62 matrix or PAM250 matrix can be used to determine the gap weights of 16, 14, 12, 10, 8, 6, or 4 and the length weights of 1, 2, 3, 4, 5, or 6.

[0107] As used herein, the term “conservative substitution” refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide, including its amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue with a similar side chain, for example, a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993), Kobayashi et al., Protein Eng. 12(10): 879-884 (1999), and Burks et al., Proc. Natl Acad. Set USA 94: 412-417 (1997) (these are incorporated herein by reference)).

[0108] The 20 conventional amino acids included herein are denoted according to their conventional use. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)) (which is incorporated herein by reference). In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. In this invention, amino acids are generally represented by one- and three-letter abbreviations known in the art. For example, alanine may be represented by A or Ala.

[0109] As used herein, the term “subject” includes, but is not limited to, a variety of animals, particularly mammals, such as humans. [Effects of the Invention]

[0110] This invention provides a kit for HBcAg detection based on a specific antibody, and a dual antibody sandwich method established based on the kit. Compared to prior art, the technical solution of this invention can achieve detection sensitivity comparable to that of DNA methods, enabling rapid, high-throughput detection with significant clinical value.

[0111] Furthermore, the present invention also provides an anti-HBcAg monoclonal antibody that can be used in the field of immunological detection of various tissue or cell samples, such as immunohistochemistry and immunofluorescence, and has a detection effect similar to that of commercially available polyclonal antibodies, and therefore has broad applicability.

[0112] Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are used only to illustrate the invention and not to limit its scope. Various subjects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of preferred embodiments. [Brief explanation of the drawing]

[0113] [Figure 1] A schematic diagram of eukaryotic expression plasmids containing HBV antigens of different lengths is shown. [Figure 2] This figure shows the results of Western blot analysis of HBV antigens of different lengths using 2A7 as the primary antibody. [Figure 3] This figure shows the detection results of HBcAg in different samples using the enzyme immunoassay of the present invention. [Figure 4] This figure shows the correlation between the results of the chemiluminescence detection method of the present invention and the results of PCR detection. [Figure 5] This figure shows the results of immunofluorescence detection of a cell sample using 2A7 as the HBcAg immunofluorescence detection antibody. [Figure 6] This figure shows the results of immunohistochemical detection of tissue sections using 2A7 as the HBcAg immunohistochemical detection antibody. [Modes for carrying out the invention]

[0114] Sequence information Information on some of the sequences mentioned in this application is listed in the table below.

[0115] [Table 1]

[0116] Deposit of biological materials This invention relates to the following biological substances deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China). 1) Hybridoma cell line 18B2-2, with deposit number CCTCC C2019303 and deposit date November 28, 2019. 2) Hybridoma cell line 2A7, with deposit number CCTCC C2019302 and a deposit date of November 28, 2019. [Examples]

[0117] The present invention will now be described with reference to the following examples, but these examples are intended to illustrate the present invention and are not intended to limit it.

[0118] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are based on J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Refined Laboratory Manual for Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995, and the use of restriction enzymes in accordance with the conditions recommended by the product manufacturer. All reagents whose sources are not indicated in the examples are either conventional reagents in the art or commercially available reagents. Those skilled in the art will recognize that the examples illustrate the invention by example and are not intended to limit the scope of the invention to any extent that may be sought to be protected.

[0119] Example 1: Preparation of c183 antigen 1.1 The C183 clone (whose sequence is shown in Sequence ID No. 17) was constructed, and the C183B antigen was prepared using an E. coli expression system.

[0120] 1.2 Purification of C183 antigen The bacterial solution was collected, subjected to sonication, and the sonicated liquid was centrifuged at 12,000 rpm and 10°C for 10 minutes. The supernatant was then collected. Next, it was allowed to stand in a water tank at 65°C for 20 minutes, and the supernatant was collected again.

[0121] The samples were dialyzed against 1×PB7.4, followed by DEAE-FF chromatography (GE medium) at moderate pressure.

[0122] After collecting the breakthrough peak solution (target protein), it was purified using Capto Core700. Then, the first sample peak was collected.

[0123] Example 2: Preparation of anti-HBcAg mouse monoclonal antibody 2.1 Mouse Immunity 2.1.1 Preparation of immunogen. The immunogen was recombinant HBcAg (C183 antigen) protein expressed in Escherichia coli. The recombinant antigen was diluted to 0.4 mg / mL and mixed with an equal volume of Freund's adjuvant to form a water-in-oil emulsion (method for determining whether the mixture was completely emulsified: a drop of the mixture was dropped onto the surface of water, and if the mixture remained aggregated and did not disperse, it was considered substantially homogenized). Freund's complete adjuvant was used for the initial immunization, while Freund's incomplete adjuvant was used for subsequent booster immunizations, and no adjuvant was added for the final booster immunization 72 hours before fusion.

[0124] 2.1.2 Basic immunization of mice. 6-8 week old BALB / c female mice were immunized by subcutaneous multi-point injection of the above immunogen at an injection dose of 500 μL / mouse / time point. 200 μL of ocular venous blood was collected before each immunization for subsequent titer determination. Booster immunization was administered every two weeks. Serum titer was measured by indirect ELISA. Once the serum titer of the mice reached a plateau, immunization was stopped, and the mice were rested for two months until fusion occurred.

[0125] 2.1.3 Booster immunization 72 hours before fusion (final boost). Booster immunization of the spleen was performed 72 hours before the fusion of mouse spleen cells and mouse myeloma cells. The immunogen for this booster was adjuvant-free, and 100 μl of 0.5 mg / mL recombinant protein was injected. Prior to spleen immunization, mice were anesthetized with ether, then the abdominal skin was opened to expose the spleen, 100 μL of antigen was injected along the long axis of the spleen, and then the abdominal incision was rapidly sutured.

[0126] 2.2 Preparation and screening of fusion hybridomas After booster immunization 72 hours prior to fusion, mouse spleens were harvested to prepare cell suspensions, which were then fused with mouse myeloma cells Sp2 / 0 to obtain hybridoma cells. Prior to this, feeder cells were prepared. During hybridoma cell culture, numerous myeloma and spleen cells died successively in 1640-HAT medium, and single cells or small dispersed cells did not easily survive; therefore, it was necessary to add other cells to ensure their survival. These added surviving cells were referred to as feeder cells. In this laboratory, mouse peritoneal macrophages or 13-day-old mouse thymocytes were used as feeder cells.

[0127] 2.2.1 Preparation of mouse macrophages. The following procedure was followed: (i) One 6-week-old BALB / c mouse was sacrificed by cervical dislocation (stretching neck), rinsed with tap water, and immersed in a 75% ethanol solution for 5 minutes. The mouse was placed on an ultra-clean workbench with its abdomen facing upward, the abdominal skin of the mouse was lifted with forceps and cut into small pieces, and the skin was split upward and downward with larger forceps to fully expose the abdomen. (ii) The peritoneum was lifted with sterile ophthalmic forceps, and an appropriate amount of culture medium was injected into the peritoneal cavity with a 5 mL syringe. The mouse's limb was slightly lifted with another sterile ophthalmic forceps, and finally the culture medium was aspirated using a syringe and placed in a centrifuge tube. (iii) Peritoneal cell fluid was lysed in HAT medium or HT medium and 2 × 10⁻⁶ cells were collected. 5Macrophage feeder cells were formed at a concentration of / mL. (iv) 0.1 mL was added per well to a 96-well cell culture plate and cultured in an incubator, or it was possible to mix it directly with fusion cells and add it to the 96-well cell culture plate.

[0128] 2.2.2 Preparation of mouse thymus. The following procedure was followed: (i) One 13-day-old BALB / c mouse was sacrificed by cervical dislocation, rinsed with tap water, and immersed in a 75% ethanol solution for 5 minutes. The mouse was placed on an ultra-clean workbench with its abdomen facing upward. (ii) The abdominal skin of the mouse was lifted with forceps, and the abdominal and thoracic skin was cut. (iii) The thoracic cavity was opened with another pair of clean scissors, and the milky white thymus was pulled out with forceps and subjected to homogenization and filtration through a 200-mesh cell sieve to obtain thymic feeder cell saturation.

[0129] 2.2.3 Preparation of mouse myeloma cells. The following steps were followed: (i) The mouse myeloma cell line Sp2 / 0-Ag14 (Sp2 / 0) is currently the most ideal fusion cell line because it is easy to culture and has a high fusion rate. However, the Sp2 / 0 hybridoma cell line is more sensitive to culture conditions than NS-1, and excessive dilution (3 × 10⁻¹⁰) 5 (ii) Growth was poor under conditions of density less than / mL and alkaline pH (pH greater than 7.3). (ii) Cells in the logarithmic growth phase were selected for fusion. (iii) Before fusion, myeloma cells were transferred from the culture flask to a centrifuge tube and washed three times with RPMI-1640 medium (1000 rpm × 5 minutes). The cells were resuspended in RPMI-1640 medium and counted. (iv) Generally, mouse myeloma cells should be revived 5 days before fusion, and each fusion should consist of approximately 6 flasks of 35 cm³. 2 Sp2 / 0 cells were needed.

[0130] 2.2.4 Preparation of immunospleen cells. The following steps were taken: (i) After removing the eyeballs of BALB / C mice for fusion, the mice were sacrificed by bleeding, and the collected blood samples were converted into antiserum, which could be used as a positive control for antibody detection. The mice were rinsed with tap water, immersed in a 75% ethanol solution for 5 minutes, and then placed in the right lateral position on a mouse dissection plate in an ultraclean bench. (ii) The abdominal cavity was aseptically opened, the spleen was removed, cut into small pieces with scissors, placed on a 200-mesh cell screen, squeezed, and simultaneously ground with a grinding rod (the inner core of a syringe) while adding RPMI-1640 medium drop by drop with a pipette. (iii) An appropriate amount of RPMI-1640 medium was added, and after standing for 3-5 minutes, 2 / 3 of the upper suspension was transferred to a 50 mL plastic centrifuge tube. The above process was repeated 2-3 times. (iv) The cells were washed three times with RPMI-1640 medium (1000 rpm × 10 minutes). (v) The cells were resuspended in RPMI-1640 medium and counted.

[0131] 2.2.5 Preparation of hybridomas by PEG-mediated fusion. The following steps were followed: (1) Before fusion, 1 mL of PEG-1500, 10 mL of RPMI-1640 serum-free medium, and 200 mL of complete medium were preheated to 37°C. (2) The prepared myeloma cells and spleen cells were mixed in a 50 mL centrifuge tube (1 × 10⁻¹⁶). 8 Spleen cells +1 × 10 7 Myeloma cells (approximately 10:1 ratio) were centrifuged at 1500 rpm for 8 minutes. After centrifugation, the bottom of the tube was gently flicked to loosen the cells and form a paste. (iii) 0.8 mL (1 × 10 8Splenocytes + 0.8 mL PEG) were added to a centrifuge tube using a 1 mL pipette, and the mixture was gently stirred while adding the PEG, with an average addition time of within 60 seconds. Then, 10 mL of RPMI-1640 complete medium, preheated to 37°C, was gently added while stirring. Finally, the RPMI-1640 medium was replenished to 40 mL, and the mixture was centrifuged at 1000 rpm for 5 minutes. (iv) The supernatant was discarded, and the cells were carefully dispersed with a small amount of HT medium. The cells were transferred to the prepared HT medium and added to a 96-well cell culture plate at a rate of 0.1 mL per well, and cultured in a CO2 incubator. (v) After 12 hours, an appropriate amount of HAT complete medium was prepared, and 0.1 mL was added to each well. After 5 days, 50% to 100% of the cell supernatant in the wells was replaced with HT complete medium. After approximately 9 to 14 days, the supernatant was collected for detection.

[0132] 2.2.6 Screening of hybridomas. Indirect ELISA was used for screening, with 0.1 mL of recombinant antigen per well coated with 100 ng / mL, followed by the addition of 50 μL of cell supernatant for detection, and positive clone wells were selected.

[0133] 2.2.7 Cloning of Hybridoma Cells. Using the limiting dilution method, cells were first serially diluted according to a specific concentration and then inoculated into each well of a 96-well cell culture plate so that only single cells proliferated in as many wells as possible. Positive hybridoma clones generally required 2-3 cloning cycles until they were 100% positive and confirmed as stable clones.

[0134] 2.3 Production of monoclonal antibodies in ascites 0.5 mL of liquid paraffin oil was injected intraperitoneally into 2-3 BALB / c mice. After one week, hybridoma cells in the logarithmic growth phase were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The hybridoma cells were suspended in serum-free medium, and the number of cells was (1-2) × 10⁻⁶. 6The solution was adjusted to 0.5 mL, and 0.5 mL of cells were injected into the peritoneal cavity of each mouse. After 7 to 10 days, the mice's abdomens were clearly enlarged. The mice were sacrificed by cervical dislocation, rinsed with tap water, immersed in 75% ethanol for 5 minutes, and then fixed to a dissection table with the limbs secured with a needle, with the abdomen facing upwards. The abdominal skin of the mouse was lifted with forceps and cut to create a small opening. Then, an incision was made by cutting from both sides of the mouse to the back, and the skin was torn up and down with large forceps to fully expose the abdomen. The peritoneum was lifted with sterile ophthalmic forceps, a small slit was made in the center of the peritoneum, and then all the ascites fluid in the peritoneal cavity was collected through the small slit using a 1 mL pipette. The collected ascites fluid was mixed and centrifuged in a centrifuge tube at 3000 rpm for 20 minutes. After centrifugation, the supernatant was collected.

[0135] 2.4 Purification of monoclonal antibody-induced ascites Purified monoclonal antibodies were obtained by ammonium sulfate precipitation and purification using protein A affinity chromatography (purchased from GE, USA).

[0136] Using the method described above, the following monoclonal antibodies were obtained: 1B11, 2A7, 6E1, 14C6, 18B2-2, 14C7, 5H4, and 1F9. Of these, the obtained hybridoma cell lines 2A7 and 18B2-2 were deposited with the China Center for Type Culture Collection (CCTCC) as described above.

[0137] Example 3: In vitro epitope identification of anti-HBcAg mouse monoclonal antibody 3.1 Peptide Synthesis Thirty-one polypeptides were synthesized using the HBV sequence GenBank ID:CAA59669.1 as the reference sequence (commissioned to Shanghai Sangon Biotechnology Co., Ltd). All 31 polypeptides (s1-s31) contained the full 183 amino acids of HBcAg. Polypeptide information for S1-S31 is shown in Table 1 below, and the full amino acid sequence of HBcAg is shown in GenBank:GU357842.1.

[0138] [Table 2]

[0139] 3.2 Analysis of the reactivity of polypeptides S1-S31 with anti-HBcAg mouse monoclonal antibodies 3.2.1 Preparation of reaction plate Polypeptides were diluted to a final concentration of 5 μg / mL in 50 mM CB buffer (NaHCO3 / Na2CO3 buffer, final concentration 50 mM, pH 9.6), pH 9.6. 100 μL of the coating solution was added to each well of a 96-well ELISA plate, and coating was performed at 2°C to 8°C for 16 to 24 hours, followed by 37°C for 2 hours. A single wash was performed with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), and then 200 μL of blocking solution (20 mM Na2HPO4 / NaH2PO4 buffer solution containing 20% ​​bovine serum and 1% casein, pH 7.4) was added to each well. The plates were allowed to stand at 37°C for 2 hours for blocking. The blocking solution was discarded. After drying, the plates were stored in an aluminum foil bag at 2°C to 8°C for later use.

[0140] 3.2.2 ELISA detection of anti-HBcAg mouse monoclonal antibodies The anti-HBcAg mouse monoclonal antibody obtained in 2.1 was diluted to 1 μg / mL in a PBS solution containing 20% ​​newborn bovine serum for qualitative ELISA detection.

[0141] Sample reaction. 36 polypeptide-coated ELISA plates were taken, 100 μL of diluted sample was added to each well, and the plates were incubated at 37°C for 30 minutes.

[0142] Enzyme labeling reaction. After the sample reaction step was completed, the ELISA plate was washed five times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), 100 μL of HRP-labeled goat anti-mouse IgG (GAM) reaction solution was added to each well, and the plate was placed in an incubator and reacted at 37°C for 30 minutes.

[0143] Chromogenic reaction. After the enzyme labeling reaction step was completed, the ELISA plate was washed five times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), 50 μL of TMB chromogenic agent (purchased from Beijing Wantai Bio-pharmaceutical Co., Ltd.) was added to each well, and the plate was placed in an incubator and reacted at 37°C for 15 minutes.

[0144] Reaction termination and measurement of readings. After the colorimetric reaction step was completed, 50 μL of stop solution (purchased from Beijing Wantai Bio-pharmaceutical Co., Ltd.) was added to each well of the ELISA plate, and the OD450 / 630 values ​​of each well were measured using a microplate reader.

[0145] The reactivity of 36 different polypeptides with anti-HBcAg mouse monoclonal antibodies was determined. The results were determined based on the post-reaction readings. A ratio of measured value to background value greater than 5 was considered positive.

[0146] 3.2.3 Analysis of the recognition characteristics of anti-HBcAg mouse monoclonal antibodies The results are shown in Table 2. The recognition types of the obtained anti-HBcAg mouse monoclonal antibodies can be divided into five groups (according to their recognition characteristics): sA, sB, sC, sD, and sE. Of these, the polypeptide recognized by the sA group antibody was S29 / S30, and 2A7, 14C6, and 14C7 belonged to the sA group. The polypeptide recognized by the sB group antibody was S31, and 1F9 and 18B2-2 belonged to the sB group. The polypeptide recognized by the sC group antibody was S1, the polypeptides recognized by the sD group antibody were s26 and s27, and the polypeptides recognized by the sE group antibody were s15 and s16.

[0147] [Table 3]

[0148] Table 3 shows the corresponding epitopes and the detection results for antibodies 2A7 and 18B2-2. This table indicates that the epitope for 2A7 is located at 141aa~152aa, and the epitope for 18B2-2 is located at 150aa~183aa.

[0149] [Table 4]

[0150] Example 4: In vivo epitope identification 4.1 Eukaryotic plasmid construction The sequence from a fragment containing a total of 222 amino acids, consisting of the HBcAg sequence of the HBV gene and its N-terminal sequence from -29 to -1, was constructed downstream of the CMV promoter in a eukaryotic expression vector (EHRP vector, obtained from Xiamen University, National Research Center of Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center). A schematic diagram of this structure is shown in Figure 1, where C149 to C183 refer to the truncated HBcAg sequence (starting from position +1) from the C-terminus to the amino acid positions corresponding to each number, pca-C183 refers to the -29 to -183 fragment, pcb-C183 refers to the -20 to -183 fragment, and pcc-C183 refers to the -10 to -183 fragment.

[0151] 4.2 Eukaryotic expression and Western blot evaluation 293β5 cells were placed in a 6-well plate. After 12 hours of adhesion, when the cell density reached approximately 80%-90%, transfection was performed. The constructed eukaryotic expression plasmid was transfected into the 293β5 cells using lipo3000 transfection reagent. After 12 hours of transfection, the culture medium was replaced with DMEM + 10% Gibco FBS, and the cells were cultured for 48 hours. The cell supernatant was discarded, the cells were washed once with PBS, and 300 μL of cell lysis solution was added to each well. Lysis was performed by standing at 4°C for 1 hour. The lysates were collected in 1.5 ml EP test tubes, centrifuged at 12000 rpm at 4°C for 10 minutes, and the supernatant was collected in a clean 1.5 ml EP test tube. The lysed samples were subjected to Western blot analysis (the secondary antibody was goat anti-mouse HRP purchased from Proteintec).

[0152] 4.3 Analysis of Results Monoclonal antibodies were evaluated using antigens of different lengths constructed by in vivo eukaryotic expression, and the results are shown in Figure 2. The results confirmed that the recognition site of 2A7-21 is 141aa~152aa.

[0153] Example 5: Screening of magnetic bead-coated monoclonal antibodies and acridinium ester-labeled monoclonal antibodies using the double antibody sandwich method. In this example, the experimental conditions of a conventional double antibody sandwich method were adopted to screen for optimal pairing of monoclonal antibodies for coating magnetic beads and monoclonal antibodies for labeling with acridinium ester.

[0154] 5.1 Preparation of HBcAg magnetic bead-coated monoclonal antibody A magnetic microparticle solution was prepared. The magnetic microparticles were magnetic beads with a particle size of 1.5 μm to 3 μm, coated on the surface with a hydrophilic polymer and carboxyl groups. The preparation method was as follows: Magnetic microparticles, EDC, and NHS in a mass ratio of 1:1:1 were mixed with a 50 mM MES solution at pH 5.0 to adjust the magnetic microparticle concentration to 4 mg / mL. This was then loaded onto a vertical rotating apparatus for activation at an ambient temperature of 25°C for 20 minutes. The activated magnetic microparticles and anti-HBcAg monoclonal antibody were loaded onto the vertical rotating apparatus for labeling at a ratio of 15 μg of anti-HBcAg monoclonal antibody per mg of magnetic microparticles, and the reaction was carried out at an ambient temperature of 25°C for 3 hours. The magnetic microparticles after the reaction were washed three times with a washing solution, and then a pH 7.4 phosphate buffer containing glycine, 0.5% bovine serum albumin, and 0.05% Triton X-100 was added to bring the magnetic microparticles to a concentration of 4 mg / mL. To terminate the reaction, the microparticles were loaded onto a vertical rotating apparatus at a reaction temperature of 25°C for 2 hours. After termination, the magnetic microparticles were washed three times with the washing solution, and a pH 7.4 phosphate buffer containing 0.5% (W / V) bovine serum albumin, 0.5% (W / V) casein, 0.05% (W / V) Triton X-100, and a preservative was added to bring the magnetic microparticles to a concentration of 4 mg / mL. The microparticles were then stored at 2°C to 8°C for later use.

[0155] A magnetic microparticle solution was prepared by coating MS300 magnetic beads with monoclonal antibodies (18B2-2, 1F9) that recognize aa150~aa183, according to the method described above.

[0156] 5.2 Preparation of HBcAg acridinium ester-labeled monoclonal antibody An acridinium ester-labeled antibody solution was prepared using the following method: 50 μg of anti-HBcAg monoclonal antibody to be labeled was added to phosphate buffer containing NaCl until the volume reached 300 μL. Then, 5 μL of acridinium ester stock solution was added, the mixture was shaken and mixed, and the reaction was carried out in the dark at room temperature for 30 minutes. After the reaction, 200 μL of phosphate buffer containing NaCl and glycine was added, and the mixture was mixed by manually inverting the bag 20 times. The reaction was carried out in the dark at room temperature for 30 minutes. After the reaction, the product was transferred to a dialysis bag and dialyzed in 20 mM PBS buffer, pH 7.4, at 2°C to 8°C in the dark. The PBS buffer was changed every 2 hours for a total of 3 times to remove the unlabeled acridinium ester. To achieve a final concentration of 0.1% (V / V, 1:100) bovine serum albumin, 10% (W / V) bovine serum albumin was added to the labeled product according to its actual volume, followed by the addition of an equal volume of glycerol. The mixture was then mixed by manually inverting the contents and stored in the dark at -15°C for later use.

[0157] Six HBcAg monoclonal antibodies (1B11, 2A7, 6E1, 14C6, 14C7, 5H4) were labeled with acridinium ester using the method described above.

[0158] 5.3 Experimental Method 5.3.1 Sample: Prepare HBV virus-positive (PCR detected) clinical serum samples, 1 × 10⁶ 7 , 1 x 10 6 , 1 x 10 5 , 1 x 10 4 , 1 x 10 3 Positive samples for detection were obtained by diluting the samples in 20% NBS to achieve different DNA loads. HBV virus-negative (PCR detected) clinical serum samples were also prepared.

[0159] 5.3.2 Loading the sample: 12.5 μl of 20% LDS was added to 25 μl of the sample, mixed, and incubated at 37°C for 30 minutes. 30 μl of 10% CHAPS was added, mixed, and neutralized. Then, 50 μl of magnetic bead-coated monoclonal antibody was added and incubated at 37°C for 15 minutes. After incubation, the sample was washed with phosphate buffer containing 0.05% to 0.08% Tween 20, then 50 μl of acridinium ester-labeled monoclonal antibody was added, shaken, mixed, and incubated at 37°C for 10 minutes. After incubation, the sample was washed with phosphate buffer containing 0.05% to 0.08% Tween 20, and pre-triggering was performed by adding 100 μl to 200 μl of pre-trigger solution. After removing the pre-trigger solution, triggering and detection were performed by adding 100 μl to 200 μl of trigger solution.

[0160] Using the method described above, orthogonal detection was performed on each magnetic bead-coated monoclonal antibody paired with each acridinium ester-labeled monoclonal antibody, and the P / N ratio (ratio of the mean value of positive samples to the mean value of negative samples) was calculated. The results are shown in the table below.

[0161] [Table 5]

[0162] [Table 6]

[0163] Tables 4-1 and 4-2 show the detection results of samples containing different DNA loads by coating magnetic beads with antibodies that recognize HBcAg aa150~aa183 (i.e., the arginine-rich domain (ARD) of HBcAg) and using monoclonal antibodies that recognize different epitopes in HBcAg 1aa~149aa as labeling antibodies (P / N>3 corresponds to a positive result). The results showed that when three antibodies that recognize HBcAg 141aa~154aa epitopes (2A7, 14C6, and 14C7) were used as labeling antibodies, the detection effect of samples containing different HBV DNA loads was significantly better than that of other antibody pairs.

[0164] Example 6: Enzyme immunoassay and detection reagent for detecting HBcAg Monoclonal antibody 18B2-2 was diluted in phosphate buffer (20 mmol / LPB, pH 7.4) and coated onto a polyvinyl chloride plate. Monoclonal antibody 2A7 was then labeled with horseradish peroxidase (Beijing Wantai Bio-pharmaceuticals, Co., Ltd.). The sample to be tested contained a 1 μg / ml dilution of C183 antigen, a 1 μg / ml dilution of C149 antigen (developed by the Laboratory of National Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center, Xiamen University), half positive sample, half negative sample, and 20% nbs.

[0165] The sample was treated in the same manner as in Example 5 to lyse the virus. Subsequently, 2A7-HRP (1 / 500 dilution) was added and incubated for 40 minutes. The plate was washed five times, and 50 μl each of dye stock solutions A and B (Beijing Wantai Bio-Pharmaceuticals, Co., Ltd.) were added and incubated for 15 minutes. Finally, stop solution (2M H2SO4) was added, the mixture was gently shaken and thoroughly mixed, and wavelength values ​​between 450 and 620 Hz were read on a microplate reader. The results are shown in Figure 3, demonstrating that the enzyme immunoassay detection reagent of the present invention was able to specifically detect HBcAg but not c149 (i.e., HBeAg), showing excellent specificity.

[0166] Example 7: Chemiluminescence detection method and reagents for detecting HBcAg 7.1 Preparation of the detection kit 7.1.1 Preparation of magnetic bead-coated monoclonal antibodies The magnetic microparticles were magnetic beads with a particle size of 1.5 μm to 3 μm, coated on the surface with a hydrophilic polymer and carboxyl groups. The preparation method was as follows: Magnetic microparticles, EDC, and NHS in a mass ratio of 1:1:1 were mixed with a 50 mM MES solution at pH 5.0 to adjust the magnetic microparticle concentration to 4 mg / mL. These were then loaded onto a vertical rotating apparatus for activation at an ambient temperature of 25°C for 20 minutes. The activated magnetic microparticles and 18B2-2 monoclonal antibody were loaded onto the vertical rotating apparatus for labeling at a ratio of 15 μg of HBcAg monoclonal antibody per mg of magnetic microparticles, and the reaction was carried out at an ambient temperature of 25°C for 3 hours. The reacted magnetic microparticles were washed three times with a washing solution, and a pH 7.4 phosphate buffer containing glycine, 0.5% bovine serum albumin, and 0.05% Triton X-100 was added to bring the magnetic particles to a concentration of 4 mg / mL. For termination, the mixture was loaded onto a vertical rotating apparatus at a reaction environment temperature of 25°C for 2 hours. After termination, the magnetic particles were washed three times with the washing solution, and a pH 7.4 phosphate buffer containing 0.5% (w / v) bovine serum albumin, 0.5% (w / v) casein, 0.05% (w / v) Triton X-100, and a preservative was added to bring the magnetic microparticles to a concentration of 4 mg / mL. The mixture was then stored at 2°C to 8°C for later use.

[0167] 7.1.2 Preparation of acridinium ester-labeled monoclonal antibodies The preparation method was as follows: 50 μg of 2A7 monoclonal antibody to be labeled was added to phosphate buffer containing NaCl until the volume reached 300 μL. Then, 5 μL of acridinium ester stock solution was added, and the mixture was shaken and thoroughly mixed. The reaction was carried out in the dark at room temperature for 30 minutes. After the reaction, 200 μL of phosphate buffer containing NaCl and glycine was added and mixed by manually inverting the mixture 20 times. The reaction was carried out in the dark at room temperature for 30 minutes. After the reaction, the product was transferred to a dialysis bag and dialyzed in 20 mM PBS buffer, pH 7.4, at 2°C to 8°C in the dark. The PBS buffer was changed every 2 hours for a total of 3 times to remove the unlabeled acridinium ester. To achieve a final concentration of 0.1% (V / V, 1:100) bovine serum albumin, 10% (W / V) bovine serum albumin was added to the labeled product according to its actual volume, an equal volume of glycerol was added, and the mixture was mixed by manually inverting the contents. The mixture was then stored in the dark at -15°C for later use.

[0168] 7.2 Detection Method 1. Preparation. The kit obtained in 7.1 was allowed to stand and equilibrate at room temperature (18°C to 30°C) for 15 to 30 minutes. 2. Liquid preparation. For later use, 50 ml of concentrated washing solution (20×) was diluted to 1000 ml with distilled water or deionized water. 3. Sample addition. 25 μl of the sample to be tested was added to each corresponding well. 4. Dissolution. The virus was dissolved using the same method as described in Example 5. 5. Reaction. 50 μl of magnetic bead-coated monoclonal antibody 18B2-2 was added to the sample well and mixed thoroughly. The plate was then sealed with a sealing film and incubated at 37±1°C for 15 minutes. After incubation for 15-20 minutes, the plate was washed with phosphate buffer containing 0.05%-0.08% Tween 20. Then, 50 μl of acridinium ester-labeled antibody 2A7 was added and incubated for 10-15 minutes. After incubation, the plate was washed with phosphate buffer containing 0.05%-0.08% Tween 20. Then, 100-200 μl of pre-trigger solution was added to pre-trigger. The pre-trigger solution was then removed, and 100-200 μl of trigger solution was added for triggering and detection.

[0169] Result judgment Threshold: Cutoff (CO) = 9000 Result determination: (S = luminescence value of each well) Negative result: (S / CO<1): If the luminescence value of the sample is below the cutoff value, it is determined to be negative, meaning that HBV core antigen was not detected in the sample. Positive result: (S / CO≧1): A positive result was determined if the luminescence value of the sample was above the cutoff value, which meant that the HBV core antigen was detected in the sample.

[0170] Example 8: Specificity and sensitivity analysis of HBcAg detection kit 8.1 Specificity analysis of core antigen detection kits 8.1.1 Kit Preparation A luminescence diagnostic kit (luminescence detection reagent method) for detecting HBV core antigen was prepared according to the method described in Example 7.

[0171] 8.1.2 Sample Detection A total of 80 samples collected from April 2019 to the present, all of which showed negative results for all items in the hepatitis B serological test (HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb5), were frozen and stored at -20°C.

[0172] 8.1.3 Detection Items Each serum sample was subjected to chemiluminescent detection for hepatitis B virus core antigen, a method shown in Example 7.

[0173] 8.1.4 Detection Results After detecting all samples, the results were analyzed regarding the specificity of the kit. The detection results for each sample are shown in the table below.

[0174] [Table 7]

[0175] 8.1.5 Results and Analysis The results in Table 7 show that S / CO < 1 indicates that the HBV core antigen was not detected in the sample, demonstrating excellent specificity.

[0176] 8.2 Sensitivity analysis of core antigen detection kits 8.2.1 Kit Preparation A luminescence diagnostic kit for detecting HBV core antigen (by luminescence detection reagent method) was prepared according to the method described in Example 7.

[0177] 8.2.2 Sample Detection 8.2.2.1. One fresh serum sample containing a DNA load of 1.60E+08 copies / mL, detectable by hepatitis B virus nucleic acid quantitative PCR detection reagent, was subjected to linear dilution in 20% NBS with a 3x gradient and 11 points, with 20% NBS used as a negative control. Detection was performed according to the method described in Example 8, and a reference curve was constructed.

[0178] 8.2.2.2. The C183B antigen was diluted to 1 μg / ml with 20% NBS and subjected to dilution at 11 points on a 3-fold gradient. 20% NBS without the C183B antigen was used as a negative control. Detection was performed according to the method described in Example 8, and the values ​​corresponding to antigen detection at each point were converted using the above reference curve. The results are shown in the table below.

[0179] [Table 8]

[0180] 8.2.2.3. Results and Analysis In the data in Table 8, an S / CO greater than 1 was considered positive, while a value less than 1 was considered negative. The results showed that the antigen detection sensitivity for c183 was 0.05 ng / ml, and the sample detection sensitivity was approximately 10 4 This indicates that it is a copy / ml (DNA loaded).

[0181] Example 9: Comparison of HBcAg detection kit and PCR detection method 9.1 Kit Preparation 9.1.1 A luminescence diagnostic kit for detecting HBV core antigen (by luminescence detection reagent method) was prepared according to the method described in Example 7.

[0182] 9.1.2 Hepatitis B virus nucleic acid quantitative PCR detection reagent was purchased from Shenzhen Piji Bioengineering Co., Ltd.

[0183] 9.2 Detection Sample A total of 82 hepatitis B virus-infected serum samples collected from April 2019 to the present were frozen and stored at -20°C.

[0184] 9.3 Detection Items Quantitative PCR detection of hepatitis B virus nucleic acid was performed on each serum sample.

[0185] Chemiluminescence detection of hepatitis B virus core antigen was performed on each serum sample.

[0186] 9.4 Detection Results The correlation between HBV core antigen detection and HBV viral nucleic acid detection was analyzed by comparing the detection results for all items. The results are shown in the table below.

[0187] [Table 9-1] [Table 9-2]

[0188] 9.5 Analysis of Results In Table 9, S / CO > 1 corresponds to the detection of the core antigen in the sample, and S / CO < 1 corresponds to the non-detection of the core antigen in the sample. Correlation analysis was performed between the HBcAg detection results and the DNA loading results obtained by PCR. In particular, linear correlation analysis was performed using the logarithm of the virus content and the luminescence intensity of each sample. As shown in Figure 4, R 2 The value was 0.8368, and this result indicates that the HBcAg detection method of the present invention has excellent detection performance and can be used to evaluate DNA loading in a sample.

[0189] Example 10: Use of 2A7 monoclonal antibody in other immunoassays 10.1 Use of 2A7 as an HBcAg immunofluorescence detection antibody HepG2 cells (obtained from the Laboratory of National Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center, Xiamen University) and HepG2-N10 cells (obtained from the Laboratory of National Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center, Xiamen University) stably incorporating a 1.1x HBV genome were plated in 24-well plates at a density of 60,000 cells per well. After 12 hours for cell adhesion, the medium was removed and the cells were washed once with 20 mM PBS. After fixing with 4% paraformaldehyde for 15 minutes, the cells were permeabilized with 0.02% Triton x-100 for 10 minutes and blocked with 2% BSA for 1 hour. Subsequently, 2A7 monoclonal antibody (1 mg / ml) diluted at a 1:1000 dilution ratio with 2% BSA was added, the cells were incubated at room temperature for 1 hour, and washed four times with PBS. A fluorescent secondary antibody, goat anti-mouse Alexa488 (Beyotime, catalog number A0428), was added, and the cells were incubated at room temperature for 40 minutes. After washing four times with PBS, the nuclei were stained with DAPI. After the experiment was completed, the cells were photographed using an Opera Phenix laser confocal high-content imaging system with a 63× water immersion objective lens.

[0190] The results are shown in Figure 5. 2A7 exhibited a clear immune response in the cytoplasm of HepG2-N10 cells with the HBV genome incorporated, but showed no binding to cells without the HBV genome incorporated, demonstrating excellent specificity. These results indicate that 2A7 can be used as an immunofluorescent antibody for HBcAg for accurate detection.

[0191] 10.2 Use of 2A7 as a monoclonal antibody for HBcAg immunohistochemical detection Currently, anti-HBcAg antibodies used for HBcAg immunohistochemical detection are polyclonal antibodies. However, polyclonal antibodies often exhibit high background and low specificity, making it difficult to standardize immunohistochemical results obtained using them. There are no reports on the use of anti-HBcAg monoclonal antibodies for immunohistochemical detection. In this experiment, we investigated the performance of 2A7 monoclonal antibody as an immunohistochemical detection antibody.

[0192] Liver tissue paraffin sections from HBV transgenic mice (HBV-TG, obtained from the Laboratory of National Infectious Disease Diagnostic Reagent and Vaccine Engineering Technology Research Center, Xiamen University) and normal C57BL / 6 mice (obtained from Shanghai SLAC Laboratory Animal Co., Ltd.) were dewaxed, rehydrated, antigen recovered, washed, and blocked. Then, commercially available anti-HBc polyclonal antibody and 2A7 monoclonal antibody were added for reaction at room temperature for 1 hour. After washing, secondary antibody was added and reacted at room temperature for 10 minutes. After washing, staining was performed with a chromogenic solution, followed by hydrochloric acid differentiation, counterstaining, and mounting.

[0193] The results are shown in Figure 6. Similar to commercially available polyclonal antibodies, the 2A7 monoclonal antibody was able to accurately detect tissue paraffin sections from HBV transgenic mice and did not bind to tissue paraffin sections from normal mice, demonstrating that 2A7 can also be used as an immunohistochemical antibody against HBcAg for accurate detection.

[0194] While specific embodiments of the present invention are described in detail, those skilled in the art will understand that various modifications and changes can be made in detail in light of all the disclosed teachings, and that all such changes fall within the scope of the present invention. The entire scope of the present invention is given by the appended claims and equivalents.

Claims

1. (i) A first antibody selected from an antibody or an antigen-binding fragment thereof that can specifically bind to an epitope located at positions 150 to 183 of the HBcAg protein, (ii) A second antibody selected from an antibody or its antigen-binding fragment that is specifically capable of binding to the epitope located at positions 141 to 154 of the HBcAg protein, A kit that includes this.

2. The kit according to claim 1, wherein the second antibody is selected from an antibody or antigen-binding fragment thereof that can specifically bind to an epitope located at positions 141 to 152 of the HBcAg protein.

3. The first antibody is one of the following antibodies or its antigen-binding fragments: (i) An antibody or its antigen-binding fragment comprising the following three complementarity-determining regions (CDRs): a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 3, 4, and 5, and a light chain variable region (VL) having the sequence shown in SEQ ID NO: 6, 7, and 8, and 9. (ii) An antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) containing three CDRs in the VH shown in SEQ ID NO: 1, and a light chain variable region (VL) containing three CDRs in the VL shown in SEQ ID NO: 2, (iii) A monoclonal antibody produced by hybridoma cell line 18B2-2, which is deposited with the China Center for Type Culture Collection (CCTCC) and has deposit number C2019303, either an antibody or its antigen-binding fragment. A kit according to claim 1 or 2, which is more selected.

4. The kit according to claim 3, wherein the CDR described in (ii) is defined by Kabat, Chothia, or IMGT numbering system.

5. The antibody or its antigen-binding fragment (a) A heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared with (i) the sequence shown in SEQ ID NO: 1, and (ii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared with the sequence shown in SEQ ID NO: 3, HCDR1 having the sequence shown in SEQ ID NO: 4, and HCDR3 having the sequence shown in SEQ ID NO:

5. And, (b) A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared with the sequence shown in (iii) Sequence ID No. 2, and including an LCDR1 having the sequence shown in Sequence ID No. 6, an LCDR2 having the sequence shown in Sequence ID No. 7, and an LCDR3 having the sequence shown in Sequence ID No.

8. The kit according to claim 3, including the following:

6. The kit according to claim 5, wherein the antibody or antigen-binding fragment thereof comprises VH having the sequence shown in SEQ ID NO: 1 and VL having the sequence shown in SEQ ID NO:

2.

7. The second antibody is the following antibody or its antigen-binding fragment: (i) An antibody or its antigen-binding fragment comprising the following three complementarity-determining regions (CDRs): a heavy chain variable region (VH) having the sequence shown in SEQ ID NO: 11, HCDR2 having the sequence shown in SEQ ID NO: 12, and HCDR3 having the sequence shown in SEQ ID NO: 13, and a light chain variable region (VL) having the following three complementarity-determining regions (CDRs): LCDR1 having the sequence shown in SEQ ID NO: 14, LCDR2 having the sequence shown in SEQ ID NO: 15, and LCDR3 having the sequence shown in SEQ ID NO: 16, (ii) An antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) containing three CDRs in the VH shown in SEQ ID NO: 9, and a light chain variable region (VL) containing three CDRs in the VL shown in SEQ ID NO: 10, (iii) A monoclonal antibody produced by hybridoma cell line 2A7, which is deposited with the China Center for Type Culture Collection (CCTCC) and has deposit number C2019302, either an antibody or its antigen-binding fragment. A kit according to any one of claims 1 to 6, which is selected from the above.

8. The kit according to claim 7, wherein the CDR described in (ii) is defined by Kabat, Chothia, or IMGT numbering system.

9. The antibody or its antigen-binding fragment (a) A heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared with (i) the sequence shown in SEQ ID NO: 9, and (ii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared with the sequence shown in SEQ ID NO: 11, HCDR1 having the sequence shown in SEQ ID NO: 12, and HCDR3 having the sequence shown in SEQ ID NO:

13. And, (b) A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared with the sequence shown in (iii) Sequence ID No. 10, and including an LCDR1 having the sequence shown in Sequence ID No. 14, an LCDR2 having the sequence shown in Sequence ID No. 15, and an LCDR3 having the sequence shown in Sequence ID No.

16. The kit according to claim 7, including the following:

10. The kit according to claim 9, wherein the antibody or antigen-binding fragment thereof comprises VH having the sequence shown in SEQ ID NO: 9 and VL having the sequence shown in SEQ ID NO:

10.

11. The kit according to any one of claims 1 to 10, wherein the first antibody and / or the second antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL).

12. The kit according to any one of claims 1 to 10, wherein the first antibody and / or the second antibody comprises a mouse heavy chain constant region and a mouse light chain constant region.

13. The kit according to any one of claims 1 to 12, wherein the first antibody and / or the second antibody is an IgG, IgM, IgE, IgD, or IgA antibody.

14. The antigen-binding fragment is Fab, Fab', (Fab') ２ A kit according to any one of claims 1 to 13, wherein the antibody is selected from the group consisting of Fv, disulfide-linked Fv, scFv, diabody, and single-domain antibody (sdAb), and / or the antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.

15. The kit according to any one of claims 1 to 14, wherein the second antibody has a detectable label, or the kit further comprises a third antibody that is specifically capable of binding to the second antibody, and the third antibody has a detectable label.

16. The kit according to claim 15, wherein the detectable label is selected from an enzyme, a chemiluminescent reagent, a fluorescent dye, or biotin.

17. The kit according to any one of claims 1 to 16, wherein the kit further comprises a solid carrier.

18. The kit according to claim 17, wherein the solid carrier is selected from magnetic beads or microtiter plates.

19. The kit according to claim 17, wherein the first antibody is coated on the surface of the solid carrier.

20. The kit according to any one of claims 1 to 19, further comprising one or more reagents or devices selected from the group consisting of a standard, a positive control sample, a negative control sample, a solubilant used to dissolve the HBV virus, and a device for collecting and storing the sample to be tested.

21. The kit according to claim 20, having one or more features selected from the group consisting of (a) to (d) below. (a) The standard is a series of samples containing different known amounts of HBcAg. (b) The positive control sample is a sample containing a known amount of HBcAg. (c) The negative control sample is a sample that does not contain HBcAg. (d) The device is a blood collection device.

22. (i) a first antibody, an isolated nucleic acid molecule encoding the first antibody, a vector comprising the isolated nucleic acid molecule, or a recombinant cell expressing the first antibody, wherein the first antibody is defined as in any one of claims 1 to 14, (ii) A second antibody, an isolated nucleic acid molecule encoding the second antibody, a vector comprising the isolated nucleic acid molecule, or a recombinant cell expressing the second antibody, wherein the second antibody is defined as in any one of claims 1 to 14. A kit that includes this.

23. The recombinant cell expressing the first antibody is a host cell containing an isolated nucleic acid molecule encoding the first antibody or a vector containing the isolated nucleic acid molecule. The recombinant cell expressing the second antibody is a host cell containing an isolated nucleic acid molecule encoding the second antibody or a vector containing the isolated nucleic acid molecule. The kit according to claim 22.

24. The recombinant cells expressing the first antibody are hybridoma cell line 18B2-2, which is deposited with the China Center for Type Culture Collection (CCTCC) and has the CCTCC deposit number C2019303. The recombinant cells expressing the second antibody are hybridoma cell line 2A7, which is deposited with the China Center for Type Culture Collection (CCTCC) and has the CCTCC deposit number C2019302. The kit according to claim 22.

25. A method for detecting the presence or level of HBcAg protein in a sample, (1) A step of contacting a first antibody with the sample to form an antibody-antigen complex, wherein the first antibody is defined as in any one of claims 1 to 14; (2) A step of contacting a second antibody with the antibody-antigen complex to form an antibody-antigen-antibody complex, wherein the second antibody is defined as in any one of claims 1 to 14. (3) A step of determining the amount of the antibody-antigen-antibody complex, Methods that include...

26. The method of claim 25, wherein the second antibody has a detectable label, or the determination as described in step (3) includes using a third antibody that includes a detectable label.

27. ​​The detectable label is selected from an enzyme, a chemiluminescent reagent, a fluorescent dye, or biotin. The method according to claim 26.

28. The method according to any one of claims 25 to 27, wherein in step (3), the determination is selected from an enzyme immunoassay or a chemiluminescence immunoassay.

29. The method according to any one of claims 25 to 28, wherein the first antibody is coated onto the surface of a solid carrier.

30. The solid carrier is selected from magnetic beads or microtiter plates. The method according to claim 29.

31. The method according to any one of claims 25 to 30, wherein the sample is selected from the group consisting of whole blood, plasma, and serum.

32. Prior to step (1), the method further comprises a step of processing the sample, wherein the processing includes mixing the sample with a solvent so as to dissolve the virus, and / or Prior to step (2) and / or step (3), the method further includes a washing step. The method according to any one of claims 25 to 31.

33. A monoclonal antibody or its antigen-binding fragment that can specifically bind to HBcAg, (i) The monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region (VH) comprising the following three complementarity-determining regions (CDRs): HCDR1 having the sequence shown in SEQ ID NO: 11, HCDR2 having the sequence shown in SEQ ID NO: 12, and HCDR3 having the sequence shown in SEQ ID NO: 13, and a light chain variable region (VL) comprising the following three complementarity-determining regions (CDRs): LCDR1 having the sequence shown in SEQ ID NO: 14, LCDR2 having the sequence shown in SEQ ID NO: 15, and LCDR3 having the sequence shown in SEQ ID NO: 16, or (ii) An antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) containing three CDRs included in the heavy chain variable region shown in SEQ ID NO: 9, and a light chain variable region (VL) containing three CDRs included in the light chain variable region shown in SEQ ID NO: 10, (iii) A monoclonal antibody produced by hybridoma cell line 2A7, which is deposited with the China Center for Type Culture Collection (CCTCC) and has deposit number C2019302, either an antibody or its antigen-binding fragment. A monoclonal antibody or its antigen-binding fragment.

34. The monoclonal antibody or antigen-binding fragment thereof according to claim 33, wherein the CDR described in (ii) is defined by the Kabat, Chothia, or IMGT numbering system.

35. The monoclonal antibody or its antigen-binding fragment, (a) A heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared with the sequence shown in SEQ ID NO: 9, and including HCDR1 having the sequence shown in SEQ ID NO: 11, HCDR2 having the sequence shown in SEQ ID NO: 12, and HCDR3 having the sequence shown in SEQ ID NO: 13, And, (b) A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity when compared with the sequence shown in (iii) Sequence ID No. 10, and including an LCDR1 having the sequence shown in Sequence ID No. 14, an LCDR2 having the sequence shown in Sequence ID No. 15, and an LCDR3 having the sequence shown in Sequence ID No. 16, A monoclonal antibody or antigen-binding fragment thereof according to claim 33, comprising:

36. The monoclonal antibody or antigen-binding fragment according to claim 35, wherein the monoclonal antibody or antigen-binding fragment comprises VH having the sequence shown in SEQ ID NO: 9 and VL having the sequence shown in SEQ ID NO:

10.

37. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 33 to 36, wherein the monoclonal antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), and / or the monoclonal antibody is an IgG, IgM, IgE, IgD, or IgA antibody.

38. The antigen-binding fragment is Fab, Fab', (Fab') ２ A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 33 to 37, wherein the monoclonal antibody is selected from the group consisting of Fv, disulfide-linked Fv, scFv, diabody, and single-domain antibody (sdAb), and / or the monoclonal antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.

39. Use of a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 33 to 38 in the preparation of a reagent for detecting HBcAg in a sample.

40. The use according to claim 39, having one or more features selected from the group consisting of (i) to (iii) below. (i) The sample is a tissue sample or a cell sample. (ii) The detection is an immunological detection selected from immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), and Western blotting. (iii) The monoclonal antibody or the antigen-binding fragment thereof has a detectable label, or the reagent further comprises a secondary antibody having a detectable label, wherein the secondary antibody is an anti-immunoglobulin antibody.

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